United States

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.


AMA Research Challenge 2024 

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

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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.


Fetal Brain Changes from In-Utero Exposure to SSRIs

Background
The pathogenesis of allergic asthma is classically understood to involve allergens crosslinking Immunoglobulin E (IgE) bound to mast cells and basophils, leading to the release of inflammatory mediators. However, omalizumab, an anti-IgE antibody, reduces exacerbation rates in only about 40% of adult patients with severe asthma. These findings highlight the need for further research to fully address the role of IgE in allergic diseases like asthma. This study aims to explore which allergen induces Th2-associated airway inflammation through an IgE-dependent mechanism. 

Methods
We used IgE deficient mice (IgE KO) to generate asthma mouse models induced by two highly clinically relevant allergens house dust mite extract (HDM), cockroach extract (CRE) and one classic allergen ovalbumin (OVA). Hematoxylin and Eosin (HE) staining and Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining was performed on lung tissue to evaluate inflammatory responses and mucus production, respectively. The count of inflammatory cells in Bronchial Alveolar Lavage Fluids (BALF) was determined using flow cytometry. Passive Cutaneous Anaphylaxis (PCA) was conducted to quantify IgE involvement in the anaphylactic reaction, and Toluidine Blue (TB) staining of dorsal skin tissue was used to visualize mast cell recruitment.

Results
All mice treated with allergens exhibited significant signs of airway inflammation, as evidenced by the recruitment of inflammatory cells to the lung parenchyma and goblet cell hyperplasia. However, IgE KO mice treated with different allergens demonstrated significantly variable degrees of inflammation compared to the WT control. Of these, HDM-treated IgE KO mice showed an attenuated inflammatory profile with reduced inflammatory cells, particularly eosinophils. These mice had lower serum levels of HDM-specific IgE1 (sIgE1) and IgG1 (sIgG1) and reduced levels of Th2 cytokines IL-4 and IL-5 in BALF. While CRE-treated IgE KO mice also displayed a reduced inflammation compared to WT mice, the only statistically significant difference was found for eosinophils. No significant differences were observed between OVA-treated IgE KO and WT mice. These findings were further supported by our well-established PCA models. Both HDM and CRE-induced PCA model showed significantly less Evan’s blue dye leakage and mast cell recruitment in the ears of IgE KO mice compared to WT mice. No differences were observed for OVA-induced PCA and mast cell recruitment.

Conclusion
The results highlight the variability in IgE pathway dependence among different allergens, emphasizing the need for further research is to delineate the mechanisms and develop more effective therapeutic strategies for allergic diseases.

Allergic Asthma and Anaphylaxis Induced by Different Allergens Show Distinctive IgE Dependence

Abstract Title:
Antimicrobial Effects of Inhaled Nitric Oxide Therapy on Pseudomonas Aeruginosa Lung Colonization in a Swine Model of Independent Lung Ventilation.

Background:
Pseudomonas Aeruginosa is the most common cause of gram-negative nosocomial pneumonia, and up to 26% of ventilator-associated pneumonia globally. Recent findings indicate that inhaled nitric oxide (iNO) at 300 parts-per-million (ppm) effectively reduces lung bacterial colonization of Klebsiella pneumonia and Pseudomonas aeruginosa (P. aeruginosa). Unlike inhaled antibiotics, iNO300 therapy might enhance the immune response in non-ventilated lung regions due to consolidated parenchyma. 

Methods:
Twelve swine were kept under general anesthesia and mechanical ventilation for 72 hours. All pigs were challenged with P. aeruginosa via bronchoscopy and twice via nebulization at 24 and 48 hours. The control group (n=6) received standard ventilation through a single-lumen endotracheal tube. The iNO300 group (n=6) was ventilated using a double-lumen endotracheal tube (DLT), with one lung receiving iNO300 and the other standard ventilation. Lung isolation was confirmed through respiratory mechanics, bronchoscopy, and lung imaging. iNO300 was administered for 30 minutes every 4 hours, to right (n=3) and left (n=3) lungs in different animals. After 72 hours, lung tissue samples were collected. Measurements of Colony Forming Units (CFUs)/gram of P. aeruginosa in each lung lobe, and pathohistological scoring of infection and inflammation (0=none/minimal, 1=focal/mild, 2=moderate, 3=diffuse/severe), were obtained. Percent methemoglobin (MetHb), mean arterial pressure, and PaO2/FiO2 were measured during treatments.

Results:
Lung tissue samples demonstrated that animals treated with iNO300 had statistically lower bacterial loads in both lungs compared to the control group (2.3±1.0 vs. 4.4±0.6 Log10 CFU/g, p-value=0.045). In the iNO group there was no difference between the treated and non-treated lungs (2.3±1.0 vs 2.3±1.4 Log10 CFU/g, p-value=0.98).

