Synthesis of (2R,4S)-4-Amino-5-hydroxybicyclo[3.1.1]heptane-2-carboxylic Acid via an Asymmetric Intramolecular Mannich Reaction.

Inhibition of human ornithine aminotransferase interferes with glutamine and proline metabolism in hepatocellular carcinoma, depriving tumors of essential nutrients. A proposed mechanism-based inhibitor containing a bicyclo[3.1.1]heptanol warhead is reported herein. The proposed inactivation mechanism involves a novel α-iminol rearrangement. The synthesis of the proposed inhibitor features an asymmetric intramolecular Mannich reaction, utilizing a chiral sulfinamide. This study presents a novel approach toward the synthesis of functionalized bicyclo[3.1.1]heptanes and highlights an underutilized method to access enantiopure exocyclic amines.

Epidemiology of Injury and Illness in North American Professional Men's Soccer: Comparing COVID-19 Lockdown With Previous Seasons.

BACKGROUND: After COVID-19 lockdown, studies across Europe and Asia examined its effect on professional soccer injury rates and severity; however, COVID-19 lockdown influence on injuries in United States men's professional soccer has not been evaluated. HYPOTHESIS: Injury and illness rates during the 2020 season were higher than the previous 2 seasons. STUDY DESIGN: Retrospective observational cohort study. LEVEL OF EVIDENCE: Level 3. METHODS: Medical staff from participating Major League Soccer (MLS) clubs entered injuries and athletic exposures during the study period into a league-wide electronic medical record system. Injury rate and severity were analyzed to examine differentials between the 2020 COVID season and historical controls. RESULTS: The injury incidence rate per 1000 hours in 2020 was 10.8, ie, higher than 2018 (5.4) and 2019 (5.0) (P < 0.05). Training injury incidence rate increased in 2020 (8.9) compared with 2018 (2.5) and 2019 (2.6) (P < 0.05), whereas match injury incidence decreased in 2020 (18.3) compared with 2018 (24.0) and 2019 (22.7) (P < 0.05). Incidence rates of lower extremity muscle injuries (6.04), anterior cruciate ligament (ACL) injuries (0.17), and concussions (0.49) were also higher in 2020 compared with 2018 (2.5, 0.07, 0.27) and 2019 (2.36, 0.05, 0.22) (P < 0.05). More injured players in 2020 missed >90 days (17.7%) than in 2018 (10.2%) and 2019 (10.1%) (P < 0.05). Incidence of all non-COVID-19 illness was higher in 2020 (3.93) than 2018 (1.53) and 2019 (1.32) (P < 0.05). CONCLUSIONS: During 2020, there were significant increases in incidence rates of overall injuries, training injuries, lower extremity muscular injuries, ACL injuries, concussions, and non-COVID illness, along with a higher percentage of players missing >90 days compared with the 2 previous seasons. CLINICAL RELEVANCE: These results may help clarify the effects of future MLS inseason work stoppages and periods of restricted training.

The 11+ injury prevention programme decreases rate of hamstring strain injuries in male collegiate soccer players.

OBJECTIVES: To investigate if the 11+ injury prevention programme decreases the risk of hamstring injury and improves recovery time and determine whether compliance with the 11+ affects hamstring injury risk. METHODS: This study is a secondary analysis from a prospective cluster randomised controlled trial that included 65 National Collegiate Athletic Association (NCAA) division I and II men's soccer teams over the fall 2012 season. Thirty-one teams were randomised to the intervention group that were using the 11+ as their warm-up and 35 teams to the control group that continued to use their traditional warm-up. Each certified athletic trainer (ATC) collected data on demographics, hamstring injury (HSI), mechanism of injury, position, playing surface, time lost due to injury and compliance to the 11+ programme. RESULTS: The 11+ decreased the risk of HSI by 63% compared with the control group (RR=0.37, 95% CI 0.21 to 0.63). Difference in return to play after HSI between the control (9.4±11.2 days) and intervention groups (10.2±11.3 days) was not significant (p=0.8). High compliance (>2 or more doses on average per week) reduced the risk of HSI by 78% (RR=0.22, 95% CI 0.06 to 0.87) compared with low compliance (<1 dose on average per week), and moderate compliance (1 to <2 doses on average per week) decreased the risk of HSI by 67% (RR=0.33, 95% CI 0.11 to 0.97) compared with low compliance. There was no significant difference between high and moderate compliance. CONCLUSION: The 11+ decreased the risk of HSI by 63% but did not improve recovery time. High to moderate compliance is essential and makes the programme more effective at reducing HSI.

