Glycosylation of Sodium/Iodide Symporter (NIS) Regulates Its Membrane Translocation and Radioiodine Uptake

Purpose

Human sodium/iodide symporter (hNIS) protein is a membrane glycoprotein that transports iodide ions into thyroid cells. The function of this membrane protein is closely regulated by post-translational glycosylation. In this study, we measured glycosylation-mediated changes in subcellular location of hNIS and its function of iodine uptake.

Methods

HeLa cells were stably transfected with hNIS/tdTomato fusion gene in order to monitor the expression of hNIS. Cellular localization of hNIS was visualized by confocal microscopy of the red fluorescence of tdTomato. The expression of hNIS was evaluated by RT-PCR and immunoblot analysis. Functional activity of hNIS was estimated by radioiodine uptake. Cyclic AMP (cAMP) and tunicamycin were used to stimulate and inhibit glycosylation, respectively. In vivo images were obtained using a Maestro fluorescence imaging system.

Results

cAMP-mediated Glycosylation of NIS resulted in increased expression of hNIS, stimulating membrane translocation, and enhanced radioiodine uptake. In contrast, inhibition of glycosylation by treatment with tunicamycin dramatically reduced membrane translocation of intracellular hNIS, resulting in reduced radioiodine uptake. In addition, our hNIS/tdTomato fusion reporter successfully visualized cAMP-induced hNIS expression in xenografted tumors from mouse model.

Conclusions

These findings clearly reveal that the membrane localization of hNIS and its function of iodine uptake are glycosylation-dependent, as our results highlight enhancement of NIS expression and glycosylation with subsequent membrane localization after cAMP treatment. Therefore, enhancing functional NIS by the increasing level of glycosylation may be suggested as a promising therapeutic strategy for cancer patients who show refractory response to conventional radioiodine treatment.     

Occurrence of a Hybrid Between Taenia saginata and Taenia asiatica Tapeworms in Cambodia

Human infection with Taenia asiatica or a hybrid between Taenia saginata and T. asiatica has not been reported in Cambodia. We detected for the first time a hybrid form between T. saginata and T. asiatica in Preah Vihear Province, Cambodia. An adult tapeworm specimen, i.e., 75 cm long strobila without scolex, was expelled from a 27-year-old man after praziquantel medication and purging. It was morphologically indistinguishable between T. saginata and T. asiatica. Several proglottids were molecularly analyzed to confirm the tapeworm species. The mitochondrial gene encoding cytochrome c oxidase subunit 1 (cox1) and nuclear genes encoding elongation factor-1α (ef1) and ezrin-radixin-moesin (ERM)-like protein (elp) were sequenced, and a single-allele analysis was performed to confirm the haploid genotype. The results revealed that our sample showed a discrepancy between the mitochondrial and 2 nuclear genes. It possessed homozygous sequences typical of T. saginata at cox1 and ef1 loci. However, it was heterozygous at the elp locus, with 1 allele in T. asiatica (elpA) and 1 in T. saginata (elpC), which indicates that it is a hybrid between T. saginata and T. asiatica. The present results confirmed the presence of a hybrid between T. saginata and T. asiatica in Cambodia and strongly suggest the existence of also ‘pure’ T. asiatica in Cambodia.     

Larval Gnathostomes and Zoonotic Trematode Metacercariae in Fish from a Local Market in Yangon City,Myanmar

