Bi-allelic variants in NAE1 cause intellectual disability, ischiopubic hypoplasia, stress-mediated lymphopenia and neurodegeneration.

Neddylation has been implicated in various cellular pathways and in the pathophysiology of numerous diseases. We identified four individuals with bi-allelic variants in NAE1, which encodes the neddylation E1 enzyme. Pathogenicity was supported by decreased NAE1 abundance and overlapping clinical and cellular phenotypes. To delineate how cellular consequences of NAE1 deficiency would lead to the clinical phenotype, we focused primarily on the rarest phenotypic features, based on the assumption that these would best reflect the pathophysiology at stake. Two of the rarest features, neuronal loss and lymphopenia worsening during infections, suggest that NAE1 is required during cellular stress caused by infections to protect against cell death. In support, we found that stressing the proteasome system with MG132-requiring upregulation of neddylation to restore proteasomal function and proteasomal stress-led to increased cell death in fibroblasts of individuals with NAE1 genetic variants. Additionally, we found decreased lymphocyte counts after CD3/CD28 stimulation and decreased NF-κB translocation in individuals with NAE1 variants. The rarest phenotypic feature-delayed closure of the ischiopubic rami-correlated with significant downregulation of RUN2X and SOX9 expression in transcriptomic data of fibroblasts. Both genes are involved in the pathophysiology of ischiopubic hypoplasia. Thus, we show that NAE1 plays a major role in (skeletal) development and cellular homeostasis during stress. Our approach suggests that a focus on rare phenotypic features is able to provide significant pathophysiological insights in diseases caused by mutations in genes with pleiotropic effects.

Identification of novel benzothiazole derivatives as inhibitors of NEDDylation pathway to inhibit the progression of gastric cancer.

The overexpression of neddylation modification is frequently observed in human tumor cells. Targeting the neddylation pathway has been recognized as a promising anticancer therapeutic strategy, thus discovering potent and selective neddylation inhibitors is of great importance. In this study, we designed and synthesized a series of novel neddylation inhibitors bearing benzothiazole scaffolds by molecular hybridization strategy and all compounds were evaluated for antiproliferative activity against MGC-803, MCF-7, A549 and KYSE-30 cell lines. In vitro anti-tumor studies showed that the most promising compound X-10, effectively suppressed MGC-803 cells growth and migration, induced apoptosis and arrested cell cycle at G2/M phase. Importantly, by directly interacting with NAE1, X-10 blocked NAE1 activity, specifically preventing neddylation of Cullin 3 and Cullin 1, and produced a dose-response decline in the level of UBC12-NEDD8 complex. Overall, our data indicate that X-10 inhibits the process of neddylation, making it a potentially agent for both cancer prevention and therapy purposes.

Discovery of the 2,4-disubstituted quinazoline derivative as a novel neddylation inhibitor for tumor therapy.

Overactivation of neddylation has been found in a number of common human tumor-related diseases. In recent years, targeting the neddylation pathway has become an appealing anti-cancer strategy, and it is critical to find neddylation inhibitors with novel structures and higher efficacy. Here, we present the discovery of novel inhibitors of the NEDD8-activating enzyme (NAE) and their antitumor activity in vitro. All synthesized 1,4-disubstituted piperidine compounds were evaluated for antiproliferative activity against MGC-803, MCF-7, A549, and KYSE-30 cells. Among five representative compounds, III-26 bearing a quinazoline motif was identified as the lead one due to the fact that it significantly hindered the neddylation of Cullin1. Cellular mechanisms elucidated that III-26 inhibited the proliferation, migration, and invasion of UBC12-overexpressed MGC-803 cell lines, as well as induced apoptosis and arrested the cell cycle at G2/M phase. Importantly, III-26 reduced NAE activity, thus selectively preventing neddylation of Cullin3 and Cullin1 over other Cullin members. At a dose of 4 µM, III-26 virtually entirely blocked UBC12-NEDD8 conjugation in MGC-803 cells. Our molecular modeling and kinetic investigation suggested that this compound may function as a non-covalent inhibitor of NAE.