Design, Synthesis, and Mechanistic Studies of (R)-3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.

Virtual Reality and Surgical Simulation Training for Orthopaedic Surgery Residents: A Qualitative Assessment of Trainee Perspectives.

Background: The demonstrated benefits of virtual reality (VR) in orthopaedic surgical training are numerous. However, it is relatively unknown how best to implement VR into an already established orthopaedic resident education curriculum and how trainees will engage and use these technologies longitudinally. Methods: This was an exploratory, qualitative research study performed in accordance with Consolidated Criteria for Reporting Qualitative Research guidelines. Orthopaedic surgery residents at a single institution were recruited during the 2022 to 2023 academic year. Semistructured interviews were conducted. Data were analyzed through grounded theory methodology, beginning with open coding, followed by axial coding, and concluding with selective coding that describes orthopaedic surgery residents' current perceptions of VR as a training tool. Results: Six residents participated in interviews before thematic saturation was achieved. Average interview length was 13:27 (±2:59) minutes. Residents felt that currently, VR is most useful for interns and junior residents as an educational adjunct for learning anatomy, surgical exposures, and the steps of a procedure in a risk- and judgment-free arena. There seems to be a "ceiling effect" with VR given current technological limitations, and residents remarked that there is an associated "opportunity cost" with using VR technology. Some residents may find it more time-efficient to study texts, videos, or surgical guides rather than use VR. Cost (limited number of headsets) and technological barriers (i.e., hardware, software, and Wi-Fi issues) were some of the described barriers to VR utilization. Residents felt that there needs to be dedicated technological support to help with these issues. At this time, given these limitations of VR, many preferred VR as an optional educational adjunct rather than as a required curricular tool or assessment of surgical competency. Conclusions: There is current utility for VR in orthopaedic surgical training. Future technological advances may make VR more central to resident education. This study describes resident perceptions about the technology and best use practices for the technology. Level of Evidence: Qualitative Study, Level V Evidence.

Crystallographic and Computational Insights into Isoform-Selective Dynamics in Nitric Oxide Synthase.

In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.

From TgO/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma.

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

Target Identification of a Class of Pyrazolone Protein Aggregation Inhibitor Therapeutics for Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure, and current treatment options are very limited. Previously, we performed a high-throughput screen to identify small molecules that inhibit protein aggregation caused by a mutation in the gene that encodes superoxide dismutase 1 (SOD1), which is responsible for about 25% of familial ALS. This resulted in three hit series of compounds that were optimized over several years to give three compounds that were highly active in a mutant SOD1 ALS model. Here we identify the target of two of the active compounds (6 and 7) with the use of photoaffinity labeling, chemical biology reporters, affinity purification, proteomic analysis, and fluorescent/cellular thermal shift assays. Evidence is provided to demonstrate that these two pyrazolone compounds directly interact with 14-3-3-E and 14-3-3-Q isoforms, which have chaperone activity and are known to interact with mutant SOD1G93A aggregates and become insoluble in the subcellular JUNQ compartment, leading to apoptosis. Because protein aggregation is the hallmark of all neurodegenerative diseases, knowledge of the target compounds that inhibit protein aggregation allows for the design of more effective molecules for the treatment of ALS and possibly other neurodegenerative diseases.

Orthopaedic Surgery Training and Education During COVID-19: A Systematic Review.

