United States

Background: Meningiomas are the most common primary intracranial tumor, accounting for over 30% of all primary brain tumors. Although most are benign, up to 20% recur and can be considered aggressive. Based on RNA and whole-exome sequencing data, we identified three molecular subtypes of meningioma. Meningioma Group B and C (MenG B/C) are characterized by deletion of Neurofibromatosis Type 2 (NF2) gene. Simultaneously, the deactivation of the Hippo pathway has been linked to meningioma growth. NF2 inactivation has previously been linked to deregulating the Hippo signaling pathway; however, the mechanism of this relationship is unclear in meningioma. This study aims to investigate the impact of common meningiomal NF2 mutations on Hippo pathway activity and identify which specific NF2-Hippo signaling interactions contribute to these effects. 

Methods: We conducted an extensive literature review to identify the most common NF2 mutations in meningioma. Six of these mutants were cloned and overexpressed in 293 cells with a N-terminal mono-GFP tag. Localization was assessed via fluorescence imaging and compared to wild-type (WT) NF2 localization. We then generated a split-luciferase Hippo pathway biosensor to quantify mutant NF2’s effects on downstream Hippo pathway activity. In this biosensor, one of two luciferase domains is attached to a small amino acid stretch of the YAP protein that harbors the phosphorylation site for degradation. The other part is attached to the phospho-dependent interactor 14-3-3. When Hippo is on, the YAP site gets phosphorylated, binds to 14-3-3 and completes a functional luciferase, which emits a luminescence signal upon substrate binding. WT and mutant NF2s were co-transfected with this biosensor and corresponding signals were quantified. In a second line of experiments WT and mutants were co-expressed with Hippo pathway kinases, MST1 and LATS1, then immunoprecipitated to investigate if these interact. 

Results: We found 19 articles and one database describing 205 NF2 mutations in meningioma, most of which were nonsense mutations. We investigated six of the most common mutants along with the three most common WT NF2 variations (v1, v2, v7). NF2 var7 and NF2 mutants showed aberrant protein aggregation compared to var1 or var2 NF2 which correlated with severity of truncation. Luciferase assay revealed that var7 NF2 as well as mutant NF2 decreased YAP phosphorylation and subsequent Hippo pathway activity. Immunoprecipitation and Western Blot results found that although NF2 v1 and v2 show normal MST1 and LATS interaction, var7 lost interaction with MST1. Interestingly, the NF2 mutants lacked LATS1 interaction, but MST1 interacted even with the smallest truncation of NF2. 

Conclusion: We propose that NF2 forms a tertiary complex with MST1 and LATS1 to activate the Hippo kinase cascade and prevent proliferation. NF2 nonsense mutations create aberrant non-functional proteins that disrupt this complex formation and attenuate Hippo signaling. The naturally occurring var7 NF2 loses MST1 interaction while the NF2 mutations lose LATS1 interaction. These findings have strong therapeutic implications in targeting the MST1-LATS interaction to suppress oncogenicity. Future directions suggest investigating the role of TRAF7 in Hippo signaling, enabling a complete picture of the Hippo pathway in meningioma.

AMA Research Challenge 2024 

NF2 mutations found in meningioma disrupt the kinase cascade of the Hippo pathway