The OsWRKY72-OsAAT30/OsGSTU26 module mediates reactive oxygen species scavenging to drive heterosis for salt tolerance in hybrid rice.

Hybrid rice (Oryza sativa) generally outperforms its inbred parents in yield and stress tolerance, a phenomenon termed heterosis, but the underlying mechanism is not completely understood. Here, we combined transcriptome, proteome, physiological, and heterosis analyses to examine the salt response of super hybrid rice Chaoyou1000 (CY1000). In addition to surpassing the mean values for its two parents (mid-parent heterosis), CY1000 exhibited a higher reactive oxygen species scavenging ability than both its parents (over-parent heterosis or heterobeltiosis). Nonadditive expression and allele-specific gene expression assays showed that the glutathione S-transferase gene OsGSTU26 and the amino acid transporter gene OsAAT30 may have major roles in heterosis for salt tolerance, acting in an overdominant fashion in CY1000. Furthermore, we identified OsWRKY72 as a common transcription factor that binds and regulates OsGSTU26 and OsAAT30. The salt-sensitive phenotypes were associated with the OsWRKY72paternal genotype or the OsAAT30maternal genotype in core rice germplasm varieties. OsWRKY72paternal specifically repressed the expression of OsGSTU26 under salt stress, leading to salinity sensitivity, while OsWRKY72maternal specifically repressed OsAAT30, resulting in salinity tolerance. These results suggest that the OsWRKY72-OsAAT30/OsGSTU26 module may play an important role in heterosis for salt tolerance in an overdominant fashion in CY1000 hybrid rice, providing valuable clues to elucidate the mechanism of heterosis for salinity tolerance in hybrid rice.

FOXO1-regulated lncRNA CYP1B1-AS1 suppresses breast cancer cell proliferation by inhibiting neddylation.

PURPOSE: Overactivated neddylation is considered to be a common event in cancer. Long non-coding RNAs (lncRNAs) can regulate cancer development by mediating post-translational modifications. However, the role of lncRNA in neddylation modification remains unclear. METHODS: LncRNA cytochrome P450 family 1 subfamily B member 1 antisense RNA 1 (CYP1B1-AS1) expression in breast cancer tissues was evaluated by RT-PCR and TCGA BRCA data. Gain and loss of function experiments were performed to explore the role of CYP1B1-AS1 in breast cancer cell proliferation and apoptosis in vitro and in vivo. Luciferase assay, CHIP-qPCR assay, transcriptome sequencing, RNA-pulldown assay, mass spectrometry, RIP-PCR and Western blot were used to investigate the regulatory factors of CYP1B1-AS1 expression and the molecular mechanism of CYP1B1-AS1 involved in neddylation modification. RESULTS: We found that CYP1B1-AS1 was down-regulated in breast cancer tissues and correlated with prognosis. In vivo and in vitro functional experiments confirmed that CYP1B1-AS1 inhibited cell proliferation and induced apoptosis. Mechanistically, CYP1B1-AS1 was regulated by the transcription factor, forkhead box O1 (FOXO1), and could be upregulated by inhibiting the PI3K/FOXO1 pathway. Moreover, CYP1B1-AS1 bound directly to NEDD8 activating enzyme E1 subunit 1 (NAE1) to regulate protein neddylation. CONCLUSION: This study reports for the first time that CYP1B1-AS1 inhibits protein neddylation to affect breast cancer cell proliferation, which provides a new strategy for the treatment of breast cancer by lncRNA targeting neddylation modification.

Neddylation is critical to cortical development by regulating Wnt/ß-catenin signaling.