COVID-19 forced surgical resident training programs to adapt to meet educational requirements within the constraints of various guidelines. Some of the changes implemented during the pandemic have imparted a lasting effect on orthopaedic education. As such, the purpose of this article was to review how orthopaedic training and education were affected during the COVID-19 pandemic. Methods: The published literature was queried using search strategies devised by a medical librarian, according to the Preferred Reporting Items for Systematic Review and Meta-Analyses guidelines. Studies eligible for inclusion were studies related to COVID-19, orthopaedic surgical training, and medical education. Studies were excluded if they (1) were abstracts, conference proceedings, letters, perspective pieces, reviews, or editorials; (2) evaluated medical student education; (3) included other specialties; or (4) were unrelated to COVID-19 and/or orthopaedic training. Results: Eighty-three (n = 83) studies were included. Five themes emerged including (I) Fellowship Application, Interview, and Match Processes; (II) Social Media and Websites for Program Information; (III) Changes in Trainee Surgical Volume; (IV) Trainee Mental Health and Well-being; and (V) Innovations in Education. The pandemic decreased opportunities for medical students to gain exposure to orthopaedic surgery. Social media use, particularly Instagram, among orthopaedic residencies increased during the pandemic. Between the cancellation of away rotations and in-person interviews, applicants saved over $6,000; however, both residency applicants and interviewers preferred in-person interviews. The pandemic led to decreased surgical volume and in-person didactics for trainees, thus relying more on virtual learning. Orthopaedic trainees had mixed feelings regarding online virtual education. Although some respondents reported that they preferred the convenience of online learning, others expressed dissatisfaction with the quality of virtual education. Conclusions: The shift to virtual learning affected how applicants learned about residency programs, with many relying on virtual away rotations and social media to compare different programs. The pandemic also highlighted issues of diversity and accessibility within orthopaedic surgery, with cost savings from virtual interviews and canceled away rotations potentially benefiting applicants from lower socioeconomic backgrounds. Although some innovative approaches and adaptations to orthopaedic education and training have shown promise and may continue to be used in the future after the COVID-19 pandemic, the role of others, such as virtual interviews, is less clear.

Potent, Selective, and Membrane Permeable 2-Amino-4-Substituted Pyridine-Based Neuronal Nitric Oxide Synthase Inhibitors.

A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS), based on a difluorobenzene ring linked to a 2-aminopyridine scaffold with different functionalities at the 4-position, is reported. In our efforts to develop novel nNOS inhibitors for the treatment of neurodegenerative diseases, we discovered 17, which showed excellent potency toward both rat (Ki 15 nM) and human nNOS (Ki 19 nM), with 1075-fold selectivity over human eNOS and 115-fold selectivity over human iNOS. 17 also showed excellent permeability (Pe = 13.7 × 10-6 cm s-1), a low efflux ratio (ER 0.48), along with good metabolic stability in mouse and human liver microsomes, with half-lives of 29 and >60 min, respectively. X-ray cocrystal structures of inhibitors bound with three NOS enzymes, namely, rat nNOS, human nNOS, and human eNOS, revealed detailed structure-activity relationships for the observed potency, selectivity, and permeability properties of the inhibitors.

Small molecules targeting different cellular pathologies for the treatment of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease in which the motor neuron circuitry displays progressive degeneration, affecting mostly the motor neurons in the brain and in the spinal cord. There are no effective cures, albeit three drugs, riluzole, edaravone, and AMX0035 (a combination of sodium phenylbutyrate and taurursodiol), have been approved by the Food and Drug Administration, with limited improvement in patients. There is an urgent need to build better and more effective treatment strategies for ALS. Since the disease is very heterogenous, numerous approaches have been explored, such as targeting genetic mutations, decreasing oxidative stress and excitotoxicity, enhancing mitochondrial function and protein degradation mechanisms, and inhibiting neuroinflammation. In addition, various chemical libraries or previously identified drugs have been screened for potential repurposing in the treatment of ALS. Here, we review previous drug discovery efforts targeting a variety of cellular pathologies that occur from genetic mutations that cause ALS, such as mutations in SOD1, C9orf72, FUS, and TARDP-43 genes. These mutations result in protein aggregation, which causes neuronal degeneration. Compounds used to target cellular pathologies that stem from these mutations are discussed and comparisons among different preclinical models are presented. Because the drug discovery landscape for ALS and other motor neuron diseases is changing rapidly, we also offer recommendations for a novel, more effective, direction in ALS drug discovery that could accelerate translation of effective compounds from animals to patients.