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INTRODUCTION
Marine organisms produce a wide variety of metabolites with unique scaffolds that are biologically active. Manzamines are marine-derived polycyclic alkaloids. Approximately 100 manzamine-type alkaloids have been isolated from 16 marine sponge species. Some of these compounds are reported to have anti-inflammatory and neuritogenic properties. Previous neurite outgrowth screens have identified manzamine A and 8-methoxymanzamine A for further analyses. 
METHODS
To assess the neuritogenic potential, this study conducted neurite outgrowth assays to build dose-response curves for manzamine A and 8-methoxymanzamine A in murine primary cortical cultures. Sytox assays were performed in the Celloger to assess cell toxicity. 
RESULTS
The neurite outgrowth analyses showed that 8-methoxymanzamine A induced neurite outgrowth in a statistically significant manner at 1 micromolar dose. Likewise, that dose and others demonstrated a statistically significant increase in neuronal complexity via number of processes and number of branches. However, at 10 micromolar dosage, 8-methoxymanzamine appears to be largely toxic, with reduced neurite outgrowth, number of processes and number of branches, accompanied by some nuclear blebbing indicative of apoptosis. In contrast, manzamine A doesn’t show increased neurite outgrowth or complexity at any dose. Moreover, like 8-methoxymanzamine A, it appears potentially toxic at the 10 micromolar dose. Initial studies with Sytox indicate substantial toxicity for manzamine A and 8-mehthoxymanzamine A between 1 and 10 micromolar dosage.
CONCLUSIONS
Together, these data suggest 8-methoxymanzamine A has the potential to increase neurite outgrowth, and shows similar indications of toxicity at the 10 micromolar dose as manzamine A. Further studies will be performed with Sytox to refine the concentration at which it is toxic to 50% of cells (LC50). Together, these studies will help identify novel neuroactive manzamine compounds for the marine natural products preclinical pipeline.
Study conducted with intramural funding from Midwestern University.

8-methoxymanzamine A, but not manzamine A, induced neurite outgrowth in murine cortical cultures

Background: Stuttering is a neurodevelopmental disorder of unclear etiology characterized by involuntary disruptions in speech fluency. Recently, stuttering has been shown to be related to a point mutation in GNPTAB, a housekeeping gene involved in cellular lysosomal enzyme-targeting pathway, though it remained unclear how such a mutation might cause the stuttering phenotype. Transcranial ultrasounds in adults who stutter detected mesencephalic hyperechogenicity, likely associated with iron deposits. The current study aims to confirm, assess, and alleviate iron accumulation in a transgenic mouse model engineered with a mutation associated with Gnptab. 
Methods: 
Animal studies: All procedures in this study were approved by the Animal Care and Use Committee of the Intramural Research Program of the National Institute of Neurological Disorders and Stroke. 
Vocalization studies: Ultrasonic vocalizations (USVs) in male mice were recorded via Avisoft Bioacoustics and SASLab Pro software, and the complexity and geometry of USVs were analyzed in Sonotrack (Metris Inc). 
Iron chelation therapy: Adult male Gnptab-mutant (n = 10) and control littermates (n = 10) were treated by 3- Hydroxy-1,2-dimethyl-4(1H)-pyridone (Deferiprone; 0.1 g/L) orally for 1 month via water supply. Animals' vocal behaviors were recorded and compared before and after the treatment. 
Histological processing: Mice were transcardially perfused with 4% paraformaldehyde (PFA) fixative. The brain was extracted and post-fixed for 3-5 days in PFA. Iron was detected by iron-sensitive DAB-enhanced Perl’s staining on coronal brain sections. Bright field images were taken and imported to Fiji, where regional iron content was quantified. 
Statistical analysis: All data was exported to Prism 9.0. Statistical significance was taken at p < 0.05. 
Results: Gnptab-mutant animals show higher percentage of complex USVs and higher iron deposition in the medial lateral striatum, dorsolateral striatum, and central striatum when analyzed for percent area of iron deposition following Perl’s staining. Alleviation of this iron accumulation via chelation therapy was confirmed by Perl’s staining. Strikingly, the percentage of complex vocal bouts decreased following iron chelator therapy. 
Conclusion: The mutation in Gnptab gene, associated with stuttering, leads to an accumulation of CNS iron that may be alleviated with iron chelation, ultimately improving the stuttering phenotype in mice. Accordingly, we posit a novel therapeutic option and long-awaited etiology of the disorder.

