A molecular switch for neuroprotective astrocyte reactivity.

The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.

Improving interface quality for 1-cm2 all-perovskite tandem solar cells.

All-perovskite tandem solar cells provide high power conversion efficiency at a low cost1-4. Rapid efficiency improvement in small-area (<0.1 cm2) tandem solar cells has been primarily driven by advances in low-bandgap (approximately 1.25 eV) perovskite bottom subcells5-7. However, unsolved issues remain for wide-bandgap (> 1.75 eV) perovskite top subcells8, which at present have large voltage and fill factor losses, particularly for large-area (>1 cm2) tandem solar cells. Here we develop a self-assembled monolayer of (4-(7H-dibenzo[c,g]carbazol-7-yl)butyl)phosphonic acid as a hole-selective layer for wide-bandgap perovskite solar cells, which facilitates subsequent growth of high-quality wide-bandgap perovskite over a large area with suppressed interfacial non-radiative recombination, enabling efficient hole extraction. By integrating (4-(7H-dibenzo[c,g]carbazol-7-yl)butyl)phosphonic acid in devices, we demonstrate a high open-circuit voltage (VOC) of 1.31 V in a 1.77-eV perovskite solar cell, corresponding to a very low VOC deficit of 0.46 V (with respect to the bandgap). With these wide-bandgap perovskite subcells, we report 27.0% (26.4% certified stabilized) monolithic all-perovskite tandem solar cells with an aperture area of 1.044 cm2. The certified tandem cell shows an outstanding combination of a high VOC of 2.12 V and a fill factor of 82.6%. Our demonstration of the large-area tandem solar cells with high certified efficiency is a key step towards scaling up all-perovskite tandem photovoltaic technology.

Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing.

The mRNA 5' cap structure serves both to protect transcripts from degradation and promote their translation. Cap removal is thus an integral component of mRNA turnover that is carried out by cellular decapping enzymes, whose activity is tightly regulated and coupled to other stages of the mRNA decay pathway. The poxvirus vaccinia virus (VACV) encodes its own decapping enzymes, D9 and D10, that act on cellular and viral mRNA, but may be regulated differently than their cellular counterparts. Here, we evaluated the targeting potential of these viral enzymes using RNA sequencing from cells infected with wild-type and decapping mutant versions of VACV as well as in uninfected cells expressing D10. We found that D9 and D10 target an overlapping subset of viral transcripts but that D10 plays a dominant role in depleting the vast majority of human transcripts, although not in an indiscriminate manner. Unexpectedly, the splicing architecture of a gene influences how robustly its corresponding transcript is targeted by D10, as transcripts derived from intronless genes are less susceptible to enzymatic decapping by D10. As all VACV genes are intronless, preferential decapping of transcripts from intron-containing genes provides an unanticipated mechanism for the virus to disproportionately deplete host transcripts and remodel the infected cell transcriptome.

Quantitative BONCAT Allows Identification of Newly Synthesized Proteins after Optic Nerve Injury.

Retinal ganglion cells (RGCs) die after optic nerve trauma or in degenerative disease. However, acute changes in protein expression that may regulate RGC response to injury are not fully understood, and detailed methods to quantify new protein synthesis have not been tested. Here, we develop and apply a new in vivo quantitative measure of newly synthesized proteins to examine changes occurring in the retina after optic nerve injury. Azidohomoalanine, a noncanonical amino acid, was injected intravitreally into the eyes of rodents of either sex with or without optic nerve injury. Isotope variants of biotin-alkyne were used for quantitative BONCAT (QBONCAT) mass spectrometry, allowing identification of protein synthesis and transport rate changes in more than 1000 proteins at 1 or 5 d after optic nerve injury. In vitro screening showed several newly synthesized proteins regulate axon outgrowth in primary neurons in vitro This novel approach to targeted quantification of newly synthesized proteins after injury uncovers a dynamic translational response within broader proteostasis regulation and enhances our understanding of the cellular response to injury.SIGNIFICANCE STATEMENT Optic nerve injury results in death and degeneration of retinal ganglion cells and their axons. The specific cellular response to injury, including changes in new protein synthesis, is obscured by existing proteins and protein degradation. In this study, we introduce QBONCAT to isolate and quantify acute protein synthesis and subsequent transport between cellular compartments. We identify novel candidate protein effectors of the regenerative response and uncover their regulation of axon growth in vitro, validating the utility of QBONCAT for the discovery of novel regulatory and therapeutic candidates after optic nerve injury.

