Ellagic acid inhibits cell proliferation, migration, and invasion of anaplastic thyroid cancer cells via the Wnt/ß-catenin and PI3K/Akt pathways.

Anaplastic thyroid cancer (ATC) is a rare but lethal human malignant cancer with no known cure. Ellagic acid (EA), a natural plant extract, has shown antitumor activity against multiple cancers; however, its effects on the malignant phenotypes of ATC cells remain unknown. This study aimed to evaluate the effects of EA on proliferation, migration, and invasion of ATC cells and further explore the associated signaling mechanisms. The normal human thyroid cell line Nthy-ori3-1 and ATC cell line BHT-101 were used. Cytotoxicity assay was performed using the Cell Counting kit-8 (CCK-8) assay. Cell proliferation, migration, and invasion assays were performed using the CCK-8 and colony formation, wound healing, and Transwell invasion assays, respectively. Western blotting was used to detect the levels of related proteins. ß-catenin nuclear protein levels were measured to evaluate the Wnt/ß-catenin pathway. The phosphorylation level of the Akt protein was measured and calculated to evaluate the PI3K/Akt pathway. LiCl and IGF-1 were used as pathway agonists to determine the involvement of the corresponding pathway. The results showed that EA inhibited the proliferation, migration, and invasion of ATC cells. Furthermore, both the Wnt/ß-catenin and PI3K/Akt pathways were suppressed by EA treatment, and activation of these two pathways reversed the EA-induced inhibition of the pathological phenotypes of ATC cells. These findings demonstrate that EA inhibits proliferation, migration, and invasion of ATC cells by suppressing the Wnt/ß-catenin and PI3K/Akt pathways, suggesting that EA is a potential drug candidate for treating ATC and provides a theoretical basis for further in vivo experiments and clinical applications.

Case report: Excessive daytime sleepiness as a presenting manifestation of autoimmune glial fibrillary acidic protein astrocytopathy.

Autoimmune glial fibrillary acidic protein astrocytopathy (GFAP-A) is a recently discovered autoimmune inflammatory disease of the central nervous system. It presents with a variety of clinical symptoms, including fever, seizures, psychiatric symptoms, limber weakness, and sensory symptoms. However, the symptoms of sleep disorders have not been sufficiently addressed. Here, we report a case of GFAP-A in which the patient complained of excessive daytime sleepiness and an excessive need for sleep. Our patient was a 58-year-old male who experienced excessive daytime sleepiness for 50 days following SARS-CoV-2 infection. He was diagnosed with coronavirus disease 2019 on June 1st. On the 7th of June, he experienced excessive daytime sleepiness, nausea, reduced food intake, lower limb weakness, and dysuria. Subsequently, his sleepiness significantly deteriorated on July 21st. Five months prior, the patient underwent laparoscopic partial right nephrectomy for clear-cell renal cell carcinoma. Brain MRI revealed abnormal hyperintense lesions in the pontine brain and around the mesencephalic aqueduct on T2 and T2-fluid attenuated inversion recovery (T2-FLAIR) sequences However, these lesions did not exhibit any pathological enhancement. Spinal cord MRI revealed lesions in the C6-C7 and T2-T3 segments on the T2 sequence. His Epworth Sleepiness Scale (ESS) score was 16 (reference range, <10), and 24-hour polysomnography supported the diagnosis of rapid-eye-movement sleep disorder and severe sleep apnea-hypopnea syndrome. Glial fibrillary acidic protein IgG antibodies were detected in the cerebrospinal fluid (1:32, cell-based assay) but not in the serum. The level of hypocretin in the cerebrospinal fluid was 29.92 pg/mL (reference range ≥110 pg/mL), suggesting narcolepsy type 1. After treatment with corticosteroids for approximately 1 month, the patient showed considerable clinical and radiological improvement, as well as an increase in hypocretin levels. Although repeated polysomnography and multiple sleep latency tests suggested narcolepsy, his ESS score decreased to 8. Our findings broaden the range of clinical manifestations associated with GFAP-A, thereby enhancing diagnostic and therapeutic strategies for this disease. Additionally, our results indicate a potential common autoimmune mechanism involving GFAP-A and orexin system dysregulation, warranting further investigation.

A Diagnostic Nitrosamine Detection Approach for Pharmaceuticals by Using Tandem Mass Spectrometry Based on Diagnostic Gas-Phase Ion-Molecule Reactions.

