United States

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. 


AMA Research Challenge 2024 

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

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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. 


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

Background: Infectious keratitis (IK) is a sight-threatening corneal infection that causes inflammation, pain, and in severe cases blindness. Risk factors include contact lens use, trauma, surgery, and systemic disease. A recent outbreak of IK highlighted the potential danger of contaminated artificial tears. This incident involved a specific strain of drug-resistant Pseudomonas aeruginosa transmitted to patients. This critical event led to Food and Drug Administration (FDA) recalls of various artificial tear products. The need to understand microbial growth in artificial tears is clear. While preservatives are used to control contamination, their effectiveness varies, and the underlying mechanisms remain unclear. Understanding how the composition of artificial tears influences the growth rate of microbes causing IK is crucial. This study investigates the in vitro growth kinetics of IK isolates within artificial tears. 

Methods: Twenty different artificial tears were chosen for this study to cover a broad range of common ingredients (i.e. viscosity enhancing agents, electrolytes, buffers, osmoprotectants, etc.). These tears fell into three categories: preservative-free single-dose vials, preservative-free multidose bottles, and preservative-containing tears. Artificial tears were inoculated with a variety of microbes isolated from patients with IK including: bacterial (Staphylococcus spp., Mycobacterium spp., Corynebacterium spp., Serratia spp., Pseudomonas spp.), fungal (Candida spp., Fusarium spp., Aspergillus spp.), and protozoal (Acanthamoeba spp.) isolates. Artificial tear solutions were aliquoted in quadruplicate into a 96-well microplate and inoculated with microbial suspension. Microbial controls in tryptic soy broth and saline, as well as saline-only controls, were included. The turbidity of the samples was recorded periodically for 24 hours in a microplate reader at 37°C (Varioskan, ThermoFisher Scientific) to determine growth kinetics. Cell counts for each group were also performed using serial dilutions on Muller Hinton agar.

Results: Microbial growth varied across artificial tear formulations and microbe types. Preservative-free solutions generally supported higher bacterial growth compared to preservative-containing tears. Cell count data confirmed the trends observed through turbidity measurements. Artificial tear only and saline only samples did not show any microbial growth.

Conclusion: Preservative-free formulations appear to be particularly susceptible to supporting bacterial growth. This in vitro data highlights the importance of understanding how artificial tear composition influences microbial proliferation. Further research is warranted to explore the specific mechanisms by which tear ingredients promote or inhibit microbial growth. These findings can inform the development of safer artificial tear formulations with improved resistance to microbial contamination, ultimately contributing to the prevention and management of IK.

Tears For Fears? Investigating The Link Between Artificial Tears And Infection Risk

Background 
This study aims to investigate how the anterior cruciate ligament (ACL) compensates for the change in knee joint stability and contact pressures due to partial horizontal cleavage tears (HCTs) in the meniscus and subsequent surgical interventions, such as partial meniscectomy and partial transplantation on knee joint stability and contact pressures. 
Methods 
Seven freshly frozen human cadaveric knees were used in a study to investigate the effects of different meniscal conditions and surgical interventions on the meniscus itself. The knees were carefully thawed, examined, and prepared for experimentation. Four testing scenarios were established: intact knees, knees with partial horizontal cleavage tears (HCT) of the meniscus, knees with partial meniscectomy, and knees with partial transplantation. Axial loading was applied to simulate single-leg standing, and contact pressures of the medial meniscus were measured using digital sensors. An axial load was applied at 0° and 30° for each testing condition. Additionally, a mathematical 3D finite element model was created to evaluate the behavior of the ACL under different meniscus scenarios, which could not have been measured experimentally. This model was used to assess the ACL's response and validate its contact and pressure response at the meniscus level. 
Results 
ACL contact pressure and stress analysis across various meniscal conditions demonstrated substantial variability. Horizontal cleavage tears (HCTs) resulted in heightened contact pressures and diminished joint stability, as evidenced by increased ACL stress attributed to compensatory mechanisms in the presence of meniscal tears. Conversely, transplantation procedures exhibited a mitigating effect, maintaining joint mechanics closer to intact conditions and minimizing alterations in ACL forces. These trends persisted at 30 degrees of knee flexion, where significant increases in ACL forces were observed in partial and complete HCT conditions, contrasting with minimal changes observed in transplantation scenarios. 
Conclusion 
This study uncovers the biomechanical impacts of meniscal injuries and surgical conditions on the 
ACL, demonstrating how the ACL compensates for various meniscus conditions. Due to the ACL’s role in providing stability, it takes on additional stress in conditions of increased contact pressure on the meniscus, as seen in HCT conditions. In contrast, transplantation and repair conditions only slightly increase the stress on the ACL, putting much less strain on the ACL and supporting structures of the knee joint than an unrepaired tear. It is important to note that the contact area and stress measure vary depending on the degree of flexion in the knee.

