Psychological Status as an Effect Modifier of the Association Between Allergy Symptoms and Allergy Testing.

OBJECTIVE: Patient-reported outcome measures, while valuable, may not correlate with diagnostic test results. To better understand this potential discrepancy, our objective was to determine whether psychological health is an effect modifier of the association between patient-reported allergy outcome scores and allergy test results. STUDY DESIGN: Prospective outcomes study. SETTING: Tertiary care hospital and community-based clinic. METHODS: This study included 600 patients at least 18 years of age who presented for symptoms related to allergic rhinitis and completed the related sinonasal outcome test (SNOT), which includes validated nasal, allergy, and psychological domains. Stratified analyses of odds ratios and Spearman correlation coefficients were utilized to assess for effect modification by psychological status. RESULTS: Worse patient-reported allergic rhinitis symptoms were significantly associated with positive allergy test results (odds ratio [OR] 1.69, 95% confidence interval [CI] 1.22-2.34, P = .002) in patients with better psychological health. In contrast, there was no association in patients with worse psychological health (OR 1.06, 95% CI 0.36-3.10, P = .92). These findings were corroborated by assessments of correlation: allergy domain scores were positively correlated with allergy testing scores (Spearman rho 0.18, 95% CI 0.10-0.25, P < .001) in patients with better psychological health, while there was no correlation in patients with worse psychological health (-0.02, 95% CI -0.16-0.12, P = .77). CONCLUSIONS: Psychological status was an effect modifier of the association between allergy domain and allergy testing data. When assessing the relationship between subjective measures, such as sinonasal validated instruments, and objective measures, such as allergy test results, accounting for effect modifiers such as psychological state can provide clinical and research-related insights.

Mitigation of Effect Modification by Psychological Status in Patients with Hearing Loss.

Importance: Although patient-reported outcomes provide valuable insights, these subjective data may not align with objective test results. Hearing loss is a pervasive problem, such that concordance between subjective perceptions of hearing ability and objective audiogram assessments would be beneficial. Objectives: To determine (1) whether psychological status is an effect modifier of the association between subjective patient reports of hearing ability and objective audiometry results, and (2) whether any effect modification observed in standard static questionnaires would be either mitigated or exacerbated by adaptive testing based on Item Response Theory analyses. Design, Setting, and Participants: This diagnostic study at a tertiary care center and community-based practice included consecutive adults who presented with queries related to hearing loss. Participants were recruited and enrolled and data analyses occurred from 2022 to 2024. Exposures: Participants prospectively reported their hearing-specific abilities through either a standard static or adaptive version of the Inner Effectiveness of Auditory Rehabilitation (EAR) scale, alongside validated measures of their mental health and audiometry. Word recognition scores (WRS) and pure tone averages (PTA) were used to analyze audiometric testing. Main Outcomes and Measures: The association between subjective Inner EAR results and audiometry was evaluated. Stratified analyses were used to assess for effect modification by psychological status. The results of standard static and adaptive testing were compared. Results: In this study of 395 patients (mean [range] age, 55.9 [18-89] years; 210 [53.2%] female), standard static Inner EAR mean scores were appropriately higher in patients with higher (better) WRS (50.7, 95% CI, 46.4-54.9), compared with patients with lower (worse) WRS (34.7, 95% CI, 24.3-45.1). However, among patients with worse mental health, there was no association between standard static Inner EAR scores and WRS. In contrast, adaptive Inner EAR mean scores were significantly higher for those with better WRS, regardless of mental health status. Thus, effect modification was observed in standard static assessments, whereas adaptive testing remained durably associated with audiometry, regardless of mental health. Conclusions and Relevance: Psychological status was an effect modifier of the association between standard Inner EAR scale scores and audiometry, with a positive association observed only in those with better mental health. Adaptive testing scores, however, remained significantly associated with audiometry, even when mental status was worse. Adaptive testing may stabilize the association between subjective and objective hearing outcomes.

Risk Prediction of Post-Endoscopic Submucosal Dissection Coagulation Syndrome.