N-Nitrosamines are strictly regulated in pharmaceutical products due to their carcinogenic nature. Therefore, the ability to rapidly and reliably identify the N-nitroso functionality is urgently needed. Unfortunately, not all ionized N-nitroso compounds produce diagnostic fragment ions and hence tandem mass spectrometry based on collision-activated dissociation (CAD) cannot be used to consistently identify the N-nitroso functionality. Therefore, a more reliable method was developed based on diagnostic functional-group selective ion-molecule reactions in a linear quadrupole ion trap mass spectrometer. 2-Methoxypropene (MOP) was identified as a reagent that reacts with protonated N-nitrosamines in a diagnostic manner by forming an adduct followed by the elimination of 2-propenol (CH3C(OH)═CH2]). From 18 protonated N-nitrosamine model compounds studied, 15 formed the diagnostic product ion. The lack of the diagnostic reaction for three of the N-nitrosamine model compounds was rationalized based on the presence of a pyridine ring that gets preferentially protonated instead of the N-nitroso functionality. These N-nitrosamines can be identified by subjecting a stable adduct formed upon ion-molecule reactions with MOP to CAD. Further, the ability to use ion-molecule reactions followed by CAD to differentiate protonated O-nitroso compounds with a pyridine ring from analogous N-nitrosamines was demonstrated This methodology is considered to be robust for the identification of the N-nitroso functionality in unknown analytes. Lastly, HPLC/MS2 experiments were performed to determine the detection limit for five FDA regulated N-nitrosamines.

Immune-Related RNA-Binding Protein-Based Signature With Predictive and Prognostic Implications in Patients With Lung Adenocarcinoma.

Background: Dysregulation of RNA-binding proteins (RBPs) in cancers is associated with immune and cancer development. Here, we aimed to profile immune-related RBPs in lung adenocarcinoma (LUAD) and construct an immune-related RBP signature (IRBPS) to predict the survival and response to immunotherapy. Methods: A correlation analysis was performed to establish a co-expression network of RBPs and immune-related genes (IRGs) to characterize immune-related RBPs in the TCGA-LUAD cohort (n = 497 cases). Then, a combination of the Random survival forest (RSF) and Cox regression analysis was performed to screen the RBPs and establish IRBPS. This was followed by independent validation of IRBPS in GSE72094 (n = 398 cases), GSE31210, (n = 226 cases), and GSE26939 (n = 114 cases). Differences between the low- and high-risk groups were compared in terms of gene mutations, tumor mutation burden, tumor-infiltrating lymphocytes, and biomarkers responsive to immunotherapy. Results: DDX56, CTSL, ZC3H12D, and PSMC5 were selected and used to construct IRBPS. The high-risk scores of patients had a significantly worse prognosis in both training and testing cohorts (p < 0.0001 and p < 0.05, respectively), and they tended to be older and have an advanced TNM stage. Furthermore, IRBPS was a prognostic factor independent of age, gender, smoking history, TNM stage, and EGFR mutation status (p = 0.002). In addition, high-risk scores of IRBPS were significantly correlated with tumor-infiltrating lymphocytes (p < 0.05). They also had a high level of PD-L1 protein expression (p < 0.01), number of neoantigens (p < 0.001), and TMB (p < 0.001), implying the possible prediction of IRBPS in the immunotherapy of LUAD. Conclusion: The currently established IRBPS encompassing immune-related RBPs might serve as a promising tool to predict survival, reflect the immune microenvironment, and predict the efficacy of immunotherapy among LUAD patients.

ATG12 is involved in the antiviral immune response in large yellow croaker (Larimichthys crocea).

ATG12, a core autophagy protein, forms a conjugate with ATG5 to promote the formation of autophagosome membrane, and plays an important role in antiviral immunity. However, little is known about the function of ATG12 in fish. Here, we cloned the open reading frame (ORF) of large yellow croaker (Larimichthys crocea) ATG12 (LcATG12), which is 354 nucleotides long and encodes a protein of 117 amino acids. The deduced LcATG12 possesses a conserved APG12 domain (residues 31 to 117), and shares 91.45% identities with ATG12 in orange-spotted grouper (Epinephelus coioides). LcATG12 was constitutively expressed in all examined tissues, with the highest level in intestine. Its transcript was also detected in primary head kidney granulocytes (PKG), primary head kidney macrophages (PKM), primary head kidney lymphocytes (PKL), and large yellow croaker head kidney (LYCK) cell line, and was significantly up-regulated by poly(I:C). LcATG12 was regularly distributed in both cytoplasm and nucleus of LYCK and epithelioma papulosum cyprinid (EPC) cells. Overexpression of LcATG12 in EPC cells significantly inhibited the replication of spring viremia of carp virus (SVCV). Further studies reveled that LcATG12 could induce the occurrence of autophagy in LYCK cells. Furthermore, overexpression of LcATG12 in LYCK cells increased the expression levels of large yellow croaker type I interferons (IFNs, IFNc, IFNd, and IFNh), IFN regulatory factors (IRF3 and IRF7), and IFN-stimulated genes (PKR, Mx, and Viperin). All these data indicated that LcATG12 plays a role in the antiviral immunity possibly by inducing both autophagy and type I IFN response in large yellow croaker.