An etiology and novel therapeutic opportunity: iron dysregulation in a mouse model of stuttering disorder

Abstract Title 
Hyperexcitation of Mediobasal Hypothalamic Glutamatergic Neurons Induces a Phenotype Resembling Anorexia Nervosa

Background
Anorexia Nervosa (AN) is an eating disorder characterized by a voluntary restriction in food intake resulting in a sustained critically low body weight, hyperactivity, and increased anxiety. AN is among the deadliest psychiatric disorders, and the primary method of assessing its neurobiological basis is with animal models. However, current animal models restrict feeding, exogenously induce stress, and/or show unrelated phenotypes like normoactivity, dissimilar from the hyperactivity and self-induced starvation seen in AN. 
The Mediobasal hypothalamus (MBH) is known to be involved in the regulation of feeding and anxiety-related behaviors. In particular, once activated, glutamatergic neurons within the MBH promote these behaviors. However, these approaches acutely activated the neurons, and the behavioral changes were usually transient. Whether chronic activation of glutamatergic MBH neurons is involved in AN is unknown. 
Previous studies identified a bacterial sodium channel (NachBac), which is more readily activated and takes more time to inactivate than mammalian counterparts. This leads to constantly increased activity when expressed in mammalian neurons. Recent treatment-based rationale suggested chronic glutamatergic signaling may contribute to AN. Based on these observations, we hypothesized that chronic activation of glutamatergic neurons in the MBH will recapitulate the self-induced starvation observed in AN.

Methods
We used the Cre-LoxP technique to achieve neuron-specific gene expression, where genes flanked with LoxP were expressed in the presence of Cre recombinase. We used mice expressing Cre driven by the promoter of vesicular glutamate transporter 2 (Vglut2, a marker gene for glutamatergic neurons), Vglut2-Cre, to target hypothalamic glutamatergic neurons. We stereotaxically injected an Adeno-Associated Virus with a FLip-EXcision vector, AAV-FLEX-NaChBac-GFP, into the MBH to express NaChBac in a Cre-dependent manner.

Results
Interestingly, not only did we observe decreased feeding behavior, but we also observed increased locomotion, increased anxiety, and drastically decreased body weight, classically resembling AN.

Conclusion
These results demonstrate that constitutive activation of glutamatergic MBH neurons recapitulates the AN phenotype. In contrast to the most-used AN mouse models, our model exhibits AN phenotype without feeding restriction or stress induction. Importantly, these results complement the growing rationale of increased glutamatergic signaling in the pathophysiology of AN, based on the use of the glutamate receptor antagonist, ketamine, in treating AN in mice and humans. These findings reveal neurobiological evidence of glutamatergic signaling hyperexcitation contributing to the AN phenotype and provide a more clinically relevant animal model to examine mechanistic insights on AN pathogenesis.

Chronic Activation of Ventral Hypothalamic Glutamatergic Neurons Induces Anorexia Nervosa Phenotype in Mice

Background
Glaucoma is a group of optic neuropathies that affects approximately 2.7 million people in the United States and 76 million people worldwide. Glaucoma is irreversible and can result in permanent blindness if it is not treated. The main risk factor is elevated intraocular pressure (IOP) which predominantly affects retinal ganglion cells (RGC), resulting in cell death and permanent vision loss. Current therapeutics for glaucoma involve reducing IOP and halting progression of disease, but no current treatments can revive degenerated RGCs. Our project aims to evaluate the therapeutic effect of a neurotrophic factor, human Neuritin 1 (NRN1), in regenerating and protecting RGCs lost due to glaucomatous disease progression. Human NRN1 has been proven to enhance plasticity and maturation of RGC axons and dendrites in an acute glaucoma mouse model. The present study utilized non-glaucomatous and glaucomatous induced pluripotent stem cells (iPSC) derived RGCs seeded within in vitro collagen scaffolds. By administering NRN1 to the scaffolds, we hope to elucidate the ability of the therapeutic to increase survival of glaucomatous RGCs.