INTRODUCTION: Endoscopic submucosal dissection (ESD) has been popular worldwide to treat laterally spreading tumors and large polyps. Post-ESD coagulation syndrome (PECS) is more common than the two major ESD-related complications, perforation, and bleeding. The aim of this study was to assess the prevalence of PECS, identify the risk factors for PECS, and create a risk prediction model for PECS. METHODS: Retrospective cross-sectional study analyzed a total of 986 patients who underwent colorectal ESD. Logistic regression models were used to assess risk factors with PECS. Each risk factor was scored, and the 3-step risk stratification index of prediction model was assessed. RESULTS: The prevalence of PECS was 21.4% (95% confidence interval [CI] = 18.9-24.1%). The risk factors of PECS in the multivariate logistic regression were tumor size (+1 cm: odds ratio [OR], 1.29; 95% CI, 1.16-7.09), cecal lesion (OR, 1.96; 95% CI, 1.09-1.53), procedure time (+30 min: OR, 1.19; 95% CI, 1.02-1.39), and ESD with snaring (OR, 0.64; 95% CI, 0.43-0.95). Applying a simplified weighted scoring system based on adjusted OR increments of 1, the risk of PECS was 12.3% (95% CI, 0.3-16.0%) for the low-risk group (score ≤4) and was 36.0% (95% CI = 29.4-43.2%) for the high-risk group (score ≥8). Overall discrimination (C-statistic = 0.629; 95% CI = 0.585-0.672) and calibration (p = 0.993) of the model were moderate to good. CONCLUSION: PECS occurs frequently, and the prediction model can be helpful for effective treatment and prevention of PECS.

CRISPR/Cas9 targeting of passenger single nucleotide variants in haploinsufficient or essential genes expands cancer therapy prospects.

CRISPR/Cas9 technology has effectively targeted cancer-specific oncogenic hotspot mutations or insertion-deletions. However, their limited prevalence in tumors restricts their application. We propose a novel approach targeting passenger single nucleotide variants (SNVs) in haploinsufficient or essential genes to broaden therapeutic options. By disrupting haploinsufficient or essential genes through the cleavage of DNA in the SNV region using CRISPR/Cas9, we achieved the selective elimination of cancer cells without affecting normal cells. We found that, on average, 44.8% of solid cancer patients are eligible for our approach, a substantial increase compared to the 14.4% of patients with CRISPR/Cas9-applicable oncogenic hotspot mutations. Through in vitro and in vivo experiments, we validated our strategy by targeting a passenger mutation in the essential ribosomal gene RRP9 and haploinsufficient gene SMG6. This demonstrates the potential of our strategy to selectively eliminate cancer cells and expand therapeutic opportunities.

Validation of a Novel Allergy-Specific Domain for the 22-Item Sino-Nasal Outcomes Test.

OBJECTIVES: To develop and assess the validity of a novel allergy-specific domain for the 22-item sino-nasal outcomes test (SNOT-22), to provide a new tool that efficiently quantifies the impact of allergic rhinitis (AR) concurrent with chronic rhinosinusitis. STUDY DESIGN: Prospective validation study. SETTING: Tertiary care hospital and community-based clinic. METHODS: Proposed items were developed based on clinician and patient input, and further assessed via factor analysis and for internal consistency (n = 1987). Items were then additionally assessed for convergent and discriminant validity (n = 415), applying data from concurrent completions of the Nasal Obstruction and Septoplasty Effectiveness Scale (NOSE), Mini-Rhinoconjunctivitis Quality-of-Life Questionnaire (MiniRQLQ), and validated global health assessments. Assessments of intra-rater reliability, responsiveness to change, and qualitative input were also performed. RESULTS: Factor analysis demonstrated that proposed allergy items mapped to a single domain. Items were internally consistent (Cronbach α: 0.80 within domain, 0.91 within all SNOT). In assessments of convergent validity, domain scores were associated with MiniRQLQ (Spearman's ρ: 0.46, 95% confidence interval [CI]: 0.30-0.59) and NOSE scores (0.36, 95% CI: 0.27-0.44). The novel items also discriminated among clinical states: a 1-point increase in domain score was associated with an 8.32 (95% CI: 5.43-12.75) increase in the odds of prompting a visit for allergy-related symptoms and a 1.52 (95% CI: 1.13-2.05) increase in the odds of positive allergy testing. Intra-rater reliability was substantial (Cohen's κ: 0.8, 95% CI: 0.8-0.9), and responsiveness to change was demonstrated (mean difference: -0.6, 95% CI: -0.8 to -0.4). CONCLUSIONS: This novel domain is a valid, efficient measure of AR alongside rhinosinusitis.

