Ningning Zhao

Interests

Teaching

Nutrition and Metabolism;Mineral Nutrition;Nutrient and Gene Interaction;

Research

The research in my laboratory has been focused on advancing molecular mechanisms for the function and regulation of plasma membrane metal transporters. These transporters play fundamental roles in regulating cellular metabolism and cellular function. Mutations and malfunctioning of these transporters are directly pertinent to the initiation and the progression of an increasing number of human diseases, including iron deficiency, hemochromatosis, cancer, and childhood on-set neurodegeneration. We identify and characterize the genes and factors that are involved in determining the structure and function of these metal transporters. We also examine the intracellular trafficking and degradation of these proteins. In our research, we combine cell-line and mouse models, and employ a variety of biochemical and molecular biology techniques. We also utilize the cutting-edge genome engineering technologies, including Adeno-Associated Virus-mediated genomic modification and CRISPR/Cas9-mediated genome editing. We hope that our research will advance the understanding of disease mechanisms, identify therapeutic target genes, and improve the life quality of patients.

Courses

Scholarly Contributions

Journals/Publications

• Lewis, S. A., Shetty, S., Gamble, S., Heim, J., Zhao, N., Stitt, G., Pankratz, M., Mangum, T., Marku, I., Rosenberg, R. B., Wilfong, A. A., Fahey, M. C., Kim, S., Myers, S. J., Appavu, B., & Kruer, M. C. (2023). Intrathecal magnesium delivery for Mg++-insensitive NMDA receptor activity due to GRIN1 mutation. Orphanet journal of rare diseases, 18(1), 225.
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  Mutations in the NMDA receptor are known to disrupt glutamatergic signaling crucial for early neurodevelopment, often leading to severe global developmental delay/intellectual disability, epileptic encephalopathy, and cerebral palsy phenotypes. Both seizures and movement disorders can be highly treatment-refractory.
• Liu, Q., Jenkitkasemwong, S., Prami, T. A., McCabe, S. M., Zhao, N., Hojyo, S., Fukada, T., & Knutson, M. D. (2023). Metal-ion transporter SLC39A8 is required for brain manganese uptake and accumulation. The Journal of biological chemistry, 299(8), 105078.
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  Manganese (Mn) is an essential nutrient, but is toxic in excess. Whole-body Mn levels are regulated in part by the metal-ion influx transporter SLC39A8, which plays an essential role in the liver by reclaiming Mn from bile. Physiological roles of SLC39A8 in Mn homeostasis in other tissues, however, remain largely unknown. To screen for extrahepatic requirements for SLC39A8 in tissue Mn homeostasis, we crossed Slc39a8-inducible global-KO (Slc39a8 iKO) mice with Slc39a14 KO mice, which display markedly elevated blood and tissue Mn levels. Tissues were then analyzed by inductively coupled plasma-mass spectrometry to determine levels of Mn. Although Slc39a14 KO; Slc39a8 iKO mice exhibited systemic hypermanganesemia and increased Mn loading in the bone and kidney due to Slc39a14 deficiency, we show Mn loading was markedly decreased in the brains of these animals, suggesting a role for SLC39A8 in brain Mn accumulation. Levels of other divalent metals in the brain were unaffected, indicating a specific effect of SLC39A8 on Mn. In vivo radiotracer studies using Mn in Slc39a8 iKO mice revealed that SLC39A8 is required for Mn uptake by the brain, but not most other tissues. Furthermore, decreased Mn uptake in the brains of Slc39a8 iKO mice was associated with efficient inactivation of Slc39a8 in isolated brain microvessels but not in isolated choroid plexus, suggesting SLC39A8 mediates brain Mn uptake via the blood-brain barrier. These findings establish SLC39A8 as a candidate therapeutic target for mitigating Mn uptake and accumulation in the brain, the primary organ of Mn toxicity.
• McCabe, S., Limesand, K., & Zhao, N. (2023). Recent progress toward understanding the role of ZIP14 in regulating systemic manganese homeostasis. Computational and structural biotechnology journal, 21, 2332-2338.
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  ZIP14 is a metal transporter essential for the regulation of body manganese homeostasis. The physiological functions of ZIP14 have been uncovered mainly through two lines of in vivo studies that examined the phenotypes of ZIP14 loss, including studies of humans with mutations and animals with ZIP14 deficiency. This mini review aims at presenting an updated view of the important advances made towards understanding the genetic and pathological mechanisms of brain manganese overload caused by ZIP14 deficiency.