Regulation of Neuronal Survival and Axon Growth by a Perinuclear cAMP Compartment.

cAMP signaling is known to be critical in neuronal survival and axon growth. Increasingly the subcellular compartmentation of cAMP signaling has been appreciated, but outside of dendritic synaptic regulation, few cAMP compartments have been defined in terms of molecular composition or function in neurons. Specificity in cAMP signaling is conferred in large part by A-kinase anchoring proteins (AKAPs) that localize protein kinase A and other signaling enzymes to discrete intracellular compartments. We now reveal that cAMP signaling within a perinuclear neuronal compartment organized by the large multivalent scaffold protein mAKAPα promotes neuronal survival and axon growth. mAKAPα signalosome function is explored using new molecular tools designed to specifically alter local cAMP levels as studied by live-cell FRET imaging. In addition, enhancement of mAKAPα-associated cAMP signaling by isoform-specific displacement of bound phosphodiesterase is demonstrated to increase retinal ganglion cell survival in vivo in mice of both sexes following optic nerve crush injury. These findings define a novel neuronal compartment that confers cAMP regulation of neuroprotection and axon growth and that may be therapeutically targeted in disease.SIGNIFICANCE STATEMENT cAMP is a second messenger responsible for the regulation of diverse cellular processes including neuronal neurite extension and survival following injury. Signal transduction by cAMP is highly compartmentalized in large part because of the formation of discrete, localized multimolecular signaling complexes by A-kinase anchoring proteins. Although the concept of cAMP compartmentation is well established, the function and identity of these compartments remain poorly understood in neurons. In this study, we provide evidence for a neuronal perinuclear cAMP compartment organized by the scaffold protein mAKAPα that is necessary and sufficient for the induction of neurite outgrowth in vitro and for the survival of retinal ganglion cells in vivo following optic nerve injury.

Rejection of xenogeneic porcine islets in humanized mice is characterized by graft-infiltrating Th17 cells and activated B cells.

Xenogeneic porcine islet transplantation is a promising potential therapy for type 1 diabetes (T1D). Understanding human immune responses against porcine islets is crucial for the design of optimal immunomodulatory regimens for effective control of xenogeneic rejection of porcine islets in humans. Humanized mice are a valuable tool for studying human immune responses and therefore present an attractive alternative to human subject research. Here, by using a pig-to-humanized mouse model of xenogeneic islet transplantation, we described the human immune response to transplanted porcine islets, a process characterized by dense islet xenograft infiltration of human CD45+ cells comprising activated human B cells, CD4+ CD44+ IL-17+ Th17 cells, and CD68+ macrophages. In addition, we tested an experimental immunomodulatory regimen in promoting long-term islet xenograft survival, a triple therapy consisting of donor splenocytes treated with ethylcarbodiimide (ECDI-SP), and peri-transplant rituximab and rapamycin. We observed that the triple therapy effectively inhibited graft infiltration of T and B cells as well as macrophages, promoted transitional B cells both in the periphery and in the islet xenografts, and provided a superior islet xenograft protection. Our study therefore indicates an advantage of donor ECDI-SP treatment in controlling human immune cells in promoting long-term islet xenograft survival.

GeneSurrounder: network-based identification of disease genes in expression data.

BACKGROUND: A key challenge of identifying disease-associated genes is analyzing transcriptomic data in the context of regulatory networks that control cellular processes in order to capture multi-gene interactions and yield mechanistically interpretable results. One existing category of analysis techniques identifies groups of related genes using interaction networks, but these gene sets often comprise tens or hundreds of genes, making experimental follow-up challenging. A more recent category of methods identifies precise gene targets while incorporating systems-level information, but these techniques do not determine whether a gene is a driving source of changes in its network, an important characteristic when looking for potential drug targets. RESULTS: We introduce GeneSurrounder, an analysis method that integrates expression data and network information in a novel procedure to detect genes that are sources of dysregulation on the network. The key idea of our method is to score genes based on the evidence that they influence the dysregulation of their neighbors on the network in a manner that impacts cell function. Applying GeneSurrounder to real expression data, we show that our method is able to identify biologically relevant genes, integrate pathway and expression data, and yield more reproducible results across multiple studies of the same phenotype than competing methods. CONCLUSIONS: Together these findings suggest that GeneSurrounder provides a new avenue for identifying individual genes that can be targeted therapeutically. The key innovation of GeneSurrounder is the combination of pathway network information with gene expression data to determine the degree to which a gene is a source of dysregulation on the network. By prioritizing genes in this way, our method provides insights into disease mechanisms and suggests diagnostic and therapeutic targets. Our method can be used to help biologists select among tens or hundreds of genes for further validation. The implementation in R is available at github.com/sahildshah1/gene-surrounder.