Methods
The iPSCs derived from non-glaucomatous and glaucomatous donor corneal fibroblasts were differentiated to retinal organoids (RO) to generate RGCs in vitro. After around 30 days of differentiation, the ROs were dissociated to isolate RGCs. The RGCs were seeded at one end of three different in vitro collagen scaffolds. The first scaffold received no NRN1 treatment, the second received NRN1 at the cell body, and the third received NRN1 at the opposite end of the cultured RGCs. Downstream immunofluorescence with the TRA-1-60 antibody was performed on iPSCs to confirm that they were successfully reprogrammed from donor fibroblasts. Additionally, gene expression experiments for iPSC markers were performed to further verify successful iPSC reprogramming. Karyotyping procedures were performed on iPSCs to ensure no reprogramming induced chromosomal aberrations were evident.

Results
We successfully generated iPSC-derived RGCs from both non-glaucomatous and glaucomatous donor eyes. These RGCs effectively integrated within the three collagen scaffolds. After NRN1 treatment, both cell types demonstrated differential expression of apoptotic markers. Downstream iPSC experimentation confirmed that iPSCs were successfully reprogrammed from donor fibroblasts with no chromosomal aberrations.

Conclusion
Our study demonstrates that NRN1 exhibits a therapeutic effect on glaucomatous RGCs. NRN1 could potentially be a useful intervention to restore vision in patients suffering from severe glaucoma. The results also indicate that iPSC-derived RGCs can successfully be obtained from donor eyes and used in collagen scaffolds for future research.

Determining the Restorative Role of Human Neuritin 1 in Protecting Retinal Ganglion Cells from Glaucoma Patients

Background
Glaucoma is a neurodegenerative ocular disorder involving the degeneration of retinal ganglion cells (RGCs), thinning of the retinal nerve fiber layer, and atrophy of the optic nerve. Disease progression is often characterized by increased intraocular pressure (IOP) and visual field loss. Currently, the only efficacious therapy for treating glaucoma is modification of IOP through pharmacological eye drops or surgical intervention. Even though these interventions slow disease progression, they do not prevent RGC death entirely thus emphasizing the need for targeting RGC survival. Decreased axonal transport of essential neurotrophic factors contributes to RGC death. Neurotrophic factor supplementation has shown to improve survival of RGCs and sustain retinal function. Neuritin-1 (NRN1), a neurotrophic protein downstream of the neurotrophin family, has shown to play a protective role in non-glaucomatous eyes exposed to high pressure via the ex vivo ocular perfusion model, Translaminar Autonomous System (TAS). We will investigate the effects of NRN1 in glaucomatous human donor eyes within the TAS to understand if NRN1 can protect RGC loss in diseased retinas.

Methods
Three sets of glaucomatous human donor eyes were obtained from eye banks in accordance with the Declaration of Helsinki. Eyes were dissected, and posterior cups were perfused using the TAS model for 6-7 days under high and normal pressure conditions with and without NRN1. We then assessed RGC survival by measuring apoptosis, inflammation, and retinal markers using qRT-PCR and immunohistochemistry. Retinal activity was compared between treated and untreated retinas using OcuScience® Ex Vivo electroretinogram.

Results
The posterior eye cups were successfully maintained in the TAS model under normal and high-pressure conditions for 6-7 days. NRN1 treated eyes showed differential expression of various inflammatory and apoptotic markers. NRN1 treated eyes showed no difference in extra cellular matrix deposition following perfusion. Improved retinal activity was seen within the treated glaucomatous eyes. 

Conclusion
The TAS model can mimic pressure induced pathogenesis in human glaucomatous eyes. Data from this study shows that NRN1 may serve as a potential therapeutic target by promoting RGC survival under glaucomatous conditions.


Evaluating the Therapeutic Effect of Neuritin 1 on Human Retinal Ganglion Cell Survival

Innate Immune Memory refers to the ability of innate immune cells to display altered responses upon secondary exposure to pathogens. This phenomenon is driven by dynamic and heritable histone protein modifications, which influence post-translational gene transcription. Currently, while the broader MAPK and PI3K/AKT pathways of α-MSH are well established, there is little existing research on the direct link between α-MSH and histone modifications. Our study aims to investigate the effects of alpha-melanocyte-stimulating hormone (α-MSH), a neuropeptide with notable anti-inflammatory properties, on Histone 3 (H3) post-translational markers and their epigenetic impact on mouse macrophages.