Wnt signaling plays a critical role in production and differentiation of neurons and undergoes a progressive reduction during cortical development. However, how Wnt signaling is regulated is not well understood. Here we provide evidence for an indispensable role of neddylation, a ubiquitylation-like protein modification, in inhibiting Wnt/ß-catenin signaling. We show that ß-catenin is neddylated; and inhibiting ß-catenin neddylation increases its nuclear accumulation and Wnt/ß-catenin signaling. To test this hypothesis in vivo, we mutated Nae1, an obligative subunit of the E1 for neddylation in cortical progenitors. The mutation leads to eventual reduction in radial glia progenitors (RGPs). Consequently, the production of intermediate progenitors (IPs) and neurons is reduced, and neuron migration is impaired, resulting in disorganization of the cerebral cortex. These phenotypes are similar to those of ß-catenin gain-of-function mice. Finally, suppressing ß-catenin expression is able to rescue deficits of Nae1 mutant mice. Together, these observations identified a mechanism to regulate Wnt/ß-catenin signaling in cortical development.

Effect of Bicarbonate on Net Acid Excretion, Blood Pressure, and Metabolism in Patients With and Without CKD: The Acid Base Compensation in CKD Study.

RATIONALE & OBJECTIVE: Patients with CKD are at elevated risk of metabolic acidosis due to impaired net acid excretion (NAE). Identifying early markers of acidosis may guide prevention in chronic kidney disease (CKD). This study compared NAE in participants with and without CKD, as well as the NAE, blood pressure (BP), and metabolomic response to bicarbonate supplementation. STUDY DESIGN: Randomized order, cross-over study with controlled feeding. SETTING & PARTICIPANTS: Participants consisted of 8 patients with CKD (estimated glomerular filtration rate 30-59mL/min/1.73m2 or 60-70mL/min/1.73m2 with albuminuria) and 6 patients without CKD. All participants had baseline serum bicarbonate concentrations between 20 and 28 mEq/L; they did not have diabetes mellitus and did not use alkali supplements at baseline. INTERVENTION: Participants were fed a fixed-acid-load diet with bicarbonate supplementation (7 days) and with sodium chloride control (7 days) in a randomized order, cross-over fashion. OUTCOMES: Urine NAE, 24-hour ambulatory BP, and 24-hour urine and plasma metabolomic profiles were measured after each period. RESULTS: During the control period, mean NAE was 28.3±10.2 mEq/d overall without differences across groups (P=0.5). Urine pH, ammonium, and citrate were significantly lower in CKD than in non-CKD (P<0.05 for each). Bicarbonate supplementation reduced NAE and urine ammonium in the CKD group, increased urine pH in both groups (but more in patients with CKD than in those without), and increased; urine citrate in the CKD group (P< 0.2 for interaction for each). Metabolomic analysis revealed several urine organic anions were increased with bicarbonate in CKD, including 3-indoleacetate, citrate/isocitrate, and glutarate. BP was not significantly changed. LIMITATIONS: Small sample size and short feeding duration. CONCLUSIONS: Compared to patients without CKD, those with CKD had lower acid excretion in the form of ammonium but also lower base excretion such as citrate and other organic anions, a potential compensation to preserve acid-base homeostasis. In CKD, acid excretion decreased further, but base excretion (eg, citrate) increased in response to alkali. Urine citrate should be evaluated as an early and responsive marker of impaired acid-base homeostasis. FUNDING: National Institute of Diabetes and Digestive and Kidney Diseases and the Duke O'Brien Center for Kidney Research. TRIAL REGISTRATION: Registered at ClinicalTrials.gov with study number NCT02427594.

Predictors of Net Acid Excretion in the Chronic Renal Insufficiency Cohort (CRIC) Study.