Biomimetic nanoparticles for transplantation tolerance.

PURPOSE OF REVIEW: The current review aims to provide a current landscape and future trends of biomimetic nanoparticles which have the potential to revolutionize the field of transplantation in the next decade. RECENT FINDINGS: Currently, the inability to safely induce robust donor-specific immunological tolerance makes it difficult to achieve immunosuppression-free graft survival. Despite progresses in the development of nanotherapeutics for antigen-specific immunomodulation in autoimmune diseases and in cancer treatments, few have been proposed and tested in transplantation with success. The complexity of parallel rejection mechanisms, multitude of antigen epitopes, and potential epitope spreading have challenged conventional nanodelivery systems in transplant models. Overcoming such challenges, biomimetic nanotherapeutics represent a promising alternative, as they allow better recapitulation of the complexity of the main biological players involved in tolerance. Within biomimetic nanodelivery systems, we envision that hybrid systems mimicking extracellular vesicles have the potential to bridging the gap between cell-based therapies, which are effective but costly and difficult to translate in clinical practice, and fully synthetic systems which are relatively easy to manufacture but lack the capacity to recapitulate the complexity of transplant antigens and tolerance mechanisms. SUMMARY: Next-generation nanotherapeutics for tolerance delivery is evolving toward biomimetic systems capable of capturing an increasing level of antigen complexity and exploiting multiple tolerance pathways.

Mass Spectrometry-Based Visualization of Molecules Associated with Human Habitats.

The cars we drive, the homes we live in, the restaurants we visit, and the laboratories and offices we work in are all a part of the modern human habitat. Remarkably, little is known about the diversity of chemicals present in these environments and to what degree molecules from our bodies influence the built environment that surrounds us and vice versa. We therefore set out to visualize the chemical diversity of five built human habitats together with their occupants, to provide a snapshot of the various molecules to which humans are exposed on a daily basis. The molecular inventory was obtained through untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of samples from each human habitat and from the people that occupy those habitats. Mapping MS-derived data onto 3D models of the environments showed that frequently touched surfaces, such as handles (e.g., door, bicycle), resemble the molecular fingerprint of the human skin more closely than other surfaces that are less frequently in direct contact with humans (e.g., wall, bicycle frame). Approximately 50% of the MS/MS spectra detected were shared between people and the environment. Personal care products, plasticizers, cleaning supplies, food, food additives, and even medications that were found to be a part of the human habitat. The annotations indicate that significant transfer of chemicals takes place between us and our built environment. The workflows applied here will lay the foundation for future studies of molecular distributions in medical, forensic, architectural, space exploration, and environmental applications.

Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.

Viable tumor cells actively release vesicles into the peripheral circulation and other biologic fluids, which exhibit proteins and RNAs characteristic of that cell. Our group demonstrated the presence of these extracellular vesicles of tumor origin within the peripheral circulation of cancer patients and proposed their utility for diagnosing the presence of tumors and monitoring their response to therapy in the 1970s. However, it has only been in the past 10 years that these vesicles have garnered interest based on the recognition that they serve as essential vehicles for intercellular communication, are key determinants of the immunosuppressive microenvironment observed in cancer and provide stability to tumor-derived components that can serve as diagnostic biomarkers. To date, the clinical utility of extracellular vesicles has been hampered by issues with nomenclature and methods of isolation. The term "exosomes" was introduced in 1981 to denote any nanometer-sized vesicles released outside the cell and to differentiate them from intracellular vesicles. Based on this original definition, we use "exosomes" as synonymous with "extracellular vesicles." While our original studies used ultracentrifugation to isolate these vesicles, we immediately became aware of the significant impact of the isolation method on the number, type, content and integrity of the vesicles isolated. In this review, we discuss and compare the most commonly utilized methods for purifying exosomes for post-isolation analyses. The exosomes derived from these approaches have been assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, there are pros and cons of each and there are critical issues linked with centrifugation-based methods, including co-isolation of non-exosomal materials, damage to the vesicle's membrane structure and non-standardized parameters leading to qualitative and quantitative variability. The down-stream analyses of these resulting varying exosomes can yield misleading results and conclusions.