We conducted a comprehensive analysis of H3 modifications to identify key changes, focusing on H3K4 global methylation, H3K9 dimethylation, and H3K14 acetylation levels in response to varying concentrations of α-MSH. Methylation and acetylation of histone proteins, such as H3K4, H3K9, and H3K14, regulate the accessibility of transcriptional start sites and promoters, thereby influencing gene transcription. In immune cells, these histone modifications alter the transcription of genes associated with immune responses, particularly inflammation.

Utilizing enzyme-linked immunosorbent assays (ELISA) to quantify the H3 methylation and acetylation levels, the response of histone modifications to α-MSH treatment was measured, highlighting the potential of α-MSH as a modulator of epigenetic marks in innate immune cells. Such findings enhance our understanding of how neuropeptides like α-MSH can influence immune responses through epigenetic mechanisms, providing insights into new therapeutic strategies for controlling inflammation and enhancing immune memory.

Our results demonstrate significant effects of α-MSH on histone modifications, with notable demethylation of H3K4, increased dimethylation of H3K9, and elevated acetylation of H3K14 observed in treated macrophages. Specifically, significant changes in H3K4 global methylation and H3K14 acetylation were detected at 1 ng/mL and 10 ng/mL α-MSH, while H3K9 dimethylation was significantly affected at 30 pg/mL α-MSH. Additionally, low-dose α-MSH (30 pg/mL) induced demethylation and deacetylation of H3K4 and H3K14, respectively.

These findings suggest that α-MSH exerts anti-inflammatory effects by modulating histone methylation and acetylation patterns. Changes in H3 histone modifications decrease the expression of proinflammatory molecules and enhance the expression of anti-inflammatory molecules in macrophages. This epigenetic regulation may contribute to the innate immune memory, eliciting trained responses to subsequent exposures.

In summary, α-MSH modulates histone H3 post-translational modifications in a concentration-dependent manner, altering the epigenetic landscape of macrophages to promote anti-inflammatory responses. This research underscores the significance of histone modifications in the regulation of innate immune memory and the potential for targeting these pathways in immunotherapy. 



Histone Epigenetic Effects of α-MSH

Background
Age-related cataract affects 22% of individuals over the age of 75 years, and data suggest that delaying the progression rate by 10 years would decrease cataract surgery by 50%. When rodent lenses are incubated in culture medium, they lose their transparency after 24-48 hours due to protein aggregation, which changes the index of refraction resulting in light scattering and opacity. We hypothesized that finding ways to prevent or delay this process through manipulation of the culture medium would provide important clues toward cataract prevention and present a solid foundation for a drug testing model on specific mutated mice lenses for cataract treatment.
Methods
13 culture conditions, each comprising 4-6 adult C57BL6 mice lenses, were tested. Variations in glucose, pyruvate, fructose, antioxidant, osmolality (sorbitol), and crowding agent (polyethylene glycol) were used. The extent of opacification, the type of opacity (nuclear vs cortical), and the lens diameter were determined as a function of incubation time.
Results
Results show that compared to the baseline conditions (Medium TC199), basic nutrition materials that lenses need for survival and antioxidants that prevent oxidative stress in lenses show no improvement in preventing lens opacification. Next, we mimicked the lens environment in the eye and tested hyperosmolarity. PEG 1000 stood out among the rest by successfully preventing cataract formation in all of its lenses. We also noticed the lens size difference between the PEG 1000 condition and the rest. PEG 1000 successfully prevents the lens from swelling and keeps lens volume low. Thus, from our results, we tested another set of biopolymers which could prevent massive intake of medium to the lens that we saw in previous conditions. We saw that Lymphoprep successfully kept lenses clear until day 4 (96 hours), which was a major improvement compared to the rest of the conditions that we tested.
Conclusion
This project is novel in its approach as the first to develop a system of drug testing lens model for cataract by keeping mice lenses clear and transparent for 7-14 days. While we are still in the process of reaching 7-14 days of clarity in lenses, we have already tested some strong potential conditions that improved lens transparency in vitro incubations, such as PEG 1000 and Lymphoprep. We plan to further test possible conditions for optimizing lens culture medium for in vitro lens transparency, specifically focusing on mimicking the lens environment in the eye to maintain lens homeostasis.