Anterior Cruciate Ligament Mechanical Response to Load in the Setting of Changes to the Medial Meniscus 


Background
The architecture of the native enthesis (tendon/bone junction), consists of an organized gradient of tendon, cartilage, and bone tissues. This complex tissue structure effectively transmits the interactions between dynamic muscle tissues and the rigid skeleton. Healed tendon repairs fail to regenerate the appropriate tissue architecture and composition present in the native enthesis. The objective of this study was to create a biologically compatible scaffold that recapitulates the transition of nano-mechanical properties found in the native enthesis.
Methods
Wild-type mouse Achilles tendon-bone composite tissues were harvested and flash frozen. The modulus of elasticity for each sample was evaluated using atomic force microscopy (AFM). Force curves were generated, and Young’s modulus values calculated using a Hertz-Sneddon model. Biocompatible inks (bio-inks) composed of gelatin methacryloyl (GelMA), and other physiologically relevant additives, such as alginate, hydroxyapatite, and collagen I were fabricated to generate hydrogels of different nano-mechanical properties as assessed using AFM. The gels were crosslinked using Lithium Phenyl (2,4,6-Trimethylbenzoyl) Phosphinate and UV light. The various concentrations of GelMA, additives, and photo cross-linking parameters were then formulated to more closely replicate the transition of nano-mechanical properties found in native enthesis tissues.
Results
The native mouse enthesis modulus values (n=3) for tendon, cartilage and bone tissues were 111.8 +/- 47.7 MPa, and 36.7 +/- 29.2 MPa, 6.5 +/- 2.6 GPa respectively. Several different formulations of bio-inks were tested to best replicate the modulus values of the native enthesis. Final bio-ink concentrations were created which mimic the nanomechanical properties of tendon, cartilage, and bone. The GelMA concentrations ranged from 10-30%. Hydroxyapatite content ranged from 0-10%. Alginate content ranged from 0-4%. The scaled nanomechanical modulus values for the synthesized hydrogels in each of the different tissue types were 17 +/- 6 KPa, 280 +/- 190 Pa, 87 +/- 28 KPa respectively. 
Conclusion
Three distinct bio-inks were created to form three distinct hydrogels with different concentrations of GelMA and other physiologically-relevant additives. These hydrogels differ in both composition and nano-mechanical properties, analogous to a native enthesis tissue gradient. Future directions include the 3D-printing of these novel bio-inks to create a biomimetic scaffold with a gradient of distinct layers. Each layer will have specific nano-mechanical properties and extracellular matrix components that support the differentiation of mesenchymal progenitor cells into distinct phenotypes reflective of the different tissue types making up the native enthesis.


Design of a Novel Biomimetic Scaffold for Enthesis Regeneration after Soft Tissue-to-Bone Repair

Varying Cortical Involvement of Periacetabular Metastatic Lesions Affects Bone Strain in the Pelvis 

Background
Metastatic bone disease, common in advanced cancer, significantly impacts survival and quality of life. As cancer survivorship improves, the incidence of skeletal metastases rises. Effective orthopedic constructs are crucial for enhancing patient outcomes. Currently, there is no validated method for biomechanical modeling or optimal surgical treatment of metastatic acetabular defects. Tumors near the acetabulum can weaken the pelvis, but the effect of different defects on fracture risk is unclear. This study aims to develop anatomic models of metastatic pelvic defects to predict fracture risk zones using finite element analysis.

Methods 
A short fiber-filled epoxy artificial bone was laser-scanned to create a 3D hemipelvic model, including cortical and trabecular regions. Lesions were defined by their cortical bone intersection area, based on binary subtraction. An orthopedic surgeon determined average lesion size and location, following the modified Harrington Classification System. Acetabular defect models were converted into finite element models via volumetric meshing. Analysis compared defect models' volumetric size, cortical bone loss, and stress/strain magnitudes. Peak stresses were evaluated at the medial wall, superior acetabular dome, acetabular rim, anterior, and posterior columns. Margin of safety quantified fracture risk, mapping weakened acetabular areas.