RATIONALE & OBJECTIVE: Higher urine net acid excretion (NAE) is associated with slower chronic kidney disease progression, particularly in patients with diabetes mellitus. To better understand potential mechanisms and assess modifiable components, we explored independent predictors of NAE in the CRIC (Chronic Renal Insufficiency Cohort) Study. STUDY DESIGN: Cross-sectional. SETTING & PARTICIPANTS: A randomly selected subcohort of adults with chronic kidney disease enrolled in the CRIC Study with NAE measurements. PREDICTORS: A comprehensive set of variables across prespecified domains including demographics, comorbid conditions, medications, laboratory values, diet, physical activity, and body composition. OUTCOME: 24-hour urine NAE. ANALYTICAL APPROACH: NAE was defined as the sum of urine ammonium and calculated titratable acidity in a subset of CRIC participants. 22 individuals were excluded for urine pH < 4 (n = 1) or ≥7.4 (n = 19) or extreme outliers of NAE values (n = 2). From an analytic sample of 978, we identified the association of individual variables with NAE in the selected domains using linear regression. We estimated the percent variance explained by each domain using the adjusted R2 from a domain-specific model. RESULTS: Mean NAE was 33.2 ± 17.4 (SD) mEq/d. Multiple variables were associated with NAE in models adjusted for age, sex, estimated glomerular filtration rate (eGFR), race/ethnicity, and body surface area, including insulin resistance, dietary potential renal acid load, and a variety of metabolically active medications (eg, metformin, allopurinol, and nonstatin lipid agents). Body size, as indicated by body surface area, body mass index, or fat-free mass; race/ethnicity; and eGFR also were independently associated with NAE. By domains, more variance was explained by demographics, body composition, and laboratory values, which included eGFR and serum bicarbonate level. LIMITATIONS: Cross-sectional; use of stored biological samples. CONCLUSIONS: NAE relates to several clinical domains including body composition, kidney function, and diet, but also to metabolic factors such as insulin resistance and the use of metabolically active medications.

Simultaneous-count models to estimate abundance from counts of unmarked individuals with imperfect detection.

We developed a method to estimate population abundance from simultaneous counts of unmarked individuals over multiple sites. We considered that at each sampling occasion, individuals in a population could be detected at 1 of the survey sites or remain undetected and used either multinomial or binomial simultaneous-count models to estimate abundance, the latter being equivalent to an N-mixture model with one site. We tested model performance with simulations over a range of detection probabilities, population sizes, growth rates, number of years, sampling occasions, and sites. We then applied our method to 3 critically endangered vulture species in Cambodia to demonstrate the real-world applicability of the model and to provide the first abundance estimates for these species in Cambodia. Our new approach works best when existing methods are expected to perform poorly (i.e., few sites and large variation in abundance among sites) and if individuals may move among sites between sampling occasions. The approach performed better when there were >8 sampling occasions and net probability of detection was high (>0.5). We believe our approach will be useful in particular for simultaneous surveys at aggregation sites, such as roosts. The method complements existing approaches for estimating abundance of unmarked individuals and is the first method designed specifically for simultaneous counts.

Modelos de Conteo Simultáneo para Estimar la Abundancia a partir de Conteos de Individuos No Marcados con Detección Imperfecta Resumen Desarrollamos un método para estimar la abundancia poblacional a partir de conteos simultáneos de individuos sin marcaje en múltiples sitios. Consideramos que en cada ocasión de muestreo los individuos de una población podrían ser detectados en uno de los sitios de censos o podrían permanecer sin ser detectados y usamos modelos de conteo simultáneo multinomial o binomial para estimar la abundancia, con el binomial como equivalente a un modelo de mezcla N con un solo sitio. Probamos el desempeño del modelo con simulaciones en un rango de probabilidades de detección, tamaños poblacionales, tasas de crecimiento, número de años, ocasiones de muestreo, y sitios. Después aplicamos nuestro método a tres especies de buitre que se encuentran en peligro crítico en Camboya para demostrar cuán aplicable es el modelo en el mundo real y para proporcionar las primeras estimaciones de abundancia para estas especies en Camboya. Nuestra nueva estrategia trabaja de mejor manera cuando se espera que los modelos existentes tengan un desempeño pobre (es decir, pocos sitios y una gran variación en la abundancia entre sitios) y si los individuos podrían moverse de un sitio a otro entre cada ocasión de muestreo. La estrategia tuvo un mejor desempeñó cuando hubo >8 ocasiones de muestreo y la probabilidad neta de detección fue alta (>0.5). Creemos que nuestra estrategia será especialmente útil para censos simultáneos en sitios de agregación, como los nidos. El método complementa las estrategias existentes para estimar la abundancia de individuos sin marcaje y es el primer método diseñado específicamente para conteos simultáneos.