Compared to controls, iNO300-treated swine showed less severe signs of acute bronchopneumonia (0.59±0.91, vs 1.91±0.72, p-value <0.001), interstitial inflammation (0.54±0.63 vs 1.19±0.69, p-value <0.001) and airway inflammation (0.67±0.7 vs 1.46±0.69, p-value <0.001) in both lungs. In the iNO group there was no difference in acute bronchopneumonia (p-value=0.71) or interstitial inflammation (p-value=0.76) between treated and non-treated lungs.

Baseline MetHb levels were measured (1.73±1.02%), and levels peaked 30 minutes (3.67±1.82%) during iNO treatments but returned to baseline within 30 minutes post-treatment (1.13±0.76%). Mean arterial pressure (p-value=0.97) and PaO2/FiO2 (p-value=0.22) remained stable during treatments

Conclusion:
In a swine model of P. aeruginosa pneumonia, iNO300 delivered to only one lung reduces bacterial load and lung tissue inflammation in both treated and untreated lungs, suggesting antimicrobial mechanisms beyond direct contact. The transient rise in MetHb levels and stable hemodynamic parameters indicate iNO300 does not adversely affect cardiovascular health.

Antimicrobial Effects of Inhaled Nitric Oxide Therapy on Pseudomonas Aeruginosa Lung Colonization



WHOLE TESTES CRYOPRESERVATION FOR FUTURE AUTO-TRANSPLANTATION: COMPARISON OF DIFFERENT FREEZING METHODS 



Chenchu Nagarakanti, MD1; Robert R.A. Wilson, Bsc1; Elizabeth D Greene, LATG.2; Ibrahim Hacibey, MD1; Megan Escott, MD1,4; Yalcin Kulahci1; Vijay Gorantla1; Anthony Atala, MD1,3; Kelvin G.M. Brockbank, PhD2,3; Hooman Sadri-Ardekani, MD, PhD1,3 

1Wake Forest Institute for Regenerative Medicine (WFIRM), Wake Forest School of Medicine, Winston-Salem, NC. 

2Tissue Testing Technologies LLC, North Charleston, SC 

3Department of Bioengineering, Clemson University, Clemson, SC 

4Department of Urology, Wake Forest School of Medicine, Winston-Salem, NC. 



Background: 

In the past decades, survival rates of cancer patients have increased significantly, though infertility remains a long-term complication of cancer therapy. Whole testes (WT) cryopreservation for successive transplantation is one of the potential fertility-sparing strategies in childhood cancer patients with solid tumors. 

Methods: 

The study involved eight rats, each were sacrificed and submitted to orchiectomy. WT organs from each rat were randomly assigned to four groups: Group A, fresh tissue (control); Group B, slow freezing with 10% DMSO and rewarmed at 37°C; Group C, slow freezing with 10% DMSO and rewarmed on ice, and Group D, vitrified using VS55, a complex mixture containing DMSO, formamide, and 1,2-propanedio. Diluted VS55 (4°C) was added sequentially to achieve full-strength VS55. The samples were rapidly cooled to -100°C in 2-methylbutane and then transferred to vapor phase nitrogen for slower cooling and storage below -135°C for >24 h before testing (vitrification). Vitrified testes were slowly warmed to -100°C followed by rapid warming to melting. Each group had two replicates from different rats. Each testicle was measured metrically and with an orchidometer. Histological examination was conducted to assess morphological changes in the testicular tissues. Immunohistochemical staining techniques, specifically targeting PGP, Calretinin, Vimentin and Smooth Muscle Actine (SMA) were employed to identify undifferentiated spermatogonia, Leydig cells, Sertoli cells and peritubular cells. Additionally, micro-CT scanning was used to evaluate the distribution and effectiveness of DMSO within the testicular tissue across different loading times (from 6 hours to 4 days). 

Results: 

Slight morphological alterations were observed in the experimental groups compared to the control, including testicle tubule tissue shrinkage in vitrified samples. Frozen tissue demonstrated more significant tubule tissue shrinkage, minor disruption of the epididymal tissues, and more significant disruption of the endothelial layers of the blood vessels in the vascular pedicle. Arterial vasospasm was present in all four groups. PGP 9.5 IHC identified undifferentiated spermatogonial cells. 1-way ANOVA test showed a statistically significantly lower number of cells in the three experimental groups than in the control. It found that 10% DMSO gave 10% concentration in the testes after 12 hours of loading time, which is close to optimum. 

Conclusion: 

Slow freezing with loading 10% DMSO for 12 hours could preserve WT structure cell-specific sensitivity to the freeze-thaw process has been detected. All methods revealed significantly decreased PGP9.5 and calretinin expression, and further studies are necessary to optimize PGP 9.5 and calretinin expression and confirm testicular tissue function for possible auto-transplantation

WHOLE TESTES CRYOPRESERVATION FOR FUTURE AUTO-TRANSPLANTATION:
Comparison of Different Freezing Methods

Abstract Title
Retrospective Study of the Relationship between Daily Oral Multivitamin Intake and Sleep Complaints in Post-Menopausal Women – An Analysis of NHANES Data (1999-2020)

Background
This study investigates whether daily oral multivitamin intake affects sleep patterns and sleep complaints in post-menopausal women. The hypothesis is that daily multivitamin intake may improve sleep quality in this demographic.