• Vungutur, V., Yu, S., McCabe, S., Fung, C., & Zhao, N. (2023). A simple and highly reproducible method for the detection of erythrocyte membrane ZIP metal transporters by immunoblotting. Methods in enzymology, 687, 87-102.
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  Manganese is one of the essential trace elements found in erythrocytes. Metal transporters situated on the plasma membrane generally facilitate the movement of manganese into and out of cells. This study aims at determining whether two recently discovered manganese importers, ZIP8 and ZIP14, are located in the erythrocyte membrane. We outline a simple, effective and repeatable method for the isolation of erythrocyte membrane from a minimum of 50 µL mouse blood, followed by the identification of ZIP metal transporters using immunoblotting. Our results revealed that ZIP8 is expressed within the erythrocyte membrane, in contrast to ZIP14 which is not identified using immunoblotting approach. A direct measurement of the ZIP8 protein expression in erythrocyte membranes could provide valuable information for further analyzing its biological function.
• Yu, S., & Zhao, N. (2023). The Regulation of ZIP8 by Dietary Manganese in Mice. International journal of molecular sciences, 24(6).
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  ZIP8 is a newly identified manganese transporter. A lack of functional ZIP8 results in severe manganese deficiency in both humans and mice, indicating that ZIP8 plays a crucial role in maintaining body manganese homeostasis. Despite a well-acknowledged connection between ZIP8 and manganese metabolism, how ZIP8 is regulated under high-manganese conditions remains unclear. The primary goal of this study was to examine the regulation of ZIP8 by high-manganese intake. We used both neonatal and adult mouse models in which mice were supplied with dietary sources containing either a normal or a high level of manganese. We discovered that high-manganese intake caused a reduction in liver ZIP8 protein in young mice. Since a decrease in hepatic ZIP8 leads to reduced manganese reabsorption from the bile, our study identified a novel mechanism for the regulation of manganese homeostasis under high-manganese conditions: high dietary manganese intake results in a decrease in ZIP8 in the liver, which in turn decreases the reabsorption of manganese from the bile to prevent manganese overload in the liver. Interestingly, we found that a high-manganese diet did not cause a decrease in hepatic ZIP8 in adult animals. To determine the potential reason for this age-dependent variation, we compared the expressions of liver ZIP8 in 3-week-old and 12-week-old mice. We found that liver ZIP8 protein content in 12-week-old mice decreases when compared with that of 3-week-old mice under normal conditions. Overall, results from this study provide novel insights to facilitate the understanding of ZIP8's function in regulating manganese metabolism.
• Fung, C. K., & Zhao, N. (2022). The Combined Inactivation of Intestinal and Hepatic ZIP14 Exacerbates Manganese Overload in Mice. International journal of molecular sciences, 23(12).
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  ZIP14 is a newly identified manganese transporter with high levels of expression in the small intestine and the liver. Loss-of-function mutations in can lead to systemic manganese overload, which primarily affects the central nervous system, causing neurological disorders. To elucidate the roles of intestinal ZIP14 and hepatic ZIP14 in maintaining systemic manganese homeostasis, we generated mice with single-tissue or two-tissue knockout, including intestine-specific (-In-KO), liver-specific (-L-KO), and double (intestine and liver) -knockout (-DKO) mice. mice were used as the control. Tissue manganese contents in these mice were compared using inductively coupled plasma mass spectrometry (ICP-MS) analysis. We discovered that although the deletion of intestinal ZIP14 only moderately increased systemic manganese loading, the deletion of both intestinal and hepatic ZIP14 greatly exacerbated the body's manganese burden. Our results provide new knowledge to further the understanding of manganese metabolism, and offer important insights into the mechanisms underlying systemic manganese overload caused by the loss of ZIP14.
• McCabe, S. M., & Zhao, N. (2021). The Potential Roles of Blood-Brain Barrier and Blood-Cerebrospinal Fluid Barrier in Maintaining Brain Manganese Homeostasis. Nutrients, 13(6).
  More info