Early Weight-Bearing After Fibula Free Flap Surgery.

Importance: Despite the widespread use of fibula free flap (FFF) surgery for head and neck reconstruction, there are no studies assessing if early weight-bearing (EWB) affects postoperative recovery, and the timing of weight-bearing initiation following FFF surgery varies considerably across institutions. Therefore, it is important to understand the effect of EWB in these patients and whether it could improve postoperative recovery. Objective: To assess the association of EWB after FFF surgery with donor-site complications, length of stay, and discharge to home status. Design, Setting, and Participants: This retrospective cohort study took place at Massachusetts Eye and Ear, a single tertiary care institution in Boston, Massachusetts. A total of 152 patients who received head and neck reconstruction with a fibula osteocutaneous free flap between January 11, 2010, and August 11, 2022, were included. Exposure: EWB on postoperative day 1 vs non-EWB on postoperative day 2 or later. Main Outcomes and Measures: Patient characteristics, including demographic characteristics and comorbidities, surgical characteristics, donor-site complications, length of stay, and discharge disposition, were recorded. Descriptive statistics and multivariate logistic regressions were used to compute effect sizes and 95% CIs to compare postoperative outcomes in EWB and non-EWB groups. Results: A total of 152 patients (median [IQR] age, 63 [55-70] years; 89 [58.6%] male) were included. The median (IQR) time to postoperative weight-bearing was 3 (1-5) days. Among all patients, 14 (9.2%) had donor-site complications. EWB on postoperative day 1 was associated with shorter length of stay (adjusted odds ratio [AOR], 0.10; 95% CI, 0.02-0.60), increased rate of discharge to home (AOR, 7.43; 95% CI, 2.23-24.80), and decreased donor-site complications (AOR, 0.11; 95% CI, 0.01-0.94). Conversely, weight-bearing 3 or more days postoperatively was associated with an increased risk of pneumonia (AOR, 6.82; 95% CI, 1.33-34.99). Conclusions and Relevance: In this cohort study, EWB after FFF surgery was associated with shorter length of stay, increased rate of discharge to home, and decreased donor-site complications. These findings support the role of early mobilization to optimize postoperative recovery after FFF surgery.

Outcomes of Primary Esophagectomy and Esophagectomy after Endoscopic Submucosal Dissection for Superficial Esophageal Squamous Cell Carcinoma: A Propensity-Score-Matched Analysis.

Even though the conventional treatment for T1 esophageal cancer is surgery, ESD is becoming the primary treatment. Currently, it is unknown whether secondary esophagectomy after endoscopic submucosal dissection (ESD) is comparable to primary esophagectomy when considering outcomes in patients with T1 esophageal cancer. We compared short- and long-term clinical outcomes between the two groups. Primary surgery (esophagectomy) was performed in 191 patients between 2003 and 2014, and 62 patients underwent secondary surgery (esophagectomy) after ESD for T1 esophageal cancer between 2007 and 2019. Propensity matching was performed for age, sex, Charlson Comorbidity Index (CCI), location, pathology, degree of differentiation, tumor size, and invasion depth. Lymph node metastasis (LNM), overall survival (OS), disease-specific survival (DSS), recurrence-free survival (RFS), and post-operative complications were compared between groups. Sixty-eight patients were included after propensity score matching; LNM, OS, DSS, and RFS were comparable between the two groups. Comparing primary and secondary surgery, the respective LNM rates were 23.5% and 26.5%, 6-year OS 78.0% and 89.7%, p = 0.15; DSS were 80.4% and 96.8%, p = 0.057; and RFS were 80.8% and 89.7%, p = 0.069. Comparing the adverse events between the two groups, there was no significant difference in the overall adverse events. However, more early complications were observed in the primary surgery group than in the secondary surgery group (50% vs. 20.6%, p = 0.021). Secondary surgery did not increase the risk of LNM. The long-term outcomes were comparable. Therefore, attempts to perform upfront ESD for superficial esophageal squamous cell cancers are justified.

IDF-11774 Induces Cell Cycle Arrest and Apoptosis by Inhibiting HIF-1α in Gastric Cancer.

