Functional prediction of the potential NGLY1 mutations associated with rare disease CDG.

Genetic diseases are currently diagnosed by functional mutations. However, only some mutations are associated with disease. It is necessary to establish a quick prediction model for clinical screening. Pathogenic mutations in NGLY1 cause a rare autosomal recessive disease known as congenital disorder of deglycosylation (NGLY1-CDDG). Although NGLY1-CDDG can be diagnosed through gene sequencing, clinical relevance of a detected mutation in NGLY1 needs to be further confirmed. In this study, taken NGLY1-CDDG as an example, a comprehensive and practical predictive model for pathogenic mutations on NGLY1 through an NGLY1/Glycopeptide complex model was constructed, the binding sites of NGLY1 and glycopeptides were simulated, and an in vitro enzymatic assay system was established to facilitate quick clinical decisions for NGLY1-CDDG patients. The docking model covers 42 % of reported NGLY1-CDDG missense mutations (5/12). All reported mutations were subjected to in vitro enzymatic assay in which 18 mutations were dysfunctional (18/30). In addition, a full spectrum of functional R328 mutations was assayed and 11 mutations were dysfunctional (11/19). In this study, a model of NGLY1 and glycopeptides was built for potential functional mutations in NGLY1. In addition, the effect of potential regulatory compounds, including N-acetyl-l-cysteine and dithiothreitol, on NGLY1 was examined. The established in vitro assay may serve as a standard protocol to facilitate rapid diagnosis of all mutations in NGLY1-CDDG. This method could also be applied as a comprehensive and practical predictive model for the other rare genetic diseases.

Silibinin, a potential fasting mimetic, inhibits hepatocellular carcinoma by triggering extrinsic apoptosis.

Fasting, without inducing malnutrition, has been shown to have various beneficial effects, including the inhibition of tumor initiation and progression. However, prolonged fasting poses challenges for many cancer patients, particularly those in intermediate and terminal stages. Thus, there is an urgent need for the development of fasting mimetics which harness the protective effects of fasting but more suitable for patients. In this study, we first highlighted the pivotal role of silibinin in AMP-activated protein kinase (AMPK) pathway and may serve, as a potential fasting mimetic via screening hepatoprotective drugs. Further metabolic analysis showed that silibinin inhibited the adenosine triphosphate (ATP) levels, glucose uptake and diminished glycolysis process, which further confirmed that silibinin served as a fasting mimetic. In addition, fasting synergized with silibinin, or used independently, to suppress the growth of hepatocellular carcinoma (HCC) in vivo. Mechanistically, silibinin upregulated death receptor 5 (DR5) through AMPK activation, and thus promoting extrinsic apoptosis and inhibiting HCC growth both in vitro and in vivo. Inhibition of AMPK using small interfering RNA (siRNA) or compound C, an AMPK inhibitor, significantly attenuated the upregulation of DR5 and the apoptotic response induced by silibinin. These findings suggest that silibinin holds promise as a fasting mimetic and may serve as an adjuvant drug for HCC treatment.

A Natural Compound Containing a Disaccharide Structure of Glucose and Rhamnose Identified as Potential N-Glycanase 1 (NGLY1) Inhibitors.

N-glycanase 1 (NGLY1) is an essential enzyme involved in the deglycosylation of misfolded glycoproteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, which could hydrolyze N-glycan from N-glycoprotein or N-glycopeptide in the cytosol. Recent studies indicated that NGLY1 inhibition is a potential novel drug target for antiviral therapy. In this study, structure-based virtual analysis was applied to screen candidate NGLY1 inhibitors from 2960 natural compounds. Three natural compounds, Poliumoside, Soyasaponin Bb, and Saikosaponin B2 showed significantly inhibitory activity of NGLY1, isolated from traditional heat-clearing and detoxifying Chinese herbs. Furthermore, the core structural motif of the three NGLY1 inhibitors was a disaccharide structure with glucose and rhamnose, which might exert its action by binding to important active sites of NGLY1, such as Lys238 and Trp244. In traditional Chinese medicine, many compounds containing this disaccharide structure probably targeted NGLY1. This study unveiled the leading compound of NGLY1 inhibitors with its core structure, which could guide future drug development.