  Manganese (Mn) is a trace nutrient necessary for life but becomes neurotoxic at high concentrations in the brain. The brain is a "privileged" organ that is separated from systemic blood circulation mainly by two barriers. Endothelial cells within the brain form tight junctions and act as the blood-brain barrier (BBB), which physically separates circulating blood from the brain parenchyma. Between the blood and the cerebrospinal fluid (CSF) is the choroid plexus (CP), which is a tissue that acts as the blood-CSF barrier (BCB). Pharmaceuticals, proteins, and metals in the systemic circulation are unable to reach the brain and spinal cord unless transported through either of the two brain barriers. The BBB and the BCB consist of tightly connected cells that fulfill the critical role of neuroprotection and control the exchange of materials between the brain environment and blood circulation. Many recent publications provide insights into Mn transport in vivo or in cell models. In this review, we will focus on the current research regarding Mn metabolism in the brain and discuss the potential roles of the BBB and BCB in maintaining brain Mn homeostasis.
• Wei, G., Wu, Y., & Zhao, N. (2021). Generation of a Polyclonal Antibody against the Mouse Metal Transporter ZIP8. Antibodies (Basel, Switzerland), 10(2).
  More info

  ZIP8 is a newly identified metal transporter. In human patients, mutations in result in severe manganese deficiency, suggesting a critical role for ZIP8 in regulating systemic manganese homeostasis. In mice, the deletion of ZIP8 recapitulates the symptoms of patients with mutations. However, further studies using mouse models to examine ZIP8's function were hindered by the lack of suitable antibodies to detect endogenous ZIP8 protein. In this study, we report the design, generation, and validation of a polyclonal antibody against mouse ZIP8. We have demonstrated that the newly generated antibody can be reliably used in immunoblotting analysis to detect endogenous ZIP8 protein in mouse tissues. The successful generation and validation of anti-mouse ZIP8 antibody provide opportunities to further examine the function and regulation of this metal transporter. In addition, our study may provide valuable insights into the future development of antibodies targeting polytopic membrane proteins.
• Wu, Y., Wei, G., & Zhao, N. (2021). Restriction of Manganese Intake Prevents the Onset of Brain Manganese Overload in Mice. International journal of molecular sciences, 22(13).
  More info