Antibody-free detection of cellular neddylation dynamics of Cullin1.

Neddylation is a posttranslational modification that regulates protein stability, activity, and subcellular localization. Here, we describe a new tool for exploring the neddylation cycle of cullin1 (Cul1) directly in a cellular context. This assay utilizes the NanoLuc® Binary Technology (NanoBiT) to monitor the covalent neddylation status of Cul1. A stable clonal cell line derived from HEK293 was developed that expressed a C-terminus LgBiT tagged-Cul1 and N-terminus SmBiT tagged-Nedd8. Using this cell line, we screened inhibitors that are known to disrupt Nedd8 biology and demonstrated that both inhibitors of Nedd8-activating enzyme (NAE) and Constitutive photomorphogenesis 9 signalosome (CSN) complex produce concentration and time dependent signal decreases and increases, respectively. The kinetics of both responses could be monitored in real time and demonstrated that modulation of the Nedd8 pathway occurs rapidly. Further characterization of the cellular components of this cell line was performed in order to quantify the various levels of Cul1, Nedd8 and NAE and determined to be near endogenous levels. There was no difference between control and stably transfected cell lines in viability studies of NAE and CSN inhibitors. Taken together, these results suggest that the NanoBiT assay can be used to monitor Cul1 neddylation specifically and in real time.

A phase I study of the investigational NEDD8-activating enzyme inhibitor pevonedistat (TAK-924/MLN4924) in patients with metastatic melanoma.

Purpose The therapeutic index of proteasome inhibitors may be improved through selective inhibition of a sub-component of the ubiquitin-proteasome system, such as the NEDD8-conjugation pathway. This multicenter, phase I, dose-escalation study assessed safety and the maximum tolerated dose (MTD), pharmacokinetics, pharmacodynamics, and antitumor activity of pevonedistat, an investigational NEDD8-activating enzyme (NAE) inhibitor, in patients with metastatic melanoma. Methods Patients received intravenous pevonedistat on Days 1, 4, 8, 11 (schedule A) or 1, 8, 15 (schedule B) of 21-day cycles. Results 26 patients received pevonedistat 50-278 mg/m(2) on schedule A; 11 patients received pevonedistat 157 mg/m(2) on schedule B. The schedule A MTD was 209 mg/m(2): dose-limiting toxicities (DLTs) included grade 3 hypophosphatemia and grade 3 increased blood creatinine (associated with grade 3 hyperbilirubinemia). Two schedule A patients experienced acute organ failure toxicities, one of whom experienced grade 5 acute renal failure. Dose escalation did not occur in schedule B: DLTs included grade 3 myocarditis, grade 2 acute renal failure, and grade 2 hyperbilirubinemia in a single patient. Pevonedistat pharmacokinetics were approximately dose-proportional across the dose range studied, with a biphasic disposition profile characterized by a short elimination half-life (~10 h). Pharmacodynamic studies showed increases in NAE-regulated transcripts post-treatment; all post-dose biopsy samples were positive for pevonedistat-NEDD8 adduct. One schedule A patient achieved a partial response; 15 patients had stable disease (4 lasting ≥6.5 months). Conclusions Pevonedistat was generally well tolerated at the MTD. Anticipated pharmacodynamic effects of NAE inhibition were observed with single-agent pevonedistat in peripheral blood and tumor tissue.

Co-inhibition of BET and NAE enhances BIM-dependent apoptosis with augmented cancer therapeutic efficacy.