Results
Simulated tumors' average length and cortical bone loss were 6.3 cm and 5.5 cm3, respectively. The hemipelvis with the anterior column defect had the lowest margin of safety and highest change in peak stress compared to no defect. The anterior defect and the anterior defect involving the medial wall had similar margin of safety results.

Conclusion
Anterior column and anterior column with medial wall defects were more severe than defects involving posterior & medial wall of the hemipelvis, posing greater fracture risks than posterior and medial wall defects. This study enhances our understanding of fracture risk in metastatic pelvic lesions, guiding surgical decision-making and improving patient care by decreasing unnecessary procedures.


Varying Cortical Involvement of Periacetabular Metastatic Lesions Affects 
Bone Strain in the Pelvis

Background
Pediatric mood and anxiety disorders are common and associated with significant morbidity and mortality, including suicidality. Understanding factors influencing symptom presentation and treatment response will aid in developing targeted therapies and optimizing outcomes. Neurochemical levels serve as proxies for neurobiological processes altered in depressive and anxiety disorders. While the impact of antidepressant treatment on brain metabolism has been studied in adults, there are limited data in pediatric populations. This study aims to evaluate the relationship between neurochemical concentrations and anxiety and depressive symptoms in fluoxetine-treated youth. 
Methods
Adolescents and young adults aged 12-21 years, on steady-state fluoxetine, underwent proton magnetic resonance spectroscopy (1H-MRS) at 3T to measure glutamate (Glu), glutamine (Gln), myo-inositol (mI), choline-containing products (Cho), N-acetyl-aspartate (NAA) and creatine (Cr). Neurochemicals were reported as ratios relative to Cr. Depressive and anxiety symptoms were assessed using the PHQ-9 and PROMIS Anxiety scales, respectively. Participants with PHQ-9 scores ≥ 11 or PROMIS Anxiety t-scores ≥ 60 (moderate anxiety) were considered non-responders. Statistical analyses included Student’s t-test, Cohen’s d, and Spearman’s rho, performed using JMP Pro v17.  
Results
In 46 youth (mean age 16.1 ± 1.9 years, range 12-21, 67% female), NAA was significantly higher in fluoxetine responders compared to non-responders (NAA mean 1.67 ± 0.09 vs 1.57 ± 0.11, p = 0.004, Cohen’s d = 0.94) when stratified by response to fluoxetine. NAA levels did not correlate with anxiety symptom severity (Spearman’s ρ= -0.146, p=0.333). Glx, Glu, Gln, mI, and Cho levels did not differ between responders and non-responders, nor did they correlate with anxiety or depression symptom severity scores. 
Conclusion
In fluoxetine-treated youth, fluoxetine anxiety responders had higher NAA levels compared to non-responders. NAA, a marker of neuronal integrity, is typically lower in certain psychiatric disorders relative to healthy controls, with some evidence suggesting normalization following successful treatment. Baseline levels of NAA were not measured to provide insight on whether the fluoxetine responders had higher NAA levels before starting treatment. Our pilot findings support the need for further research into NAA as a potential biomarker for fluoxetine response. 


Exploring the relationship between antidepressant treatment, neurochemicals in the brain and anxiety/depression symptoms in youth

Title: Topical senolytic treatment with ABT-263 improves delayed wound healing of aging
Authors: Joy Ha, BA; Jannat Dhillon, BA; Rex Jeya Rajkumar Samdavid Thanapaul, PhD; Maria Shvedova, MD, PhD; Jack Crouch, BS, MS; Adam Gower, PhD; Sami Gritli, MD, PhD; Daniel S. Roh, MD, PhD.
Affiliations: Boston University School of Medicine, Division of Plastic and Reconstructive
Surgery, Department of Surgery, Boston, Massachusetts, USA