A survey was performed to investigate the infection status of zoonotic helminth larvae in fish from a local market of North Dagon District in Yangon City, Myanmar. A total of 486 fish in 13 species were collected 8 times from December 2015 to December 2019. All fish were transported under ice to a laboratory in Korea and examined for helminth larvae using artificial digestion method. Larval gnathostomes and metacercariae of more than 8 zoonotic trematode species, i.e., Opisthorchis viverrini, Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus spp., Stellantchasmus falcatus, Pygidiopsis cambodiensis, and Procerovum sp., were detected. Larval gnathostomes were found in 58 (16.0%) out of 362 fish of 6 species, with mean intensity of 2.8 per fish infected. Metacercariae of O. viverrini were detected in 10 (2.9%) out of 349 fish of 5 species, with mean intensity of 16.9 per fish infected. Metacercarial prevalences of 4 intestinal flukes, H. taichui, H. pumilio, H. yokogawai, and Centrocestus spp., were 16.8%, 26.0%, 12.5%, and 15.0% in the positive fish species, respectively, and mean metacercarial intensity was 63.3, 26.8, 86.2, and 8.7 per fish infected. Metacercariae of S. falcatus and P. cambodiensis were detected only from the mullet, Chelon macrolepis. Metacercariae of Procerovum sp. were found in Channa striata and Anabas testudineus. Collectively, it was confirmed that the fish were infected with gnathostome larvae and metacercariae of O. viverrini and intestinal flukes in Yangon City, Myanmar.     

Morphological and Molecular Diagnosis of Necator americanus and Ancylostoma ceylanicum Recovered from Villagers in Northern Cambodia

Human hookworm infections caused by adult Ancylostoma spp. and Necator americanus are one of the most important tropical diseases. We performed a survey of intestinal helminths using the Kato-Katz fecal examination technique targeting 1,156 villagers residing in 2 northern provinces (Preah Vihear and Stung Treng) of Cambodia in 2018. The results revealed a high overall egg positive rate of intestinal helminths (61.9%), and the egg positive rate of hookworms was 11.6%. Nine of the hookworm egg positive cases in Preah Vihear Province were treated with 5–10 mg/kg pyrantel pamoate followed by purging with magnesium salts, and a total of 65 adult hookworms were expelled in diarrheic stools. The adult hookworms were analyzed morphologically and molecularly to confirm the species. The morphologies of the buccal cavity and dorsal rays on the costa were observed with a light microscope, and the nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were analyzed. The majority of the hookworm adults (90.7%) were N. americanus, whereas the remaining 9.3% were Ancylostoma ceylanicum, a rare hookworm species infecting humans. The results revealed a high prevalence of hookworm infections among people in a northern part of Cambodia, suggesting the necessity of a sustained survey combined with control measures against hookworm infections.     

Melatonin suppresses senescence‐derived mitochondrial dysfunction in mesenchymal stem cells via the HSPA1L–mitophagy pathway

Mesenchymal stem cells (MSCs) are a popular cell source for stem cell‐based therapy. However, continuous ex vivo expansion to acquire large amounts of MSCs for clinical study induces replicative senescence, causing decreased therapeutic efficacy in MSCs. To address this issue, we investigated the effect of melatonin on replicative senescence in MSCs. In senescent MSCs (late passage), replicative senescence decreased mitophagy by inhibiting mitofission, resulting in the augmentation of mitochondrial dysfunction. Treatment with melatonin rescued replicative senescence by enhancing mitophagy and mitochondrial function through upregulation of heat shock 70 kDa protein 1L (HSPA1L). More specifically, we found that melatonin‐induced HSPA1L binds to cellular prion protein (PrP^C), resulting in the recruitment of PrP^C into the mitochondria. The HSPA1L‐PrP^C complex then binds to COX4IA, which is a mitochondrial complex IV protein, leading to an increase in mitochondrial membrane potential and anti‐oxidant enzyme activity. These protective effects were blocked by knockdown of HSPA1L. In a murine hindlimb ischemia model, melatonin‐treated senescent MSCs enhanced functional recovery by increasing blood flow perfusion, limb salvage, and neovascularization. This study, for the first time, suggests that melatonin protects MSCs against replicative senescence during ex vivo expansion for clinical application via mitochondrial quality control.     