Agents that inhibit bromodomain and extra-terminal domain (BET) proteins have been actively tested in the clinic as potential anticancer drugs. NEDD8-activating enzyme (NAE) inhibitors, represented by MLN4924, target the only activation enzyme in the neddylation pathway that has been identified as an attractive target for cancer therapy. In this study, we focus on the combination of BET inhibitors (BETis) and NAE inhibitors (NAEis) as a cancer therapeutic strategy and investigate its underlying mechanisms to explore and expand the application scope of both types of drugs. The results showed that this combination synergistically inhibited the proliferative activity of tumor cells from different tissues. Compared to a single drug, combination therapy had a weak effect on cycle arrest but significantly enhanced cell apoptosis. Furthermore, the growth of NCI-H1975 xenografts in nude mice was significantly inhibited by the combination without obvious body weight loss. Research on the synergistic mechanism demonstrated that combination therapy significantly increased the mRNA and protein levels of the proapoptotic gene BIM. The inhibition and knockout of BIM significantly attenuated the apoptosis induced by the combination, whereas the re-expression of BIM restored the synergistic effects, indicating that BIM induction plays a critical role in mediating the enhanced apoptosis induced by the co-inhibition of BET and NAE. Together, the enhanced transcription mediated by miR-17-92 cluster inhibition and reduced degradation promoted the increase in BIM levels, resulting in a synergistic effect. Collectively, these findings highlight the need for further clinical investigation into the combination of BETi and NAEi as a promising strategy for cancer therapy.

Dual role of targeting NAE1 in nasopharyngeal carcinoma: Antitumor effects yet inducing radiotherapy resistance.

Background and objectives: The inhibitor MLN4924 of Neural Precursor Cell-Expressed Developmentally Down-Regulated 8 (NEDD8) Activating Enzyme 1 (NAE1) has been found to suppress the growth of nasopharyngeal carcinoma (NPC). However, its effect on NPC's radiotherapy sensitivity remains unclear. Methods: By integrating single-cell RNA sequencing and bulk RNA sequencing, we predict the impact of NAE1 on the cell cycle, cell death, and its relationship with radiotherapy sensitivity and prognosis in NPC. The effect of inhibiting NAE1 on NPC cell behavior and radiation sensitivity is explored through MLN4924 intervention in vitro and in vivo. We construct a prognosis prediction model based on NAE1 using machine learning methods and validate the efficacy of NAE1 and the model in clinical cohorts. Results: NPC patients with high NAE1 expression have better prognosis and higher expression in the radiotherapy-sensitive group. Inhibiting NAE1 with MLN4924 causes cell cycle arrest in NPC cells, preventing them from entering the G2/M phase, thereby inhibiting proliferation but not affecting migration and metastasis. However, in vitro and in vivo experiments demonstrate that inhibiting NAE1 with MLN4924 leads to increased resistance of NPC to radiation. Conclusions: Targeting NAE1 for NPC treatment may have dual effects, inhibiting NPC proliferation while also increasing radiation resistance.

[A case of Hashimoto's encephalopathy with acute onset of psychiatric symptoms and diffuse deep white matter lesions on brain MRI].

A 51-year-old man developed acute disturbances in consciousness and psychiatric symptoms one month prior to admission. He was referred and admitted to the Department of Psychiatry of our hospital and transferred to the neurology department because diffuse white matter lesions were found on his brain during MRI. 123I-IMP-SPECT showed extensive cerebral hypoperfusion mainly in the frontal lobes. Anti-Tg, anti-TPO, and anti-NAE antibodies were positive. These findings led to a diagnosis of Hashimoto's encephalopathy. The patient responded to steroid pulse therapy, high-dose steroid therapy, and intravenous immunoglobulin therapy, showing improvement in symptoms and imaging findings. Hashimoto's encephalopathy often presents with MRI findings similar to those of limbic encephalitis, when the patient presents with acute consciousness disturbance and psychiatric symptoms. However, this case showed diffuse white matter lesions, which may be clinically important for the differential diagnosis.

Effect of nanoarray density on enhanced electron transfer efficiency and analytical sensitivity for electrochemical immunosensors.