Characterization and Molecular Mechanism of Aminoglycoside-6-Adenyl Transferase Associated with Aminoglycoside Resistance from Elizabethkingia meningoseptica.

Purpose: Elizabethkingia meningoseptica (EM) is a multi-drug-resistant bacterium of global concern for its role in nosocomial infection and is generally resistant to aminoglycoside antibiotics. In the whole genome of an EM strain (FMS-007), an aminoglycoside-6-adenyl transferase gene (ant(6)FMS-007) was predicted. This study aimed to characterize the biochemical function of ANT(6)FMS-007 and analyze the relationship between genotype and phenotype of ant(6) in clinical EM isolates, so as to provide evidence for clinical precision drug use. This study could establish a method for the verification of known or unknown functionally resistant genes. Methods: A total of 42 EM clinical isolates were collected from clinical departments during 2015-2023. The phenotype of aminoglycoside antibiotics was analyzed by broth microdilution (BMD) and Kirby-Bauer (K-B) methods. The whole-length ant(6) from EM clinical isolates was analyzed by polymerase chain reaction (PCR) and sequencing. The biochemical function of predictive ANT(6)FMS-007 from the FMS-007 whole genome was identified by 3D plate experiment and mass spectrometry analysis. Candidate active sites were predicted by multi-species sequence alignment and molecular docking, and other important sites were identified in the comparison of ant(6) genotypes and phenotypes of EM clinical isolates. Drug susceptibility test was used to verify the function of these sites. Results: The predictive ANT(6)FMS-007 protein could inactivate STR by modifying STR with ATP to form STR-AMP. Four active sites (Asp-38, Asp-42, Lys-95, and Lys-213) of ANT(6)FMS-007 were identified. Thirty-one EM clinical isolates (74%) carried the ant(6) gene. Eight EM clinical isolates containing the ant(6) gene had MIC values (<=32µg/mL) lower by at least 16-fold than FMS-007 (512µg/mL) for STR, and N59H and K204Q were the common mutations in the ant(6) gene. Conclusion: This assay verified the biochemical function of the predictive gene ant(6)FMS-007 and could provide an alternative method to study resistant gene function in multi-drug-resistant bacteria. The inconsistency between genotype and phenotype of resistant genes indicated that the combination of resistance gene detection and functional analysis could better provide precision medicine for clinical use.

Evodiamine inhibits ESCC by inducing M-phase cell-cycle arrest via CUL4A/p53/p21 axis and activating noxa-dependent intrinsic and DR4-dependent extrinsic apoptosis.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a malignancy with high incidence in several regions of China, and the prognosis of patients with ESCC is unfavorable. Evodiamine (Evo), a small molecule derived from the traditional Chinese herb Evodia rutaecarpa, has shown anti-cancer efficacy in numerous human malignancies but not in ESCC. PURPOSE: To determine whether Evo induces cell-cycle arrest and apoptosis in ESCC in vitro and in vivo and elucidate the underlying mechanisms. METHODS: ATPlite and colony formation assay were used to validate the inhibiting effect of Evo on three ESCC cells in vitro; Two subcutaneous tumor models of ESCC cells were used to evaluate the anti-ESCC effect of Evo and assess the biosafety of Evo in vivo; RNAseq and Database of KEGG pathway analysis provided a direction for the mechanistic study of Evo; FACS was used to detect Evo-induced cell cycle arrest and cell apoptosis in ESCC cells; Western blot and QPCR were respectively used to detect the level of related genes and proteins in Evo-treated ESCC cells; SiRNA and other experimental techniques were used to identify the molecular mechanism of Evo-induced ESCC cell cycle arrest and cell apoptosis. RESULTS: Evo significantly suppressed the growth of ESCC both in vitro and in vivo. Mechanistically, Evo induced M-phase cell-cycle arrest by inactivation of CUL4A E3 ligase, which mediates degradation blockage of p53 and transcriptional activation of p21. With the prolonged treatment time, Evo triggered both Noxa-dependent intrinsic and DR4-dependent extrinsic cell apoptosis in two ESCC cell lines. CONCLUSION: Our findings revealed the anti-tumor efficacy and mechanisms of Evo, providing a solid scientific basis for Evo as an attractive choice for ESCC treatment.