Background & Objectives:
Senescent cells (SnC) accumulate in aged tissues, limiting their ability to repair and regenerate following injury. Senolytics have been shown to promote tissue regeneration and repair when targeted at resident SnCs in certain aged tissues. We hypothesized that the removal of chronic SnCs from aged skin would improve repair potential and subsequent wound healing. This study’s objectives were to determine the effects of topical ABT-263 (navitoclax) treatment on aged skin
senescence and wound healing.
Methods:
Aged male C57Bl/6 mice (22-24 months) were treated with topical ABT-263 or control DMSO daily for 5 consecutive days. Skin was harvested at two time points: 1) immediately after 5 treatment days and 2) at day 10 (allowing for 5 days of skin recovery) and analyzed using 1) bulk RNAseq, 2) qRT-PCR and 3) histology. Additional mice treated with topical ABT-263 or control DMSO subsequently underwent 1cm full-thickness dorsal skin wounds and were imaged daily
until wound closure.
Results:
Topical ABT-263 reduced expression of p16, p21 in aged but not young skin and reduced numbers of SA-β-Gal and p21-positive cells. ABT-263 downregulated genes involved in maintaining mitochondrial homeostasis and increased pro-apoptotic genes consistent with its mechanism of action. However, there was increased inflammatory processes and cell infiltration in ABT-263 skin. GSEA analysis demonstrated ABT-263 upregulated wound healing-related pathways: ↑cell migration, ↑cell proliferation, ↑extracellular matrix synthesis and organization,
↑angiogenesis. ABT-263 treated aged mice achieved more rapid wound closure compared to controls DMSO.
Conclusions:
Topical ABT-263 reduces several senescence markers in aged skin and “primes” the skin for subsequent improved wound healing. This may involve senolysis-induced inflammation. Future work is needed to determine specific cells targeted by topical ABT-263 and the senescence-dependent vs senescence-independent mechanisms by which ABT-263 improves delayed wound healing in aging.

Topical senolytic treatment with ABT-263 improves delayed wound healing of aging


Background: Caregiving and caregiving intensity is associated with increased depressive symptoms among caregivers. Nonetheless, more research is needed to examine the relationship between caregiving intensity and depressive symptoms among care recipients. 

Methods: Using data from the seventh wave of the Hispanic Established Populations for the Epidemiologic Study of the Elderly (H-EPESE)(n=550), we assessed the association between depressive symptoms in care recipients and the source of care for ADL disability in Older Mexican Americans age 75+. Depressive symptoms were measured using the Center for Epidemiological Studies Depression (CESD) scale. Caregivers were asked to name all sources of ADL care for care recipients, including the caregiver, the care recipient, another family, and other non-family. We used regression models controlling for the care recipient and caregiver characteristics, as well as the care recipient’s health. 

Results: The average CESD score of care recipients was 11.12. Care recipients' mean age was 87 years old, 68% were women, and 52% were born in the U.S. Caregivers reported sources of care included the care recipient (73%), the caregiver (32%), another family (13%) and other non-family(21%). Care recipients who provided care for themselves had significantly fewer depressive symptoms (-3.16), while those who reported non-family as sources of care had significantly higher depressive symptoms (3.33), even when controlling for the level of disability. Depressive symptoms were higher for females than males and among those that had fewer than seven years of education.

Conclusion: The source and amount of attention the care recipients receive are crucial in determining the risk of developing depressive symptoms.


Depressive Symptoms in Older Mexican Americans and the Role of Disability Care

Background
Serotonin reuptake inhibitors (SSRIs) are the most widely used antidepressants for Major Depressive Disorder (MDD), and prenatal exposure to SSRIs has been associated with brain changes in newborns. Although few human studies have reported these changes, they may explain the increased risk for depression after in-utero exposure to SSRIs. This review analyzes findings from a small number of studies to explore SSRI-induced changes in the developing hippocampus and other regions associated with depression.
Methods
A literature search using key phrases such as “perinatal SSRIs,” “hippocampal changes,” and “fetal brain development” between April-July 2024 yielded six human and 17 rodent studies exploring the potential effects of prenatal exposure to SSRIs on fetal brain areas linked to depression vulnerability.
Results
Four of the six reviewed studies in human subjects revealed significant changes in corticofugal and corticothalamic tracts after prenatal SSRI exposure. Two human studies showed increased amygdala volume and one preliminary study found decreased hippocampal volume. One study reported global brain changes, which contradicted another. Compared to only one study in human subjects, fifteen of seventeen rodent studies revealed hippocampal changes after SSRI exposure, with reduced BDNF exon mRNA. Two of the studies also reported decreased serotonin and dendritic density. Some inconsistent findings regarding hippocampal DNA methylation, cell proliferation, and synaptic density were reported. 
Conclusion
This review reinforces the connection between in-utero exposure to SSRIs and an increased risk for depression. More research is required to confirm the findings from the reviewed studies, including the risk of increased vulnerability to depression and decreased treatment response.


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

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