Hypoxia-inducible factor-1 alpha (HIF-1α) is a regulatory factor of intracellular oxygen supersession. The expression or increased activity of HIF-1α is closely related to various human cancers. Previously, IDF-11774 was demonstrated to inhibit HSP70 chaperone activity and suppress the accumulation of HIF-1α. In this study, we aimed to determine the effects of IDF-11774 on gastric cancer cell lines. Treatment with IDF-11774 was found to markedly decrease the proliferation, migration, and invasion of the gastric cancer cell lines. Furthermore, the phosphorylation levels of extracellular signal-regulated kinase 1/2, p38, and Jun N-terminal kinase in the mitogen-activated protein kinase signaling pathways were markedly increased in a dose-dependent manner, ultimately promoting apoptosis via the induction of cell cycle arrest. Our findings indicate that HIF-1α inhibitors are potent drugs for the treatment of gastric cancer.

Weight loss from diagnosis of Crohn's disease to one year post-diagnosis results in earlier surgery.

Malnutrition might play a key role in the prognosis of patients with Crohn's disease (CD). The aim of this study was to explore the impact of weight loss from diagnosis of CD to one-year post-diagnosis on disease prognosis in terms of surgery. Patients who were diagnosed with CD at Samsung Medical Center between 1995 to 2020 were included in this study. The study defined the "group with weight loss" as patients with weight loss in one year after diagnosis and the "group without body weight loss" as patients without weight loss in one year after diagnosis. Their data such as demographics, laboratory findings, and medical interventions were collected retrospectively. The primary outcome was confirmation of the difference in the incidence of surgery associated with CD between the group with weight loss and the group without body weight loss. We further analyzed factors associated with surgery outcomes. A total of 165 patients were analyzed in this study. Forty-one patients (24.8%) had body weight loss whereas 124 patients (75.2%) had no body weight loss. Body change at one year showed no significant association with direct surgical incidence. However, the patients with weight loss tended to undergo surgery earlier than patients without body weight loss. Among factors associated with outcomes of Crohn's surgery, the albumin was the only significant factor. Patients with weight loss had no statistically significant increase in the risk of surgery than patients without weight loss, although they tended to undergo surgery earlier than patients without body weight loss. A prospective study is needed to determine serial body weight changes during follow-up for patients with CD.

Discovery of a novel NAMPT inhibitor that selectively targets NAPRT-deficient EMT-subtype cancer cells and alleviates chemotherapy-induced peripheral neuropathy.

Background: Exploiting synthetic lethality (SL) relationships between protein pairs has emerged as an important avenue for the development of anti-cancer drugs. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme of the NAD+ salvage pathway, having an SL relationship with nicotinic acid phosphoribosyltransferase (NAPRT), the key enzyme in the NAD+ Preiss-Handler pathway. NAMPT inhibitor holds clinical potential not only as a promising cancer treatment but also as a means of protection against chemotherapy-induced-peripheral-neuropathy (CIPN). However, as NAD+ is essential for normal cells, the clinical use of NAMPT inhibitors is challenging. This study aimed to identify a novel NAMPT inhibitor with enhanced selective cytotoxicity against NAPRT-deficient cancer cells as well as prominent efficacy in alleviating CIPN. Methods: We began by conducting drug derivatives screening in a panel of lung cancer cell lines to select an agent with the broadest therapeutic window between the NAPRT-negative and-positive cancer cell lines. Both in vitro and In vivo comparative analyses were conducted between A4276 and other NAMPT inhibitors to evaluate the NAPRT-negative cancer cell selectivity and the underlying distinct NAMPT inhibition mechanism of A4276. Patient-derived tumor transcriptomic data and protein levels in various cancer cell lines were analyzed to confirm the correlation between NAPRT depletion and epithelial-to-mesenchymal transition (EMT)-like features in various cancer types. Finally, the efficacy of A4276 for axonal protection and CIPN remedy was examined in vitro and in vivo. Results: The biomarker-driven phenotypic screening led to a discovery of A4276 with prominent selectivity against NAPRT-negative cancer cells compared with NAPRT-positive cancer cells and normal cells. The cytotoxic effect of A4276 on NAPRT-negative cells is achieved through its direct binding to NAMPT, inhibiting its enzymatic function at an optimal and balanced level allowing NAPRT-positive cells to survive through NAPRT-dependent NAD+ synthesis. NAPRT deficiency serves as a biomarker for the response to A4276 as well as an indicator of EMT-subtype cancer in various tumor types. Notably, A4276 protects axons from Wallerian degeneration more effectively than other NAMPT inhibitors by decreasing NMN-to-NAD+ ratio. Conclusion: This study demonstrates that A4276 selectively targets NAPRT-deficient EMT-subtype cancer cells and prevents chemotherapy-induced peripheral neuropathy, highlighting its potential as a promising anti-cancer agent for use in cancer monotherapy or combination therapy with conventional chemotherapeutics.