The fabrication of ordered nanoarray electrode (NAE) using UV imprinting and their application as electrochemical (EC) immunosensor is described in this study. Especially, the influence of the array density factors on the performance of NAE was characterized electrochemically and compared with flat-electrode. Low-density (hole: 200 nm, hole space = 600 nm), medium-density (hole: 200 nm, hole space = 400 nm), and high-density NAE (hole: 200 nm, hole space = 200 nm) which have the same active area were fabricated and their redox cycling was compared with empirical results. We observed that the high-density is the optimum NAE exhibiting the lowest charge transfer resistance and the highest redox cycling performance among all NAEs. Finally, to observe the effect of their EC performance as biosensor, an EC immunoassay was performed using Interleukine-6 (IL-6), and high-density NAE has lowest a low limit of detection (LOD) of 0.45 pg/mL compared with other NAEs (medium-density: 3.91 pg/mL, low-density: 5.87 pg/mL).

The NEDD8 activating enzyme inhibitor MLN4924 mitigates doxorubicin-induced cardiotoxicity in mice.

Doxorubicin (DOX) is a widely utilized chemotherapeutic agent in clinical oncology for treating various cancers. However, its clinical use is constrained by its significant side effects. Among these, the development of cardiomyopathy, characterized by cardiac remodeling and eventual heart failure, stands as a major concern following DOX chemotherapy. In our current investigation, we have showcased the efficacy of MLN4924 in mitigating doxorubicin-induced cardiotoxicity through direct inhibition of the NEDD8-activating enzyme, NAE. MLN4924 demonstrated the ability to stabilize mitochondrial function post-doxorubicin treatment, diminish cardiomyocyte apoptosis, alleviate oxidative stress-induced damage in the myocardium, enhance cardiac contractile function, mitigate cardiac fibrosis, and impede cardiac remodeling associated with heart failure. At the mechanistic level, MLN4924 intervened in the neddylation process by inhibiting the NEDD8 activating enzyme, NAE, within the murine cardiac tissue subsequent to doxorubicin treatment. This intervention resulted in the suppression of NEDD8 protein expression, reduction in neddylation activity, and consequential manifestation of cardioprotective effects. Collectively, our findings posit MLN4924 as a potential therapeutic avenue for mitigating doxorubicin-induced cardiotoxicity by attenuating heightened neddylation activity through NAE inhibition, thereby offering a viable and promising treatment modality for afflicted patients.

Campylobacter jejuni from no antibiotics ever (NAE) broilers: prevalence, antibiotic resistance, and virulence genes analysis.

Campylobacter jejuni (C. jejuni) is a leading foodborne illness causing bacteria, and poultry is a major reservoir of this pathogen. With the recent increase in broiler production under the "no antibiotics ever" (NAE) system, this study aimed to assess the prevalence, antibiotic resistance, and virulence characteristics of C. jejuni isolated from NAE raised broilers. A total of 270 cloacal swabs were collected from the live-hang areas of 3 commercial processing plants over 9 wk. Each processing plant was visited 3 times at a 1-wk interval, and 30 samples were collected per visit. Among the total 270 cloacal swab samples, C. jejuni was isolated from 44 (16.3%) samples . Of these isolates, 65.9% possessed toxin-producing genes cdtA, cdtB, and cdtC, and invasion gene ciaB. The prevalence of antibioitc resistance genes aph (3')-IIIa, erm(B) were 59.1%, and 50%, respectively. Nine (20.45%) C. jejuni isolates were identified as multidrug resistant (MDR), and 18 (40.9%) isolates showed resistance to at least 1 tested antibiotic. The highest resistance was observed against tetracycline (29.5%), followed by nalidixic acid (25%), whereas 22.7% of isolates were resistant to 2 clinically important antibiotics, azithromycin and ciprofloxacin. These results suggest that there ishigh prevalence level of multi-drug resistant C. jejuni with toxin producing virulence genes in the NAE-raised broilers sampled in this study, indicating the potential for serious human illnesses if transmitted through the food chain.