In vitro formation of cataract as a model for age-related cataract

Purpose: Infectious keratitis caused by Aspergillus species is difficult to treat with conventional therapies. Novel treatments have been used to treat these severe infections, such as Rose Bengal (RB) photodynamic antimicrobial therapy (PDAT), which uses a photosensitizer (PS) activated with light. This activation leads to the generation of reactive oxygen species, which can kill infectious organisms. Resistant fungal strains, like Aspergillus, are not inhibited by RB. Other green light activated photosensitizers, such as Erythrosin B (EB), may inhibit fungal isolates. This study will investigate singlet oxygen (1O2) generation and fungal growth inhibition in RB and EB. 

Methods: To quantify 1O2, one mL aliquots of RB and EB (1.0, 2.0, and 4.9 mM) were plated onto SabDex agar. The plates were then measured with an optical dosimeter to detect the 1O2 luminescence signal. To evaluate in vitro inhibition, two Aspergillus strains (A. fumigatus and A. glaucus) were isolated from patients with keratitis and prepared in solutions of 1x10E4 CFU/mL. Organisms were mixed with RB or EB at the same three concentrations as the 1O2 experiments. Organism/photosensitizer solutions were plated in triplicate and irradiated with a custom green (518 nm) LED light (5.4 J/cm2). Plates were then incubated for 72 hours and photographed to analyze fungal growth.

Results: For RB and EB,1O2 signal increased as the concentration increased. Also, EB had more 1O2 signal than RB at the highest concentration (4.9 mM) (Figure 1). The 1O2 findings were supported by the in vitro inhibition experiments which showed increased growth inhibition with increased PS concentration. Furthermore, EB had more inhibition of the Aspergillus organisms than RB (Figure 2).

Conclusions: This study shows 1O2 generation alongside strain specific inhibition of PDAT in support of high concentrations of EB to kill Aspergillus species. Increasing concentrations of RB and EB boosts the 1O2 signal and Aspergillus species growth inhibition. These findings support the idea that 1O2 dose is an important metric for evaluating the efficacy of PDAT. 


Inhibition of Fungal Isolates via Singlet Oxygen Generation from Erythrosin B and Rose Bengal PDAT

Purpose:
Corneal Schwann cells (cSCs) ensheath axons and are believed to play a vital role in the health and regeneration of the cornea, including its sensory functions. Here we have exploited a nitrogen mustard (NM) corneal injury model to examine cSC and axonal degeneration/regeneration post injury and also tested a novel drug micellar formulation RM4404 that targets cSCs as a potential therapeutic.

Methods:
Transgenic mice harboring the proteolipid protein 1 promoter enhanced green fluorescent protein (PLP-eGFP) gene construct that marks cSCs were used. Topical 0.2% NM solution was applied for 5 min under sedation followed by a phosphate buffered saline washing. Over days 8 to 14, mice were treated twice per day with a topical micellar formulation RM4404 while the control group received vehicle Kolliphor HS-15. Corneal mechanosensitivity scoring by esthesiometer was performed. Mice were euthanized and corneal flat-mounts were stained with axonal β3-tubulin antibody. Fluorescently stained tissues were imaged using epifluorescence microscopy. Tiled images of the corneal staining were assembled using Leica software. Axonal and cSC networks were measured using FIJI Neuroanatomy plugin.