Corrigendum: Neurological adverse events associated with esketamine: A disproportionality analysis for signal detection leveraging the FDA adverse event reporting system.

[This corrects the article DOI: 10.3389/fphar.2022.849758.].

Daurisoline Inhibits ESCC by Inducing G1 Cell Cycle Arrest and Activating ER Stress to Trigger Noxa-Dependent Intrinsic and CHOP-DR5-Dependent Extrinsic Apoptosis via p-eIF2α-ATF4 Axis.

Esophageal squamous cell carcinoma (ESCC), one of the most malignant human cancers in clinic, requires novel treatment. Daurisoline (DAS) is a component of traditional Chinese herb, which exhibits anti-cancer effects by autophagy inhibition and metastasis suppression. However, the effect and mechanism of DAS on ESCC remain unclear. Here, we found that DAS inhibited cell proliferation and colony formation in both human ESCC cell lines EC1 and ECA109. Mechanistically, DAS induced p21-/p27-dependent G1 phase cell cycle arrest and apoptosis in a dose-dependent manner. The induction of apoptosis by DAS was largely dependent on the activation of the transcription factor ATF4 and its downstream NOXA-dependent intrinsic and CHOP-DR5-dependent extrinsic apoptotic pathway. ATF4 activation induced by DAS was due to the generation of excessive reactive oxygen species (ROS) and the subsequent activation of endoplasmic reticulum (ER) stress through the p-eIF2α-ATF4 signal pathway, which can be largely abrogated by N-acetylcysteine (NAC), a scavenger of ROS. Moreover, DAS treatment significantly inhibited tumor growth and reduced tumor weight in the tumor xenograft mouse model by up-regulating key proteins related to cell cycle arrest and apoptotic pathway. Taken together, these findings identified DAS as a novel candidate for the treatment of ESCC.

Celastrol Inhibited Human Esophageal Cancer by Activating DR5-Dependent Extrinsic and Noxa/Bim-Dependent Intrinsic Apoptosis.

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest digestive system cancers worldwide lacking effective therapeutic strategies. Recently, it has been found that the natural product celastrol plays an anti-cancer role in several human cancers by inducing cell cycle arrest and apoptosis. However, it remains elusive whether and how celastrol suppresses tumor growth of ESCC. In the present study, for the first time, we demonstrated that celastrol triggered both extrinsic and intrinsic apoptosis pathways to diminish the tumor growth of ESCC in vivo and in vitro. Mechanistic studies revealed that celastrol coordinatively induced DR5-dependent extrinsic apoptosis and Noxa-dependent intrinsic apoptosis through transcriptional activation of ATF4 in ESCC cells. Furthermore, we found that the FoxO3a-Bim pathway was involved in the intrinsic apoptosis of ESCC cells induced by celastrol. Our study elucidated the tumor-suppressive efficacy of celastrol on ESCC and revealed a previously unknown mechanism underlying celastrol-induced apoptosis, highlighting celastrol as a promising apoptosis-inducing therapeutic strategy for ESCC.

Andrographolide Inhibits ER-Positive Breast Cancer Growth and Enhances Fulvestrant Efficacy via ROS-FOXM1-ER-α Axis.

Estrogen receptor (ER)-positive breast cancer is the main subtype of breast cancer (BRCA) with high incidence and mortality. Andrographolide (AD), a major active component derived from the traditional Chinese medicine Andrographis paniculate, has substantial anti-cancer effect in various tumors. However, the antitumor efficacy and the underlying molecular mechanisms of AD on ER-positive breast cancer are poorly understood. In the present study, we demonstrated that andrographolide (AD) significantly inhibited the growth of ER-positive breast cancer cells. Mechanistically, AD suppressed estrogen receptor 1 (ESR1, encodes ER-α) transcription to inhibit tumor growth. Further studies revealed that AD induced ROS production to down-regulate FOXM1-ER-α axis. Conversely, inhibiting ROS production with N-acetylcysteine (NAC) elevated AD-decreased ER-α expression, which could be alleviated by FOXM1 knockdown. In addition, AD in combination with fulvestrant (FUL) synergistically down-regulated ER-α expression to inhibit ER-positive breast cancer both in vitro and in vivo. These findings collectively indicate that AD suppresses ESR1 transcription through ROS-FOXM1 axis to inhibit ER-positive breast cancer growth and suggest that AD might be a potential therapeutic agent and fulvestrant sensitizer for ER-positive breast cancer treatment.