Improved synthesis and anticancer activity of a potent neuronal nitric oxide synthase inhibitor.

An improved synthesis of 4-methyl-7-(3-((methylamino)methyl)phenethyl)quinolin-2-amine (1) is reported. A scalable, rapid, and efficient methodology was developed to access this compound with an overall yield of 35%, which is 5.9-fold higher than the previous report. The key differences in the improved synthesis are a high yielding quinoline synthesis by a Knorr reaction, a copper-mediated Sonogashira coupling to the internal alkyne in excellent yield, and a crucial deprotection of the N-acetyl and N-Boc groups achieved under acidic conditions in a single step rather than a poor yielding quinoline N-oxide strategy, basic deprotection conditions, and low yielding copper-free conditions that were reported in the previous report. Compound 1, which previously was shown to inhibit IFN-γ-induced tumor growth in a human melanoma xenograft mouse model, was found to inhibit the growth of metastatic melanoma, glioblastoma, and hepatocellular carcinoma in vitro.

American Academy of Orthopaedic Surgeons Clinical Practice Guideline Case Study: Management of Anterior Cruciate Ligament Injuries.

Considering the frequency of anterior cruciate ligament (ACL) tears, optimal management of these injuries was the subject of a 2022 clinical practice guideline update from The American Academy of Orthopaedic Surgeons (AAOS) with input from representatives from the American Orthopaedic Society for Sports Medicine, the Pediatric Orthopaedic Society of North America, the American Orthopaedic Society for Sports Medicine, the American Medical Society for Sports Medicine, the American Academy of Physical Medicine and Rehabilitation, and the American College of Emergency Physicians. The eight recommendations and seven options to guide orthopaedic surgeons and other physicians managing patients with these anterior cruciate ligament injuries are based on the best current available evidence. The cases presented in this article are examples designed to demonstrate the clinical application of these guidelines.

Structural and Mechanistic Basis for the Inactivation of Human Ornithine Aminotransferase by (3S,4S)-3-Amino-4-fluorocyclopentenecarboxylic Acid.

Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC. Recently, we found that (3S,4S)-3-amino-4-fluorocyclopentenecarboxylic acid (5) was a potent inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), proceeding by an enamine mechanism. Here we describe our investigations into the activity and mechanism of 5 as an inactivator of human OAT. We have found that 5 exhibits 10-fold less inactivation efficiency (kinact/KI) against hOAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with hOAT. pKa and electrostatic potential calculations were performed to further support the notion that the α,ß-unsaturated alkene of 5 is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of 5 over the corresponding saturated analogue (4). Intact protein mass spectrometry and the crystal structure complex with hOAT provide evidence to conclude that 5 mainly inactivates hOAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with hOAT is a minor component of the total inhibition which is unique relative to other monofluoro-substituted derivatives. Furthermore, based on the results of transient-state measurements and free energy calculations, it is suggested that the α,ß-unsaturated carboxylate group of PLP-bound 5 may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound 5 exhibits unusual structural conversions which are catalyzed by specific residues within hOAT, ultimately leading to an enamine mechanism-based inactivation of hOAT through noncovalent interactions and covalent modification.

2-Aminopyridines with a shortened amino sidechain as potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors.

A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS) based on the 2-aminopyridine scaffold with a shortened amino sidechain is reported. A rapid and simple protocol was developed to access these inhibitors in excellent yields. Neuronal nitric oxide synthase (nNOS) is a novel therapeutic target for the treatment of various neurological disorders. The major challenges in designing nNOS inhibitors in humans focus on potency, selectivity over other isoforms of nitric oxide synthases (NOSs), and blood-brain barrier permeability. In this context, we discovered a promising inhibitor, 6-(3-(4,4-difluoropiperidin-1-yl)propyl)-4-methylpyridin-2-amine dihydrochloride, that exhibits excellent potency for rat (Ki = 46 nM) and human nNOS (Ki = 48 nM), respectively, with 388-fold human eNOS and 135-fold human iNOS selectivity. It also displayed excellent permeability (Pe = 17.3 × 10-6 cm s-1) through a parallel artificial membrane permeability assay, a model for blood-brain permeability. We found that increasing lipophilicity by incorporation of fluorine atoms on the backbone of the inhibitors significantly increased potential blood-brain barrier permeability. In addition to measuring potency, isoform selectivity, and permeability of NOS inhibitors, we also explored structure-activity relationships via structures of key inhibitors complexed to various isoforms of nitric oxide synthases.