Gastric Cancer Incidence and Mortality After Endoscopic Resection of Gastric Adenoma: A Nationwide Cohort Study.

INTRODUCTION: Gastric adenoma is a precursor lesion of gastric cancer. We investigated whether the removal of gastric adenoma prevented gastric cancer incidence and its mortality. METHODS: Using the linkage of nationwide databases, we assessed gastric cancer incidence and mortality among patients who had gastric adenomas removed between 2011 and 2013 in Korea. These outcomes were compared primarily with those of the Korean general population by estimating the standardized incidence and mortality ratio and secondarily with internal control subjects who did not have gastric neoplasm after esophagogastroduodenoscopy and were matched for age, sex, and calendar year by calculating hazard ratios (HR) with the Cox proportional hazards model. RESULTS: We identified 44,405 adenoma removal patients. During a median follow-up of 8.4 years, 1,038 (2.34%) of them were given a diagnosis of gastric cancer and a total of 524 gastric cancers were expected for a standard incidence ratio of 1.98 (95% confidence interval [CI], 1.84-2.13). A total of 199 deaths from gastric cancer were expected and 99 were observed for a standard mortality ratio of 0.50 (95% CI, 0.40-0.60). Compared with the nonadenoma cohort (n = 39,826), the adenoma removal patients had a higher risk of gastric cancer (HR, 2.84; 95% CI, 2.51-3.21) and associated mortality (HR, 1.66; 95% CI, 1.19-2.31). DISCUSSION: Removal of gastric adenoma resulted in lower-than-expected mortality but higher-than-expected incidence due to gastric cancer than that in the general population. Our analyses indicated the importance of follow-up strategy after removal of gastric adenoma.

USP14 inhibition regulates tumorigenesis by inducing apoptosis in gastric cancer.

Deubiquitinases (DUBs) are an essential component of the ubiquitin-proteasome system (UPS). They trim ubiquitin from substrate proteins, thereby preventing them from degradation, and modulate different cellular processes. Ubiquitin-specific protease 14 (USP14) is a DUB that has mainly been studied for its role in tumorigenesis in several cancers. In the present study, we found that the protein levels of USP14 were remarkably higher in gastric cancer tissues than in the adjacent normal tissues. We also demonstrated that the inhibition of USP14 activity using IU1 (an USP14 inhibitor) or the inhibition of USP14 expression using USP14-specific siRNA markedly reduced the viability of gastric cancer cells and suppressed their migratory and invasive abilities. The reduction in gastric cancer cell proliferation due to the inhibition of USP14 activity was a result of the increase in the degree of apoptosis, as evidenced by the increased expression levels of cleaved caspase-3 and cleaved PARP. Furthermore, an experiment using the USP14 inhibitor IU1 revealed that the inhibition of USP14 activity suppressed 5-fluorouracil (5-FU) resistance in GC cells. Collectively, these findings indicate that USP14 plays critical roles in gastric cancer progression and suggest its potential to serve as a novel therapeutic target for gastric cancer treatment. [BMB Reports 2023; 56(8): 451-456].

Improvement of semantic segmentation through transfer learning of multi-class regions with convolutional neural networks on supine and prone breast MRI images.

Semantic segmentation of breast and surrounding tissues in supine and prone breast magnetic resonance imaging (MRI) is required for various kinds of computer-assisted diagnoses for surgical applications. Variability of breast shape in supine and prone poses along with various MRI artifacts makes it difficult to determine robust breast and surrounding tissue segmentation. Therefore, we evaluated semantic segmentation with transfer learning of convolutional neural networks to create robust breast segmentation in supine breast MRI without considering supine or prone positions. Total 29 patients with T1-weighted contrast-enhanced images were collected at Asan Medical Center and two types of breast MRI were performed in the prone position and the supine position. The four classes, including lungs and heart, muscles and bones, parenchyma with cancer, and skin and fat, were manually drawn by an expert. Semantic segmentation on breast MRI scans with supine, prone, transferred from prone to supine, and pooled supine and prone MRI were trained and compared using 2D U-Net, 3D U-Net, 2D nnU-Net and 3D nnU-Net. The best performance was 2D models with transfer learning. Our results showed excellent performance and could be used for clinical purposes such as breast registration and computer-aided diagnosis.