LC-MS/MS Validation Analysis of Trastuzumab Using dSIL Approach for Evaluating Pharmacokinetics.

Quantitative targeted proteomics based approaches deploy state-of-the-art Liquid chromatography tandem mass spectrometry LC-MS technologies and are evolving as a complementary technique to standard ligand-binding based assays. Advancements in MS technology, which have augmented the specificity, selectivity and sensitivity limits of detection and freedom from antibody generation, have made it amicable towards various clinical applications. In our current work, a surrogate peptide based quantitative proteomics assessment is performed by selecting specific signature peptides from the complementary determining region CDR region of trastuzumab (Herclon®, Roche products in India). We developed a double Stable Isotope Label (dSIL) approach by using two different surrogate peptides to evaluate the proteolytic digestion efficiency and accurate quantification of the target analyte peptide of Herclon® in human serum. Method validation experiments were meticulously performed as per bioanalytical method validation guidelines. The dSIL approach, using an LC-MS/MS based quantification assay demonstrated good linearity over a range of 5-500 µg/mL of Herclon®, and validation experimental data is in compliance with bioanalytical regulatory guidelines.

Maps of interaural delay in the owl's nucleus laminaris.

Axons from the nucleus magnocellularis form a presynaptic map of interaural time differences (ITDs) in the nucleus laminaris (NL). These inputs generate a field potential that varies systematically with recording position and can be used to measure the map of ITDs. In the barn owl, the representation of best ITD shifts with mediolateral position in NL, so as to form continuous, smoothly overlapping maps of ITD with iso-ITD contours that are not parallel to the NL border. Frontal space (0°) is, however, represented throughout and thus overrepresented with respect to the periphery. Measurements of presynaptic conduction delay, combined with a model of delay line conduction velocity, reveal that conduction delays can account for the mediolateral shifts in the map of ITD.

A functional circuit model of interaural time difference processing.

Inputs from the two sides of the brain interact to create maps of interaural time difference (ITD) in the nucleus laminaris of birds. How inputs from each side are matched with high temporal precision in ITD-sensitive circuits is unknown, given the differences in input path lengths from each side. To understand this problem in birds, we modeled the geometry of the input axons and their corresponding conduction velocities and latencies. Consistent with existing physiological data, we assumed a common latency up to the border of nucleus laminaris. We analyzed two biological implementations of the model, the single ITD map in chickens and the multiple maps of ITD in barn owls. For binaural inputs, since ipsi- and contralateral initial common latencies were very similar, we could restrict adaptive regulation of conduction velocity to within the nucleus. Other model applications include the simultaneous derivation of multiple conduction velocities from one set of measurements and the demonstration that contours with the same ITD cannot be parallel to the border of nucleus laminaris in the owl. Physiological tests of the predictions of the model demonstrate its validity and robustness. This model may have relevance not only for auditory processing but also for other computational tasks that require adaptive regulation of conduction velocity.

A spiking neural network with continuous local learning for robust online brain machine interface.