  As a newly identified manganese transport protein, ZIP14 is highly expressed in the small intestine and liver, which are the two principal organs involved in regulating systemic manganese homeostasis. Loss of ZIP14 function leads to manganese overload in both humans and mice. Excess manganese in the body primarily affects the central nervous system, resulting in irreversible neurological disorders. Therefore, to prevent the onset of brain manganese accumulation becomes critical. In this study, we used mice as a model for ZIP14 deficiency and discovered that these mice were born without manganese loading in the brain, but started to hyper-accumulate manganese within 3 weeks after birth. We demonstrated that decreasing manganese intake in mice was effective in preventing manganese overload that typically occurs in these animals. Our results provide important insight into future studies that are targeted to reduce the onset of manganese accumulation associated with ZIP14 dysfunction in humans.
• Morgan, S. E., Schroten, H., Ishikawa, H., & Zhao, N. (2020). Localization of ZIP14 and ZIP8 in HIBCPP Cells. Brain sciences, 10(8).
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  The blood-cerebrospinal fluid barrier (BCB) is important in maintaining brain manganese (Mn) homeostasis. This barrier consists of a single layer of epithelial cells, connected by tight junctions, that restrict the passage of nutrients to only allow molecules to be carried through the membrane by a transporter. These epithelial cells are polarized with asymmetrical blood-facing and cerebrospinal fluid-facing sides. Here, we have established a polarized model of a human choroid plexus papilloma cell line, HIBCPP. For the first time, Mn importers ZIP14 and ZIP8 were identified in HIBCPP cells and were found to be enriched at the basolateral and apical sides of the cell monolayer, respectively. The localization of each ZIP protein adds to the understanding of Mn transport across the HIBCPP BCB model to help understand the mechanism of Mn homeostasis within the brain.
• Winslow, J. W., Limesand, K. H., & Zhao, N. (2020). The Functions of ZIP8, ZIP14, and ZnT10 in the Regulation of Systemic Manganese Homeostasis. International journal of molecular sciences, 21(9).
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  As an essential nutrient, manganese is required for the regulation of numerous cellular processes, including cell growth, neuronal health, immune cell function, and antioxidant defense. However, excess manganese in the body is toxic and produces symptoms of neurological and behavioral defects, clinically known as manganism. Therefore, manganese balance needs to be tightly controlled. In the past eight years, mutations of genes encoding metal transporters (), (), and () have been identified to cause dysregulated manganese homeostasis in humans, highlighting the critical roles of these genes in manganese metabolism. This review focuses on the most recent advances in the understanding of physiological functions of these three identified manganese transporters and summarizes the molecular mechanisms underlying how the loss of functions in these genes leads to impaired manganese homeostasis and human diseases.
• Felber, D. M., Wu, Y., & Zhao, N. (2019). Regulation of the Metal Transporters ZIP14 and ZnT10 by Manganese Intake in Mice. Nutrients, 11(9).
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  The metal transporters ZIP14 and ZnT10 play key physiological roles in maintaining manganese (Mn) homeostasis. However, in vivo regulation of these two transporters by Mn is not understood. Here, we examined how dietary Mn intake regulates ZIP14 and ZnT10 by feeding mice a low-Mn diet, a control diet, or a high-Mn diet for 6 weeks. Inductively coupled plasma mass spectrometry was used to measure Mn and iron (Fe) levels. ZIP14 and ZnT10 protein levels were measured by western blot analysis. While mice on the high-Mn diet exhibited significantly higher levels of Mn in the blood, liver, and brain, the low-Mn diet group did not display matching reductions, indicating that high Mn intake is more effective in disrupting Mn homeostasis in mice. Additionally, Fe levels were only slightly altered, suggesting independent transport mechanisms for Mn and Fe. In the high-Mn diet group, ZIP14 and ZnT10 were both upregulated in the liver, as well as in the small intestine, indicating a coordinated role for these transporters in Mn excretion. Unexpectedly, this upregulation only occurred in male mice, with the exception of hepatic ZIP14, providing new insight into mechanisms behind widely observed sex differences in Mn homeostasis.
• Scheiber, I. F., Alarcon, N. O., & Zhao, N. (2019). Manganese Uptake by A549 Cells is Mediated by Both ZIP8 and ZIP14. Nutrients, 11(7).
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  The alveolar epithelia of the lungs require manganese (Mn) as an essential nutrient, but also provide an entry route for airborne Mn that can cause neurotoxicity. Transporters involved in Mn uptake by alveolar epithelial cells are unknown. Recently, two members of the Zrt- and Irt-like protein (ZIP) family of metal transporters, ZIP8 and ZIP14, have been identified as crucial Mn importers in vivo. ZIP8 is by far most abundantly expressed in the lungs, whereas ZIP14 expression in the lungs is low compared to other tissues. We hypothesized that Mn uptake by alveolar epithelial cells is primarily mediated by ZIP8. To test our hypothesis, we used A549 cells, a type II alveolar cell line. Mirroring the in vivo situation, A549 cells expressed higher levels of ZIP8 than cell models for the liver, intestines, and kidney. Quantification of ZIP8 and ZIP14 revealed a strong enrichment of ZIP8 over ZIP14 in A549 cells. Using siRNA technology, we identified ZIP8 and ZIP14 as the major transporters mediating Mn uptake by A549 cells. To our surprise, knockdown of either ZIP8 or ZIP14 impaired Mn accumulation to a similar extent, which we traced back to similar amounts of ZIP8 and ZIP14 at the plasma membrane. Our study highlights the importance of both ZIP8 and ZIP14 in Mn metabolism of alveolar epithelial cells.