Single Cell Raman Spectroscopy Deuterium Isotope Probing for Rapid Antimicrobial Susceptibility Test of Elizabethkingia spp.

Nosocomial infection by multi-drug resistance Elizabethkingia spp. is an emerging concern with severe clinical consequences, particularly in immunocompromised individuals and infants. Efficient control of this infection requires quick and reliable methods to determine the appropriate drugs for treatment. In this study, a total of 31 Elizabethkingia spp., including two standard strains (ATCC 13253 and FMS-007) and 29 clinical isolates obtained from hospitals in China were subjected to single cell Raman spectroscopy analysis coupled with deuterium probing (single cell Raman-DIP). The results demonstrated that single cell Raman-DIP could determine antimicrobial susceptibility of Elizabethkingia spp. in 4 h, only one third of the time required by standard broth microdilution method. The method could be integrated into current clinical protocol for sepsis and halve the report time. The study also confirmed that minocycline and levofloxacin are the first-line antimicrobials for Elizabethkingia spp. infection.

Neurological Adverse Events Associated With Esketamine: A Disproportionality Analysis for Signal Detection Leveraging the FDA Adverse Event Reporting System.

Esketamine was approved for the treatment of treatment-resistant depression in 2019. After the approval of esketamine, numerous concerns have been raised regarding its long-term safety and tolerability. A previous systematic pharmacovigilance study on esketamine-related adverse events (AEs) was published in 2020; however, it has not been updated 2 years later. The primary aim of this study was to detect and characterize neurological safety signals of esketamine to partially update the knowledge in this field using the FDA pharmacovigilance database. Reporting odds ratio (ROR) was calculated for esketamine-related neurological AEs from 2019 to 2021 with a signal considered when the lower limit of the 95% confidence interval (CI) of ROR (ROR025) exceeded one. Severe and non-severe cases were compared using an independent samples t-test or chi-squared (χ2) test, and a rating scale was used to prioritize the signals. The database contained 720 cases of esketamine-associated neurological AEs, with 21 signals detected, ranging from a ROR025 of 1.05 (disturbance in attention) to 204.00 (sedation). 16 latest neurological AEs emerged in the second year of marketing approval of esketamine, with eight signals detected. The associations between esketamine and nervous system disorders persisted when stratifying by sex, age, and reporter type, whereas the spectrum of neurological AEs differed in stratification regimens. Esketamine dosage, antidepressant polypharmacy, or co-prescription with benzodiazepines affected AEs severity (t = 2.41, p = 0.017; χ2 = 6.75, p = 0.009; and χ2 = 4.10, p = 0.043; respectively), while age and sex did not (p = 0.053 and p = 0.397, respectively). Three signals were categorized as moderate clinical priority [i.e., sedation, dizziness, and dysgeusia (priority points 7, 5, and 5, respectively)], showing the same early failure type profiles. Notably, seven detected disproportionality signals were not previously detected in clinical trials. Although the majority of results were in line with those obtained in the previous study, there were discrepancies in the spectrum of neurological AEs and the effects of several risk factors on AEs severity among the two studies that should be recognized and managed early in clinical treatments.

Jujuboside B Inhibits the Proliferation of Breast Cancer Cell Lines by Inducing Apoptosis and Autophagy.