Objective.Spiking neural networks (SNNs) are powerful tools that are well suited for brain machine interfaces (BMI) due to their similarity to biological neural systems and computational efficiency. They have shown comparable accuracy to state-of-the-art methods, but current training methods require large amounts of memory, and they cannot be trained on a continuous input stream without pausing periodically to perform backpropagation. An ideal BMI should be capable training continuously without interruption to minimize disruption to the user and adapt to changing neural environments.Approach.We propose a continuous SNN weight update algorithm that can be trained to perform regression learning with no need for storing past spiking events in memory. As a result, the amount of memory needed for training is constant regardless of the input duration. We evaluate the accuracy of the network on recordings of neural data taken from the premotor cortex of a primate performing reaching tasks. Additionally, we evaluate the SNN in a simulated closed loop environment and observe its ability to adapt to sudden changes in the input neural structure.Main results.The continuous learning SNN achieves the same peak correlation (ρ=0.7) as existing SNN training methods when trained offline on real neural data while reducing the total memory usage by 92%. Additionally, it matches state-of-the-art accuracy in a closed loop environment, demonstrates adaptability when subjected to multiple types of neural input disruptions, and is capable of being trained online without any prior offline training.Significance.This work presents a neural decoding algorithm that can be trained rapidly in a closed loop setting. The algorithm increases the speed of acclimating a new user to the system and also can adapt to sudden changes in neural behavior with minimal disruption to the user.

Gene regulatory roles of growth and differentiation factors in retinal development.

Retinal ganglion cell (RGC) differentiation is tightly controlled by extrinsic and intrinsic factors. Growth and differentiation factor 15 (GDF-15) promotes RGC differentiation, opposite to GDF-11 which inhibits RGC differentiation, both in the mouse retina and in human stem cells. To deepen our understanding of how these two closely related molecules confer opposing effects on retinal development, here we assess the transcriptional profiles of mouse retinal progenitors exposed to exogenous GDF-11 or -15. We find a dichotomous effect of GDF-15 on RGC differentiation, decreasing RGCs expressing residual pro-proliferative genes and increasing RGCs expressing non-proliferative genes, suggestive of greater RGC maturation. Furthermore, GDF-11 promoted the differentiation of photoreceptors and amacrine cells. These data enhance our understanding of the mechanisms underlying the differentiation of RGCs and photoreceptors from retinal progenitors and suggest new approaches to the optimization of protocols for the differentiation of these cell types.

Optineurin-facilitated axonal mitochondria delivery promotes neuroprotection and axon regeneration.

Optineurin (OPTN) mutations are linked to amyotrophic lateral sclerosis (ALS) and normal tension glaucoma (NTG), but a relevant animal model is lacking, and the molecular mechanisms underlying neurodegeneration are unknown. We found that OPTN C-terminus truncation (OPTN∆C) causes late-onset neurodegeneration of retinal ganglion cells (RGCs), optic nerve (ON), and spinal cord motor neurons, preceded by a striking decrease of axonal mitochondria. Surprisingly, we discover that OPTN directly interacts with both microtubules and the mitochondrial transport complex TRAK1/KIF5B, stabilizing them for proper anterograde axonal mitochondrial transport, in a C-terminus dependent manner. Encouragingly, overexpressing OPTN/TRAK1/KIF5B reverses not only OPTN truncation-induced, but also ocular hypertension-induced neurodegeneration, and promotes striking ON regeneration. Therefore, in addition to generating new animal models for NTG and ALS, our results establish OPTN as a novel facilitator of the microtubule-dependent mitochondrial transport necessary for adequate axonal mitochondria delivery, and its loss as the likely molecular mechanism of neurodegeneration.

Maps of ITD in the nucleus laminaris of the barn owl.

Axons from the nucleus magnocellularis (NM) and their targets in nucleus laminaris (NL) form the circuit responsible for encoding interaural time differences (ITDs). In barn owls, NL receives bilateral inputs from NM such that axons from the ipsilateral NM enter NL dorsally, while contralateral axons enter from the ventral side. These afferents and their synapses on NL neurons generate a tone-induced local field potential, or neurophonic, that varies systematically with position in NL. From dorsal to ventral within the nucleus, the best interaural time difference (ITD) of the neurophonic shifts from contralateral space to best ITDs around 0 µs. Earlier recordings suggested that in NL, iso-delay contours ran parallel to the dorsal and ventral borders of NL (Sullivan WE, Konishi M. Proc Natl Acad Sci U S A 83:8400-8404, 1986). This axis is orthogonal to that seen in chicken NL, where a single map of ITD runs from around 0 µs ITD medially to contralateral space laterally (Köppl C, Carr CE. Biol Cyber 98:541-559, 2008). Yet the trajectories of the NM axons are similar in owl and chicken (Seidl AH, Rubel EW, Harris DM, J Neurosci 30:70-80, 2010). We therefore used clicks to measure conduction time in NL and made lesions to mark the 0 µs iso-delay contour in multiple penetrations along an isofrequency slab. Iso-delay contours were not parallel to the dorsal and ventral borders of NL; instead the 0 µs iso-delay contour shifted systematically from a dorsal position in medial NL to a ventral position in lateral NL. Could different conduction delays account for the mediolateral shift in the representation of 0 µs ITD? We measured conduction delays using the neurophonic potential and developed a simple linear model of the delay-line conduction velocity. We then raised young owls with time-delaying earplugs in one ear (Gold JI, Knudsen EI, J Neurophysiol 82:2197-2209, 1999) to examine map plasticity.