Transcriptional Antagonism by CDK8 Inhibition Improves Therapeutic Efficacy of MEK Inhibitors.

Aberrant RAS/MAPK signaling is a common driver of oncogenesis that can be therapeutically targeted with clinically approved MEK inhibitors. Disease progression on single-agent MEK inhibitors is common, however, and combination therapies are typically required to achieve significant clinical benefit in advanced cancers. Here we focused on identifying MEK inhibitor-based combination therapies in neuroblastoma with mutations that activate the RAS/MAPK signaling pathway, which are rare at diagnosis but frequent in relapsed neuroblastoma. A genome-scale CRISPR-Cas9 functional genomic screen was deployed to identify genes that when knocked out sensitize RAS-mutant neuroblastoma to MEK inhibition. Loss of either CCNC or CDK8, two members of the mediator kinase module, sensitized neuroblastoma to MEK inhibition. Furthermore, small-molecule kinase inhibitors of CDK8 improved response to MEK inhibitors in vitro and in vivo in RAS-mutant neuroblastoma and other adult solid tumors. Transcriptional profiling revealed that loss of CDK8 or CCNC antagonized the transcriptional signature induced by MEK inhibition. When combined, loss of CDK8 or CCNC prevented the compensatory upregulation of progrowth gene expression induced by MEK inhibition. These findings propose a new therapeutic combination for RAS-mutant neuroblastoma and may have clinical relevance for other RAS-driven malignancies. SIGNIFICANCE: Transcriptional adaptation to MEK inhibition is mediated by CDK8 and can be blocked by the addition of CDK8 inhibitors to improve response to MEK inhibitors in RAS-mutant neuroblastoma, a clinically challenging disease.

Optimized parameters for effective SARS-CoV-2 inactivation using UVC-LED at 275 nm.

The spread of SARS-CoV-2 infections and the severity of the coronavirus disease of 2019 (COVID-19) pandemic have resulted in the rapid development of medications, vaccines, and countermeasures to reduce viral transmission. Although new treatment strategies for preventing SARS-CoV-2 infection are available, viral mutations remain a serious threat to the healthcare community. Hence, medical devices equipped with virus-eradication features are needed to prevent viral transmission. UV-LEDs are gaining popularity in the medical field, utilizing the most germicidal UVC spectrum, which acts through photoproduct formation. Herein, we developed a portable and rechargeable medical device that can disinfect SARS-CoV-2 in less than 10 s by 99.9%, lasting 6 h. Using this device, we investigated the antiviral effect of UVC-LED (275 nm) against SARS-CoV-2 as a function of irradiation distance and exposure time. Irradiation distance of 10-20 cm, < 10 s exposure time, and UV doses of > 10 mJ/cm2 were determined optimal for SARS-CoV-2 elimination (≥ 99.99% viral reduction). The UVC-LED systems have advantages such as fast-stabilizing intensity and insensitivity to temperature, and may contribute to developing medical devices capable of containing SARS-CoV-2 infection. By demonstrating SARS-CoV-2 inactivation with very short-term UVC-LED irradiation, our study may suggest guidelines for securing a safer medical environment.

Rapamycin Cannot Reduce Seizure Susceptibility in Infantile Rats with Malformations of Cortical Development Lacking mTORC1 Activation.

The mechanistic target of the rapamycin (mTOR) pathway is involved in cortical development. However, the efficacy of mTOR inhibitors in malformations of cortical dysplasia (MCD) outside of the tuberous sclerosis complex is unknown. We selected the MCD rat model with prenatal MAM exposure to test the efficacy of mTOR inhibitors in MCDs. We explored the early cortical changes of mTOR pathway protein expression in rats aged P15. We also monitored the early treatment effect of the mTOR inhibitor, rapamycin, on N-methyl-D-aspartate (NMDA)-induced spasms at P15 and their behavior in the juvenile stage. In vivo MR spectroscopy was performed after rapamycin treatment and compared with vehicle controls. There was no difference in mTORC1 pathway protein expression between MAM-exposed MCD rats and controls at P15, and prolonged treatment of rapamycin had no impact on NMDA-induced spasms despite poor weight gain. Prenatal MAM-exposed juvenile rats treated with rapamycin showed increased social approaching and freezing behavior during habituation. MR spectroscopy showed altered neurometabolites, including Gln, Glu+Gln, Tau, and Cr. Despite behavioral changes and in vivo neurometabolic alteration with early prolonged rapamycin treatment, rapamycin had no effect on spasms susceptibility in prenatal MAM-exposed infantile rats with MCD without mTORC1 activation. For MAM-exposed MCD rats without mTORC1 activation, treatment options outside of mTOR pathway inhibitors should be explored.