• Scheiber, I. F., Wu, Y., Morgan, S. E., & Zhao, N. (2019). The intestinal metal transporter ZIP14 maintains systemic manganese homeostasis. The Journal of biological chemistry, 294(23), 9147-9160.
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  ZIP14 (encoded by the solute carrier 39 family member 14 () gene) is a manganese transporter that is abundantly expressed in the liver and small intestine. Loss-of-function mutations in cause severe hypermanganesemia. Because the liver is regarded as the main regulatory organ involved in manganese homeostasis, impaired hepatic manganese uptake for subsequent biliary excretion has been proposed as the underlying disease mechanism. However, liver-specific KO mice exhibit decreased manganese only in the liver and do not develop manganese accumulation in other tissues under normal conditions. This suggests that impaired hepatobiliary excretion is not the primary cause for manganese overload observed in individuals lacking functional ZIP14. We therefore hypothesized that increased intestinal manganese absorption could induce manganese hyperaccumulation when ZIP14 is inactivated. To elucidate the role of ZIP14 in manganese absorption, here we used CaCo-2 Transwell cultures as a model system for intestinal epithelia. The generation of a ZIP14-deficient CaCo-2 cell line enabled the identification of ZIP14 as the major transporter mediating basolateral manganese uptake in enterocytes. Lack of ZIP14 severely impaired basolateral-to-apical (secretory) manganese transport and strongly enhanced manganese transport in the apical-to-basolateral (absorptive) direction. Mechanistic studies provided evidence that ZIP14 restricts manganese transport in the absorptive direction via direct basolateral reuptake of freshly absorbed manganese. In support of such function of intestinal ZIP14 , manganese levels in the livers and brains of intestine-specific KO mice were significantly elevated. Our findings highlight the importance of intestinal ZIP14 in regulating systemic manganese homeostasis.
• Wahedi, M., Wortham, A. M., Kleven, M. D., Zhao, N., Jue, S., Enns, C. A., & Zhang, A. S. (2017). Matriptase-2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway. The Journal of Biological Chemistry, 292(44), 18354-18371.
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  Systemic iron homeostasis is maintained by regulation of iron absorption in the duodenum, iron recycling from erythrocytes, and iron mobilization from the liver and is controlled by the hepatic hormone hepcidin. Hepcidin expression is induced via the bone morphogenetic protein (BMP) signaling pathway that preferentially uses two type I (ALK2 and ALK3) and two type II (ActRIIA and BMPR2) BMP receptors. Hemojuvelin (HJV), HFE, and transferrin receptor-2 (TfR2) facilitate this process presumably by forming a plasma membrane complex with BMP receptors. Matriptase-2 (MT2) is a protease and key suppressor of hepatic hepcidin expression and cleaves HJV. Previous studies have therefore suggested that MT2 exerts its inhibitory effect by inactivating HJV. Here, we report that MT2 suppresses hepcidin expression independently of HJV. In Hjv-/- mice, increased expression of exogenous MT2 in the liver significantly reduced hepcidin expression similarly as observed in wild-type mice. Exogenous MT2 could fully correct abnormally high hepcidin expression and iron deficiency in MT2-/- mice. In contrast to MT2, increased Hjv expression caused no significant changes in wild-type mice, suggesting that Hjv is not a limiting factor for hepcidin expression. Further studies revealed that MT2 cleaves ALK2, ALK3, ActRIIA, Bmpr2, Hfe, and, to a lesser extent, Hjv and Tfr2. MT2-mediated Tfr2 cleavage was also observed in HepG2 cells endogenously expressing MT2 and TfR2. Moreover, iron-loaded transferrin blocked MT2-mediated Tfr2 cleavage, providing further insights into the mechanism of Tfr2's regulation by transferrin. Together, these observations indicate that MT2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway.
• Zhao, N., Zhang, A. S., Wortham, A. M., Jue, S., Knutson, M. D., & Enns, C. A. (2017). The Tumor Suppressor, P53, Decreases the Metal Transporter, ZIP14. Nutrients, 9(12).
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  Loss of p53's proper function accounts for over half of identified human cancers. We identified the metal transporter ZIP14 (Zinc-regulated transporter (ZRT) and Iron-regulated transporter (IRT)-like Protein 14) as a p53-regulated protein. ZIP14 protein levels were upregulated by lack of p53 and downregulated by increased p53 expression. This regulation did not fully depend on the changes in ZIP14's mRNA expression. Co-precipitation studies indicated that p53 interacts with ZIP14 and increases its ubiquitination and degradation. Moreover, knockdown of p53 resulted in higher non-transferrin-bound iron uptake, which was mediated by increased ZIP14 levels. Our study highlights a role for p53 in regulating nutrient metabolism and provides insight into how iron and possibly other metals such as zinc and manganese could be regulated in p53-inactivated tumor cells.