Clinico-microbiological Features and Treatment Outcomes of Serratia Keratitis and Comparison with Pseudomonas aeruginosa Keratitis.

PURPOSE: To describe clinico-microbiological features and outcomes of Serratia keratitis and to compare them with Pseudomonas aeruginosa keratitis. METHODS: Cases of microbiologically proven Serratia keratitis and P. aeruginosa keratitis were reviewed. Data regarding demographic and clinical characteristics, and outcomes were recorded. RESULTS: 39 patients with pure Serratia keratitis were included. Median presenting vision was 1.8 logMAR (IQR, 0.8-2.4) and median infiltrate size was 5 mm (IQR 3-7.8 mm). An ocular risk factor was present in 35 (89.7%) cases. S. marcescens was the most common species (31/39, 79.5%). Medical resolution was observed in 36/39 (92.3%) cases, while three (7.7%) eyes needed penetrating keratoplasty. On comparing with P. aeruginosa keratitis (58 eyes), no difference in outcomes (p = .14) was noted. CONCLUSION: Serratia keratitis usually occurs in eyes with a compromised surface and has good resolution with medical therapy. Both Serratia and P. aeruginosa keratitis have similar outcomes.

Kif5a Regulates Mitochondrial Transport in Developing Retinal Ganglion Cells In Vitro.

Purpose: Axon transport of organelles and neurotrophic factors is necessary for maintaining cellular function and survival of retinal ganglion cells (RGCs). However, it is not clear how trafficking of mitochondria, essential for RGC growth and maturation, changes during RGC development. The purpose of this study was to understand the dynamics and regulation of mitochondrial transport during RGC maturation using acutely purified RGCs as a model system. Methods: Primary RGCs were immunopanned from rats of either sex during three stages of development. MitoTracker dye and live-cell imaging were used to quantify mitochondrial motility. Analysis of single-cell RNA sequencing was used to identify Kinesin family member 5A (Kif5a) as a relevant motor candidate for mitochondrial transport. Kif5a expression was manipulated with either short hairpin RNA (shRNA) or exogenous expression adeno-associated virus viral vectors. Results: Anterograde and retrograde mitochondrial trafficking and motility decreased through RGC development. Similarly, the expression of Kif5a, a motor protein that transports mitochondria, also decreased during development. Kif5a knockdown decreased anterograde mitochondrial transport, while Kif5a expression increased general mitochondrial motility and anterograde mitochondrial transport. Conclusions: Our results suggested that Kif5a directly regulates mitochondrial axonal transport in developing RGCs. Future work exploring the role of Kif5a in vivo in RGCs is indicated.

Determination of Mobile Ion Densities in Halide Perovskites via Low-Frequency Capacitance and Charge Extraction Techniques.

Mobile ions in perovskite photovoltaic devices can hinder performance and cause degradation by impeding charge extraction and screening the internal field. Accurately quantifying mobile ion densities remains a challenge and is a highly debated topic. We assess the suitability of several experimental methodologies for determining mobile ion densities by using drift-diffusion simulations. We found that charge extraction by linearly increasing voltage (CELIV) underestimates ion density, but bias-assisted charge extraction (BACE) can accurately reproduce ionic lower than the electrode charge. A modified Mott-Schottky (MS) analysis at low frequencies can provide ion density values for high excess ionic densities, typical for perovskites. The most significant contribution to capacitance originates from the ionic depletion layer rather than the accumulation layer. Using low-frequency MS analysis, we also demonstrate light-induced generation of mobile ions. These methods enable accurate tracking of ionic densities during device aging and a deeper understanding of ionic losses.