Au nanospheres and nanorods for enzyme-free electrochemical biosensor applications

Au nanocrystals with different morphologies were prepared and used for enzyme-free electrochemical biosensor applications. To investigate the electrocatalytic properties of Au nanocrystals as a function on their morphologies, Au nanocrystals, Au nanospheres (NSs) on silica, Au NSs, and Au nanorods (NRs) with aspect ratios of 1:3 and 1:5, were coated on the screen printed electrodes and further measure the amperometric responses to hydrogen peroxide via three-electrode system. The electrodes modified with Au nanocrystals showed biosensing properties without any enzyme being attached or immobilized at their surface. The hydrogen peroxide detection limits of the biosensors with Au NSs, Au NRs (1:3), and Au NRs (1:5) were 6.48, 8.65, and 9.38 μM (S/N = 3), respectively. The biosensors with Au NSs, Au NRs (1:3), and Au NRs (1:5) showed the sensitivities of 11.13, 54.53, and 58.51 μA/mM, respectively. These results indicate that morphologies of Au nanocrystals significantly influence the sensitivity of the biosensors. In addition, the enzyme-free biosensors with Au nanocrystals were stable for 2 months. Au nanocrystal-based enzyme-free system, which is proposed in this study, can be used as a platform for various electrochemical biosensors.     

MicroRNA-513a-5p mediates TNF-α and LPS induced apoptosis via downregulation of X-linked inhibitor of apoptotic protein in endothelial cells

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that have emerged as one of the central players of gene expression regulation. Endothelial cell apoptosis plays a fundamental role in the development of atherosclerosis. This study was designed to determine the effect of miR-513a-5p on apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs were treated with tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) and miR-513a-5p expression levels were determined. MiR-513a-5p target gene indentification, validation, and signalling pathways were investigated. Treatment of HUVECs with TNF-α and LPS up-regulated miR-513a-5p expressions more than 2-fold compared to control (P < 0.05). Inhibition of miR-513a-5p by antisense (AS) miR-513a-5p reversed TNF-α and LPS induced apoptosis (P < 0.01). Transfection of HUVECs with miR-513a-5p mimics also induced apoptosis (P < 0.01). Treatment of HUVECs with TNF-α and LPS attenuated X-linked inhibitor of apoptosis (XIAP) while increased caspase-3 expression, poly ADP-ribose polymerase (PARP) cleavage, and p53 expression. These effects were reversed by inhibition of miR-513a-5p. Of those miR-513a-5p candidate target genes, we identified and validated XIAP as a miR-513a-5p target gene. Targeting of the XIAP 3′-untranslated region by miR-513a-5p using luciferase reporter assay resulted in attenuated luciferase activity. Transfection of HUVECs with AS miR-513a-5p increased XIAP protein expression while miR-513a-5p mimics attenuated XIAP expression. These results together suggest that miR-513a-5p mediates TNF-α and LPS induced apoptosis via downregulation of XIAP in HUVECs.     

Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. In this study, we found that the expressions of anti-oxidant proteins (gamma-glutamylcysteine ligase heavy chain (gamma-GCL h), heme oxygenase-1, thioredoxin and peroxiredoxin1) in TAM-resistant MCF-7 (TAMR-MCF-7) cells were higher than control MCF-7 cells. Molecular analyses using antioxidant response element (ARE)-containing reporters and gel-shift supported the critical role of NF-E2-related factor2 (Nrf2)/ARE in the overexpression of antioxidant proteins in TAMR-MCF-7 cells. Intracellular peroxide production was significantly decreased in TAMR-MCF-7 cells and TAM resistance was partially reversed by Nrf2 siRNA. The basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase were increased in the TAMR-MCF-7 cells and the inhibition of ERK significantly decreased the activity of minimal ARE reporter and gamma-GCL h protein expression in TAMR-MCF-7 cells. However, exposure of TAMR-MCF-7 cells to 17-beta-estradiol or ICI-182,780 did not significantly change gamma-GCL h expression. These results suggest that the persistent activation of Nrf2/ARE is critical for the enhanced expression of anti-oxidant proteins in TAM-resistant breast cancer cells and the pathway of ERK, but not of estrogen receptor signaling are involved in the up-regulation of Nrf2/ARE.