A Small Peptide Increases Drug Delivery in Human Melanoma Cells.

Melanoma is the most fatal type of skin cancer and is notoriously resistant to chemotherapies. The response of melanoma to current treatments is difficult to predict. To combat these challenges, in this study, we utilize a small peptide to increase drug delivery to melanoma cells. A peptide library array was designed and screened using a peptide array-whole cell binding assay, which identified KK-11 as a novel human melanoma-targeting peptide. The peptide and its D-amino acid substituted analogue (VPWxEPAYQrFL or D-aa KK-11) were synthesized via a solid-phase strategy. Further studies using FITC-labeled KK-11 demonstrated dose-dependent uptake in human melanoma cells. D-aa KK-11 significantly increased the stability of the peptide, with 45.3% remaining detectable after 24 h with human serum incubation. Co-treatment of KK-11 with doxorubicin was found to significantly enhance the cytotoxicity of doxorubicin compared to doxorubicin alone, or sequential KK-11 and doxorubicin treatment. In vivo and ex vivo imaging revealed that D-aa KK-11 distributed to xenografted A375 melanoma tumors as early as 5 min and persisted up to 24 h post tail vein injection. When co-administered, D-aa KK-11 significantly enhanced the anti-tumor activity of a novel nNOS inhibitor (MAC-3-190) in an A375 human melanoma xenograft mouse model compared to MAC-3-190 treatment alone. No apparent systemic toxicities were observed. Taken together, these results suggest that KK-11 may be a promising human melanoma-targeted delivery vector for anti-melanoma cargo.

Palladium-Catalyzed α-Arylation of Cyclic ß-Dicarbonyl Compounds for the Synthesis of CaV1.3 Inhibitors.

Cyclic α-aryl ß-dicarbonyl derivatives are important scaffolds in medicinal chemistry. Palladium-catalyzed coupling reactions of haloarenes were conducted with diverse five- to seven-membered cyclic ß-dicarbonyl derivatives including barbiturate, pyrazolidine-3,5-dione, and 1,4-diazepane-5,7-dione. The coupling reactions of various para- or meta-substituted aryl halides occurred efficiently when Pd(t-Bu3P)2, Xphos, and Cs2CO3 were used under 1,4-dioxane reflux conditions. Although the couplings of ortho-substituted aryl halides with pyrazolidine-3,5-dione and 1,4-diazepane-5,7-dione were moderate, the coupling with barbiturate was limited. Using the optimized reaction conditions, we synthesized several 5-aryl barbiturates as new scaffolds of CaV1.3 Ca2+ channel inhibitors. Among the synthesized molecules, 14e was the most potent CaV1.3 inhibitor with an IC50 of 1.42 µM.

NU-9 improves health of hSOD1G93A mouse upper motor neurons in vitro, especially in combination with riluzole or edaravone.

Even though amyotrophic lateral sclerosis (ALS) is a disease of the upper and lower motor neurons, to date none of the compounds in clinical trials have been tested for improving the health of diseased upper motor neurons (UMNs). There is an urgent need to develop preclinical assays that include UMN health as a readout. Since ALS is a complex disease, combinatorial treatment strategies will be required to address the mechanisms perturbed in patients. Here, we describe a novel in vitro platform that takes advantage of an UMN reporter line in which UMNs are genetically labeled with fluorescence and have misfolded SOD1 toxicity. We report that NU-9, an analog of the cyclohexane-1,3-dione family of compounds, improves the health of UMNs with misfolded SOD1 toxicity more effectively than riluzole or edaravone, -the only two FDA-approved ALS drugs to date-. Interestingly, when NU-9 is applied in combination with riluzole or edaravone, there is an additive effect on UMN health, as they extend longer axons and display enhanced branching and arborization, two important characteristics of healthy UMNs in vitro.