Molecular characterization and role in virus infection of Beclin-1 in large yellow croaker (Larimichthys crocea).

Beclin-1, the ortholog of yeast autophagy-related gene 6 (Atg6), has a central role in autophagy, which has been linked to diverse biological processes including immunity, development, tumor suppression, and lifespan extension. However, understanding of function of fish Beclin-1 is limited now. In this study, the complete Beclin-1 cDNA of large yellow croaker Larimichthys crocea (LcBeclin-1) was cloned, whose open reading frame (ORF) is 1344 bp long and encodes a protein of 447 amino acids (aa). The deduced LcBeclin-1 possesses a typical Bcl-2 homology domain 3(BH3) and an APG6 domain that contains a central coiled-coil domain (CCD, residues 174 to 231) and a C-terminal evolutionarily conserved domain (ECD, residues 241 to 334). LcBeclin-1 shared a high amino acid identity of 81.66-98.66% with reported Beclin-1 molecules from other vertebrate species. LcBeclin-1 gene was constitutively expressed in all tissues tested, with the highest levels in heart. LcBeclin-1 transcripts were also detected in primary head kidney granulocytes (PKGs), primary head kidney macrophages (PKMs), primary head kidney leukocytes (PKLs), and large yellow croaker head kidney cell line (LYCK), and were significantly upregulated by poly (I:C) in PKMs and LYCK cells. Subcellular localization showed that LcBeclin-1 was evenly distributed in the cytoplasm and nucleus of LYCK cells. Overexpression of LcBeclin-1 significantly increased the replication of SVCV, as evidenced by increased severity of the cytopathic effects, enhanced viral titre, and upregulated transcriptional levels of viral genes. Further studies showed that LcBeclin-1 induced the occurrence of autophagy in LYCK cells. Additionally, LcBeclin-1 also decreased the expression levels of large yellow croaker interferons (IFNs; IFNc, IFNd, and IFNh), interferon regulatory factor 3 (IRF3) and IRF7, IFN-stimulated genes (ISGs; Mx, PKR, and Viperin) in LYCK cells. All these data suggest that LcBeclin-1 promoted the viral replication possibly by inducing autophagy or negatively modulating IFN response, which will help us to further understand the function of fish Beclin-1.

Molecular and functional identification of a ß-defensin homolog in large yellow croaker (Larimichthys crocea).

ß-defensin (BD) is a cysteine-rich cationic antibacterial peptide that is active against a wide range of bacteria. Here, a ß-defensin homolog (LcBD2) was identified in large yellow croaker (Larimichthys crocea). The open reading frame of LcBD2 contains 195 nucleotides, encoding a protein of 64 amino acids that possesses a typical arrangement of six conserved cysteine residues (C31 , C37 , C41 , C53 , C59 and C60 ). LcBD2 transcripts were constitutively expressed in all examined tissues and significantly increased in head kidney, spleen and gills by Vibrio alginolyticus. The synthetic LcBD2 peptide imparted antimicrobial effects on both Gram-negative bacteria (V. campbellii, V. parahaemolyticus, V. alginolyticus, V. harveyi and Pseudomonas plecoglossicida) and Gram-positive bacteria (Bacillus subtilis). We also observed that after treatment with synthetic LcBD2 peptide, numerous blisters appeared on the membrane of P. plecoglossicida, which in turn may result in cell membrane breakage and bacterial death. Moreover, the synthetic LcBD2 peptide significantly upregulated the expression levels of TNF-α2, IL-1ß and CXCL8_L1 in monocytes/macrophages, while downregulated expression level of IL-10. The LcBD2 peptide also remarkedly enhanced the phagocytosis of monocytes/macrophages. These results indicate that LcBD2 not only protects large yellow croaker against multiple bacterial pathogens but also plays a role in activation of monocytes/macrophages.

The role of 99mTc-MIBI SPECT/CT in patients with secondary hyperparathyroidism: comparison with 99mTc-MIBI planar scintigraphy and ultrasonography.