Jujuboside B (JB) is one of the main biologically active ingredients extracted from Zizyphi Spinosi Semen (ZSS), a widely used traditional Chinese medicine for treating insomnia and anxiety. Breast cancer is the most common cancer and the second leading cause of cancer-related death in women worldwide. The purpose of this study was to examine whether JB could prevent breast cancer and its underlying mechanism. First, we reported that JB induced apoptosis and autophagy in MDA-MB-231 and MCF-7 human breast cancer cell lines. Further mechanistic studies have revealed that JB-induced apoptosis was mediated by NOXA in both two cell lines. Moreover, the AMPK signaling pathway plays an important role in JB-induced autophagy in MCF-7. To confirm the anti-breast cancer effect of JB, the interaction of JB-induced apoptosis and autophagy was investigated by both pharmacological and genetic approaches. Results indicated that autophagy played a pro-survival role in attenuating apoptosis. Further in vivo study showed that JB significantly suppressed the growth of MDA-MB-231 and MCF-7 xenografts. In conclusion, our findings indicate that JB exerts its anti-breast cancer effect in association with the induction of apoptosis and autophagy.

Using engineering models to shorten cryoprotectant loading time for the vitrification of articular cartilage.

Osteochondral allograft transplantation can treat full thickness cartilage and bone lesions in the knee and other joints, but the lack of widespread articular cartilage banking limits the quantity of cartilage available for size and contour matching. To address the limited availability of cartilage, vitrification can be used to store harvested joint tissues indefinitely. Our group's reported vitrification protocol [Biomaterials 33 (2012) 6061-6068] takes 9.5 h to load cryoprotectants into intact articular cartilage on bone and achieves high cell viability, but further optimization is needed to shorten this protocol for clinical use. Herein, we use engineering models to calculate the spatial and temporal distributions of cryoprotectant concentration, solution vitrifiability, and freezing point for each step of the 9.5-h protocol. We then incorporate the following major design choices for developing a new shorter protocol: (i) all cryoprotectant loading solution concentrations are reduced, (ii) glycerol is removed as a cryoprotectant, and (iii) an equilibration step is introduced to flatten the final cryoprotectant concentration profiles. We also use a new criterion-the spatially and temporally resolved prediction of solution vitrifiability-to assess whether a protocol will be successful instead of requiring that each cryoprotectant individually reaches a certain concentration. A total cryoprotectant loading time of 7 h is targeted, and our new 7-h protocol is predicted to achieve a level of vitrifiability comparable to the proven 9.5-h protocol throughout the cartilage thickness.

[ADS-J1 antagonizes semen-derived enhancer of virus infection-mediated enhancement of transmitted founder HIV-1 and its matched chronic control strain infection].

OBJECTIVE: To investigate the effect of semen-derived enhancer of virus infection (SEVI) on the infection of transmitted/founder (TF) HIV-1 and its matched chronic control (CC) viruses and the antagonism of ADS-J1 on SEVI-mediated enhancement of TF and CC virus infection in vitro. METHODS: PAP248-286 self-assembling into SEVI amyloid fibrils was validated by ThT assay. We generated the virus stocks of TF and CC virus pair. TZM-bl cells were infected with the mixture of SEVI and TF or CC viruses for 72 h. Luciferase activity was used to observe the enhancement of SEVI. SEVI was treated with different concentrations of ADS-J1 and incubated with TF or CC viruses. TZM-bl cells were then infected with the mixture and luciferase activity was detected 72 h after infection to analyze the antagonism of ADS-J1 on the enhancing effect of SEVI. ADS-J1 was also incubated with TF and CC viruses directly and TZM-bl cells were infected for 72 h to evaluate the antiviral effect using luciferase assay. SEVI was treated with ADS-J1 and Zeta potential was determined to explore the antagonistic mechanism of ADS-J1. RESULTS: ThT assay showed that PAP248-286 was capable of self-assembly into SEVI amyloid fibrils. SEVI significantly accelerated TF and CC viruses infection (P<0.05), and ADS-J1 not only significantly antagonized the enhancement of SEVI (P<0.05) but also directly inhibited the infection of TF and CC viruses (P<0.05). ADS-J1 neutralized the positive charge of SEVI in a dose-dependent manner. CONCLUSIONS: SEVI promotes the infection of TF and CC strains, and ADS-J1 antagonizes SEVI-mediated enhancement of TF and CC viruses by neutralizing the positive charge of SEVI.