Snail acetylation by autophagy-derived acetyl-coenzyme A promotes invasion and metastasis of KRAS-LKB1 co-mutated lung cancer cells.

BACKGROUND: Autophagy is elevated in metastatic tumors and is often associated with active epithelial-to-mesenchymal transition (EMT). However, the extent to which EMT is dependent on autophagy is largely unknown. This study aimed to identify the mechanisms by which autophagy facilitates EMT. METHODS: We employed a liquid chromatography-based metabolomic approach with kirsten rat sarcoma viral oncogene (KRAS) and liver kinase B1 (LKB1) gene co-mutated (KL) cells that represent an autophagy/EMT-coactivated invasive lung cancer subtype for the identification of metabolites linked to autophagy-driven EMT activation. Molecular mechanisms of autophagy-driven EMT activation were further investigated by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting analysis, immunoprecipitation, immunofluorescence staining, and metabolite assays. The effects of chemical and genetic perturbations on autophagic flux were assessed by two orthogonal approaches: microtubule-associated protein 1A/1B-light chain 3 (LC3) turnover analysis by Western blotting and monomeric red fluorescent protein-green fluorescent protein (mRFP-GFP)-LC3 tandem fluorescent protein quenching assay. Transcription factor EB (TFEB) activity was measured by coordinated lysosomal expression and regulation (CLEAR) motif-driven luciferase reporter assay. Experimental metastasis (tail vein injection) mouse models were used to evaluate the impact of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) or ATP citrate lyase (ACLY) inhibitors on lung metastasis using IVIS luciferase imaging system. RESULTS: We found that autophagy in KL cancer cells increased acetyl-coenzyme A (acetyl-CoA), which facilitated the acetylation and stabilization of the EMT-inducing transcription factor Snail. The autophagy/acetyl-CoA/acetyl-Snail axis was further validated in tumor tissues and in autophagy-activated pancreatic cancer cells. TFEB acetylation in KL cancer cells sustained pro-metastatic autophagy in a mammalian target of rapamycin complex 1 (mTORC1)-independent manner. Pharmacological inhibition of this axis via CAMKK2 inhibitors or ACLY inhibitors consistently reduced the metastatic capacity of KL cancer cells in vivo. CONCLUSIONS: This study demonstrates that autophagy-derived acetyl-CoA promotes Snail acetylation and thereby facilitates invasion and metastasis of KRAS-LKB1 co-mutated lung cancer cells and that inhibition of the autophagy/acetyl-CoA/acetyl-Snail axis using CAMKK2 or ACLY inhibitors could be a potential therapeutic strategy to suppress metastasis of KL lung cancer.

Phenotypic and Genetic Complexity in Pediatric Movement Disorders.

The complex and evolving nature of clinical phenotypes have made genetically diagnosing pediatric patients with movement disorders difficult. Here, we describe this diverse complexity in the clinical and genetic features of a pediatric cohort examined by whole-exome sequencing (WES) and demonstrate the clinical benefit of WES as a diagnostic tool in a pediatric cohort. We evaluated 75 patients with diverse single or combined movement phenomenologies using WES. WES identified 42 variants in 37 genes (56.0%). The detection rate was highest in patients with dystonia (11/13, 84.6%), followed by ataxia (21/38, 55.3%), myoclonus (3/6, 50.0%), unspecified dyskinesia (1/4, 25.0%), tremor (1/1, 100%), respectively. Most genetically diagnosed patients (90.5%) were affected by other neurologic or systemic manifestations; congenital hypotonia (66.7%), and epilepsy (42.9%) were the most common phenotypes. The genetic diagnosis changed the clinical management for five patients (6.7%), including treatments targeting molecular abnormalities, and other systemic surveillance such as cancer screening. Early application of WES yields a high diagnostic rate in pediatric movement disorders, which can overcome the limitations of the traditional phenotype-driven strategies due to the diverse phenotypic and genetic complexity. Additionally, this early genetic diagnosis expands the patient's clinical spectrum and provides an opportunity for tailored treatment.