Methods
Data from the NHANES surveys (1999-2020) were analyzed to study the relationship between multivitamin use and sleep complaints. Dietary supplement usage, menopause status, and sleep data were extracted and pre-processed. Participants were categorized into two groups: those who took daily multivitamins and those who did not. Post-menopausal status was identified, and relevant sleep data from 2005-2020 was included. The final sample comprised 1,647 post-menopausal women who took daily multivitamins and 2,643 who did not.

The primary variable of interest was hours of sleep per night (SLD010/12). Outliers were removed, and the Shapiro-Wilk test was conducted to assess normality. Due to non-normal distributions (p < 0.05), the Mann-Whitney U test was used to compare the sleep duration between groups.

The secondary variable was sleep complaints (SLQ050), evaluated using a Chi-Square test of independence to assess the association between multivitamin use and reported sleep troubles. A post-hoc Fisher’s Exact test was also performed to confirm the significance of the findings.

Results
The analysis revealed that post-menopausal women who took daily multivitamins had a significantly higher mean sleep duration (7.289 hours) compared to those who did not take multivitamins (6.946 hours), with a median of 7.0 hours for both groups. The Mann-Whitney U test indicated a significant difference in sleep duration between the groups (Z = -6.754, p < 0.001). Additionally, there was a significant association between multivitamin use and reported sleep troubles, with the Chi-Square test showing (Χ² (1) = 4.167, p = .041) and the Fisher’s Exact test confirming a statistically significant difference (p < 0.001). These findings suggest that while multivitamin intake may increase sleep duration, it is also associated with a higher incidence of reported sleep troubles among post-menopausal women.

Conclusion
Daily multivitamin use may increase the number of sleep hours per night but is also associated with reported sleep troubles. These findings highlight the importance of discussing multivitamin use with healthcare providers for post-menopausal women. Further research should consider additional confounding factors and the broader implications of multivitamin use on sleep quality.

Retrospective Study of the Relationship between Daily Oral Multivitamin Intake and Sleep Complaints

Background
Endothelial Notch signaling is a key player in regulating inflammation. While Notch1 has been extensively studied in the context of vascular inflammation, the role of endothelial Notch4, remains relatively unexplored. Growing evidence suggests that Notch4 plays critical roles in human endothelial inflammatory disorders. Polymorphisms in Notch4 are associated with endothelial dysfunction in preeclampsia, HIV-triggered glomerular injury, and severity of COVID-19 endothelial inflammation. Our recent work has shown that pharmacologic inhibition of endothelial Notch4 signaling using an anti-Notch4 antibody alters macrophage number and phenotype in mouse models of breast cancer, further supporting a distinct role for Notch4 in regulation of inflammatory targets. Our preliminary work has determined that Notch4 regulates an endothelial transcriptome distinct from Notch1, which is enriched for inflammatory genes such as CCL2 and IL6. However, all Notch proteins bind DNA via a common transcriptional co-factor, CSL, and the mechanism of how different Notch proteins regulate distinct transcriptional targets remains unclear. Better methods for detection of real-time and gene-specific activation of Notch signaling are critical for dissecting the roles of different Notch proteins in endothelium.

Methods
To examine gene-specific Notch transcriptional activity, we used the NanoBiT protein-protein-interaction system, a complementation-based luciferase reporter system composed of two split luciferase fragments, LgBiT and SmBiT. The NanoBiT PPI system allows for detection of protein:protein interactions through two subunits, Large BiT (LgBit) and Small BiT (SmBit), fused to two proteins of interest. Interaction between target proteins allows the subunits to come together resulting in luminescent signal that can be quantified in live cells. Real-time quantification allows for dynamic monitoring, capturing transient interactions and providing temporal resolution that homogenate testing lacks. We designed fusions of the active intracellular domain of Notch4 (ICN4) or Notch1 (ICN1) with SmBiT, and CSL with LgBiT and expressed the fusion proteins in HeLa cells. The resulting interactions between fusion proteins resulted in luminescent signal that was quantified using the SpectraMax L Microplate Reader. 

Results
We validated that NanoBiT CSL and ICN fusion proteins were expressed at high levels and retained their ability to induce Notch pathway targets. Co-expression of CSL-LgBiT with either ICN4-SmBiT or ICN1-SmBiT resulted in robust induction of luminescent signal. Interaction between CSL and ICN4 consistently yielded a higher luminescent signal than ICN1, suggesting that Notch4 may more strongly interact with CSL. 

Conclusion
We have designed and tested a novel method for real time measurement of gene-specific Notch pathway activation. Our preliminary findings suggest that Notch4 plays a distinct role in inflammatory signaling, possibly due to its robust interaction with CSL. Our NanoBiT-based assay may be used to determine protective targets in inflammatory diseases. 


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