Circulating Endocannabinoids and N-Acylethanolamines in Individuals with Cannabis Use Disorder-Preliminary Findings.

BACKGROUND: Endocannabinoids and related N-acylethanolamines (NAEs) are bioactive lipids with important physiological functions and putative roles in mental health and addictions. Although chronic cannabis use is associated with endocannabinoid system changes, the status of circulating endocannabinoids and related NAEs in people with cannabis use disorder (CUD) is uncertain. METHODS: Eleven individuals with CUD and 54 healthy non-cannabis using control participants (HC) provided plasma for measurement by high-performance liquid chromatography-mass spectrometry of endocannabinoids (2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA)) and related NAE fatty acids (N-docosahexaenoylethanolamine (DHEA) and N-oleoylethanolamine (OEA)). Participants were genotyped for the functional gene variant of FAAH (rs324420, C385A) which may affect concentrations of AEA as well as other NAEs (OEA, DHEA). RESULTS: In overnight abstinent CUD, AEA, OEA and DHEA concentrations were significantly higher (31-40%; p < 0.05) and concentrations of the endocannabinoid 2-AG were marginally elevated (55%, p = 0.13) relative to HC. There were no significant correlations between endocannabinoids/NAE concentrations and cannabis analytes, self-reported cannabis use frequency or withdrawal symptoms. DHEA concentration was inversely related with marijuana craving (r = -0.86; p = 0.001). Genotype had no significant effect on plasma endocannabinoids/NAE concentrations. CONCLUSIONS: Our preliminary findings, requiring replication, might suggest that activity of the endocannabinoid system is elevated in chronic cannabis users. It is unclear whether this elevation is a compensatory response or a predating state. Studies examining endocannabinoids and NAEs during prolonged abstinence as well as the potential role of DHEA in craving are warranted.

Targeting NEDD8-activating enzyme for cancer therapy: developments, clinical trials, challenges and future research directions.

NEDDylation, a post-translational modification through three-step enzymatic cascades, plays crucial roles in the regulation of diverse biological processes. NEDD8-activating enzyme (NAE) as the only activation enzyme in the NEDDylation modification has become an attractive target to develop anticancer drugs. To date, numerous inhibitors or agonists targeting NAE have been developed. Among them, covalent NAE inhibitors such as MLN4924 and TAS4464 currently entered into clinical trials for cancer therapy, particularly for hematological tumors. This review explains the relationships between NEDDylation and cancers, structural characteristics of NAE and multistep mechanisms of NEDD8 activation by NAE. In addition, the potential approaches to discover NAE inhibitors and detailed pharmacological mechanisms of NAE inhibitors in the clinical stage are explored in depth. Importantly, we reasonably investigate the challenges of NAE inhibitors for cancer therapy and possible development directions of NAE-targeting drugs in the future.

Neddylation is required for perinatal cardiac development through stimulation of metabolic maturation.

Cardiac maturation is crucial for postnatal cardiac development and is increasingly known to be regulated by a series of transcription factors. However, post-translational mechanisms regulating this process remain unclear. Here we report the indispensable role of neddylation in cardiac maturation. Mosaic deletion of NAE1, an essential enzyme for neddylation, in neonatal hearts results in the rapid development of cardiomyopathy and heart failure. NAE1 deficiency disrupts transverse tubule formation, inhibits physiological hypertrophy, and represses fetal-to-adult isoform switching, thus culminating in cardiomyocyte immaturation. Mechanistically, we find that neddylation is needed for the perinatal metabolic transition from glycolytic to oxidative metabolism in cardiomyocytes. Further, we show that HIF1α is a putative neddylation target and that inhibition of neddylation accumulates HIF1α and impairs fatty acid utilization and bioenergetics in cardiomyocytes. Together, our data show neddylation is required for cardiomyocyte maturation through promoting oxidative metabolism in the developing heart.