Results:
We found that NM injury causes degeneration of cSCs and associated axons which remained significantly reduced during the 14-day post injury period (P=0.0025; P=0.0283). RM4404 treatment supported the regeneration of cSCs at 14 days post injury (dpi), showing significantly greater numbers in drug treated over vehicle-treated groups (P=0.0012), and RM4404 also supported the regeneration of axons at 14 dpi (P=0.0006). Comparisons of central versus peripheral corneal regions revealed that regeneration was greater at the peripheral regions for both cSCs and axons in RM4404 treated corneas compared to vehicle controls (P=0.0002; P=0.0002). Mechanosensory testing revealed sensory reduction at 7 dpi that remained low in vehicle treated mice at 14 dpi (P=0.0015), while RM4404 treated mice at 14 dpi showed significant restoration achieving corneal sensitivity of uninjured mice (P=0.7627).

Conclusions:
cSCs are vulnerable to DNA targeting agents, such as NM. Surviving cSCs in the corneal periphery can regenerate and promote trophic support for injured axons to regeneration. For the first time, we provide evidence that pharmacological targeting of cSCs can be exploited for axonal regeneration and restoration of corneal sensory function.

Sights Set on Corneal Schwann Cells: Restoring Corneal Sensory Function Post Nitrogen Mustard Injury


Abstract Title
Synergistic effect of silver nanoparticles and antibiotics in enhancing antimicrobial activity against ocular pathogens

Background
Ocular infections, such as endophthalmitis and keratitis, remain one of the leading causes of irreversible blindness, worldwide. Despite advances in antibiotic therapy, increasing antimicrobial resistance among pathogens limit treatment options. We sought to develop dual antibacterial nanoformulations using silver and antibiotics and evaluated their antimicrobial efficacy against ocular pathogens.

Methods
Silver nanoparticles were synthesized via a chemical reduction reaction, then loaded with vancomycin and coated with chitosan. Minimum inhibitory concentration (MIC) of silver nanoparticles against staphylococcus aureus (SA) and methicillin-resistant staphylococcus aureus (MRSA) were determined via double dilution antibacterial assay with optical density measured at 600 nanometers. Ex vivo porcine eyes, ex vivo human vitreous, cultured human retinal Müller glial (MIO-M1) cells, and in vivo mice eyes were then intravitreally infected with SA. Chitosan-silver-nanoparticles (CAgNPs), chitosan-silver-vancomycin-nanoparticles (CAgVNPs), and vancomycin alone were separately administered to the above testing groups post-6 hours of infection. The samples were harvested and plated for colony-forming units (CFUs). Statistical significance was performed using one-way ANOVA. Lastly, the inflammatory response and cytotoxicity of the nanoformulations were assessed: inflammatory markers of SA-infected mice eyes were evaluated via ELISA, cytotoxicity of nanoformulations in SA-infected MIO-M1 cells were evaluated quantitatively via LDH assay and qualitatively via Calcein assay, and cytotoxicity of nanoformulations in SA-infected mice eyes were evaluated via TUNEL assay and H&E staining.

Results
CAgVNPs had a two-fold reduction in MIC compared to vancomycin. Comparing CAgVNP to vancomycin, there was an 8-fold reduction in CFUs in ex vivo porcine eyes, a 3.5-fold reduction in ex vivo human vitreous, and a 103-fold reduction in in vivo mice eyes. Levels of inflammatory markers IL-1β, IL-6, and KC were decreased in SA-infected mice eyes treated with CAgVNPs compared to vancomycin. LDH values were the same as the control group without SA infection. Calcein assay showed that CAgVNPs were able to save MIO-M1 cells from SA infection. TUNEL assay of mice eye sections that were treated with CAgVNPs were histologically the same as the control group without SA infection. 

Conclusion
This study demonstrates that silver nanoparticles have potent antimicrobial activity against ocular pathogens ex vivo, in vitro, and in vivo. More importantly, silver nanoparticles have synergistic effects with antibiotics to reduce MIC as well as enhance antimicrobial activity. Lastly, silver nanoparticles do not demonstrate any cytotoxicity, indicating their potential use for treatment of ocular infections. 


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NF2 mutations found in meningioma disrupt the kinase cascade of the Hippo pathway

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