BACKGROUND: This study aimed to compare the sensitivity of 99mTc-MIBI SPECT/CT, 99mTc-MIBI planar scintigraphy and ultrasonography (US) in patients with secondary hyperparathyroidism (SHPT), and to explore the factors that affect the sensitivity of 99mTc-MIBI SPECT/CT. METHODS: In this retrospective study, forty-six patients with SHPT who underwent 99mTc-MIBI planar scintigraphy, 99mTc-MIBI SPECT/CT and US were enrolled. They underwent surgery within 1 month. We compared the sensitivity of the different imaging methods based on the lesions according to the pathological results. The parathyroid lesions on 99mTc-MIBI SPECT/CT images were divided into missed diagnosis group (MDG) and non-missed diagnosis group (NMDG). We compared the lesion to background ratio (LBR), maximum diameter, volume, the mean CT Hounsfield unit values (CTmean) and location of lesions between MDG and NMDG. RESULTS: The sensitivity of 99mTc-MIBI SPECT/CT, 99mTc-MIBI planar scintigraphy and US were 70.30% versus 48.48% versus 61.82%, respectively. The sensitivity of 99mTc-MIBI SPECT/CT combined US was 79.39%, which was higher than 99mTc-MIBI SPECT/CT with significant difference (P = 0.000). On 99mTc-MIBI SPECT/CT images, the LBR, maximum diameter and volume of lesions in MDG was smaller than those in NMDG with significant difference (P < 0.001). The average LBR, maximum diameter and volume of lesions in MDG and NMDG were 3.42 ± 1.28, 9.32 ± 2.69 mm, 208.51 ± 163.22 mm3 versus 6.75 ± 5.08, 15.03 ± 4.94 mm and 863.85 ± 1216.0 mm3, respectively. CONCLUSIONS: 99mTc-MIBI SPECT/CT exhibited the highest sensitivity among the three methods. When 99mTc-MIBI SPECT/CT combined with US, the sensitivity can be further improved. Lesions with lower MIBI uptake and smaller lesions on 99mTc-MIBI SPECT/CT images were easily missed.

Potential GnRH and steroidogenesis pathways in the scallop Patinopecten yessoensis.

Gonadotropin-releasing hormone (GnRH) controls synthesis of sex steroid hormones through hypothalamic-pituitary-gonadal (HPG) axis in vertebrates. But in mollusks, research on GnRH and steroidogenesis pathways is still limited. In this study, we first identified two gonadotropin receptor like genes (LGR and LGR5L) and four steroidogenesis-related genes (CYP17A, HSD17B12, HSD3B1 and HSD3B2) in the scallop Patinopecten yessoensis. By examining the expression of 11 genes in the ganglia and/or gonad as well as the concentration of progesterone, testosterone and estradiol in the gonad, we postulate that a potential GnRH signaling pathway (GnRH-GnRHR-GPB5-LGR/LGR5L) in the cerebral and pedal ganglia (CPG) and steroidogenesis pathway (CYP17A, HSD17B12 and HSD3B1) in the gonad are involved in regulating sex steroid hormones. E2/T index that indicates aromatase activity is higher in the ovary than testis and is positively correlated with the expression of FOXL2 in the gonad, implying the presence of aromatase in the scallop. In addition, we confirmed that expression of most of the downstream genes in the two pathways was significantly elevated after injection of mature py-GnRH peptide. This study would contribute to a new understanding of the molecular basis underlying reproduction regulation by GnRH in mollusks.

Expression of Cathepsin F in response to bacterial challenges in Yesso scallop Patinopecten yessoensis.

Cathepsin F is a unique papain cysteine proteinase with highly conserved structures: catalytic triad and a cystatin domain contained in the elongated N-terminal pro-region. It has been reported that cathepsin F is associated with the establishment of innate immune in several vertebrate including fish in aquaculture, but not known in bivalves. In this study, we firstly identified and characterized cathepsin F in the Yesso scallop (Patinopecten yessoensis). The protein structural and phylogenetic analyses were then conducted to determine its identity and evolutionary position. We've also investigated the expression levels of cathepsin F gene at different embryonic developmental stages, in healthy adult tissues and especially in the hemocytes and hepatopancreas after Gram-positive (Micrococcus luteus) and negative (Vibrio anguillarum) challenges using quantitative real-time PCR (qPCR). Cathepsin F was significantly up-regulated 3 h after infection of V. anguillarum in hemocytes, suggesting its participation in immune response. Our findings have provided strong evidence that cathepsin F may be a good target for enhancing the immune activity in Yesso scallop.