Factors Influencing Luciferase-Based Bioluminescent Imaging in Preclinical Models of Brain Tumor.

Bioluminescent imaging (BLI) is a powerful tool in biomedical research to measure gene expression and tumor growth. The current study examined factors that influence the BLI signal, specifically focusing on the tissue distribution of two luciferase substrates, D-luciferin and CycLuc1. D-luciferin, a natural substrate of firefly luciferase, has been reported to have limited brain distribution, possibly due to the efflux transporter, breast cancer resistance protein (Bcrp), at the blood-brain barrier. CycLuc1, a synthetic analog of D-luciferin, has a greater BLI signal at lower doses than D-luciferin, especially in the brain. Our results indicate that limited brain distribution of D-luciferin and CycLuc1 is predominantly dictated by their low intrinsic permeability across the cell membrane, where the efflux transporter, Bcrp, plays a relatively minor role. Both genetic ablation and pharmacological inhibition of Bcrp decreased the systemic clearance of both luciferase substrates, significantly increasing exposure in the blood and, hence, in organs and tissues. These data also indicate that the biodistribution of luciferase substrates can be differentially influenced in luciferase-bearing tissues, leading to a "tissue-dependent" BLI signal. The results of this study point to the need to consider multiple mechanisms that influence the distribution of luciferase substrates. SIGNIFICANCE STATEMENT: Bioluminescence is used to monitor many biological processes, including tumor growth. This study examined the pharmacokinetics, brain distribution, and the role of active efflux transporters on the luciferase substrates D-luciferin and CycLuc1. CycLuc1 has a more sustained systemic circulation time (longer half-life) that can provide an advantage for the superior imaging outcome of CycLuc1 over D-luciferin. The disparity in imaging intensities between brain and peripheral sites is due to low intrinsic permeability of these luciferase substrates across the blood-brain barrier.

Central Nervous System Distribution of an Opioid Agonist Combination with Synergistic Activity.

Novel combinations of specific opioid agonists like loperamide and oxymorphindole targeting the µ- and δ-opioid receptors, respectively, have shown increased potency with minimized opioid-associated risks. However, whether their interaction is pharmacokinetic or pharmacodynamic in nature has not been determined. This study quantitatively determined whether these drugs have a pharmacokinetic interaction that alters systemic disposition or central nervous system (CNS) distribution. We performed intravenous and oral in vivo pharmacokinetic assessments of both drugs after discrete dosing and administration in combination to determine whether the combination had any effect on systemic pharmacokinetic parameters or CNS exposure. Drugs were administered at 5 or 10 mg/kg i.v. or 30 mg/kg orally to institute for cancer research (ICR) mice and 5 mg/kg i.v. to Friend leukemia virus strain B mice of the following genotypes: wild-type, breast cancer resistance protein (Bcrp-/- ) (Bcrp knockout), Mdr1a/b-/- [P-glycoprotein (P-gp) knockout], and Bcrp-/- Mdr1a/b-/- (triple knockout). In the combination, clearance of oxymorphindole (OMI) was reduced by approximately half, and the plasma area under the concentration-time curve (AUC) increased. Consequently, brain and spinal cord AUCs for OMI in the combination also increased proportionately. Both loperamide and OMI are P-gp substrates, but administration of the two drugs in combination does not alter efflux transport at the CNS barriers. Because OMI alone shows appreciable brain penetration but little therapeutic efficacy on its own, and because loperamide's CNS distribution is unchanged in the combination, the mechanism of action for the increased potency of the combination is most likely pharmacodynamic and most likely occurs at receptors in the peripheral nervous system. This combination has favorable characteristics for future development. SIGNIFICANCE STATEMENT: Opioids have yet to be replaced as the most effective treatments for moderate-to-severe pain and chronic pain, but their side effects are dangerous. Combinations of opioids with peripheral activity, such as loperamide and oxymorphindole, would be valuable in that they are effective at much lower doses and have reduced risks for dangerous side effects because the µ-opioid receptor agonist is largely excluded from the CNS.