Presentations

• Zhao, N. (2023).

  Neurotoxicity caused by loss of ZIP metal transporter

  . Global Experts meet on Neurology, Neuroscience and Brain Disorders.
• Zhao, N. (2023).

  Regulation of ZIP metal transporters by high dietery manganese intake

  . 14th Asian Congress of Nutrition.
• Zhao, N. (2021, October). Mechanism of manganese homeostasis regulation by the metal transporter ZIP14.. Cell Biology of Metals Gordon Research Conference 2021.
• Zhao, N. (2020, December). Novel mechanism of manganese homeostasis regulation. International Conference on Cell and Experimental Biology.
• Zhao, N. (2020, January). The Metal Transporter ZIP14 and Manganese Metabolism. Basic Medical Sciences Seminar Series of College of Medicine, University of Arizona.
• Zhao, N. (2020, July). The identification of a novel mechanism underlying the control of manganese homeostasis. International Nutrition Research Virtual Conference 2020.
• Zhao, N. (2020, June). The regulation of manganese transporters in mice. 29th World Congress on Nutrition & Dietetics.
• Zhao, N. (2018, December). P53 regulates non-transferrin-bound iron uptake via modulating ZIP14. American Society for Cell Biology, Annual Meeting 2018.
• Zhao, N. (2017, August/2017). Metal transporter ZIP14 in human diseases: function, regulation and pathological implications. College of Pharmacy Drug Discovery and Developmental Therapeutics Seminar.
• Zhao, N. (2017, June/2017). Function and regulation of the metal transporter ZIP14: implications of using mouse models to study human diseases. University Animal Care Departmental Seminar.
• Zhao, N. (2017, May/2017). Neogenin Facilitates the Induction of Hepcidin Expression by Hemojuvelin in the Liver. Seventh Congress of the International Bioiron Society 2017.
• Zhao, N. (2017, October/2017). Function and regulation of the metal transporter ZIP14: implications for understanding human diseases. The Department of Immunobiology Departmental Seminar.

Poster Presentations

• Zhao, N. (2023).

  Novel insights into childhood-onset neurodegeneration associated with loss of ZIP14

  . 16th Asian Pan-Pacific Congress for Pediatric Gastroenterology, Hepatologyand Nutrition (APPSPGHAN 2023).
• Zhao, N. (2023).

  The Role of ZIP8 in K562 cells

  . The 34rd Annual UBRP Conference. Tucson, AZ.
• Zhao, N. (2023).

  The mechanism of a childhood-onset neurodegenerative disorder cause by manganese overload

  . 11th IBRO World Congress of Neuroscience.
• Zhao, N. (2022). Regulation of ZIP8 by manganese. Annual UBRP Conference of The university of Arizona.
• Zhao, N. (2022). The physiological function of ZIP14 in manganese metabolism. 22nd International Congress of Nutrition.
• Zhao, N. (2021, April). Control of systemic manganese homeostasis by metal transporter ZIP14.. Experimental Biology 2021.
• Zhao, N. (2020, December). ZIP14 and the regulation of systemic manganese metabolism. American Society for Cell Biology 2020 Annual meeting.
• Zhao, N. (2020, February). Combination of chelation therapy and low-manganese diet in a patient with SLC39A14-associated hypermanganesemia and dystonia. 1st International Symposium on Genetic Syndromes with Movement Disorders and Epilepsy.
• Zhao, N. (2020, July). The role of hepatic and intestinal ZIP14 in regulating manganese homeostasis. Arizona Academy of Nutrition & Dietetics, Annual Meeting 2020.
• Zhao, N. (2017, December/2017). Matriptase‐2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway. American Society for Cell Biology, Annual Meeting 2017.