New Insights into Depressive Disorder with Respect to Low-Grade Inflammation and Fish Oil Intake.

Unipolar depression has been recognized as one of the major diseases by the World Health Organization in the 21st century. The etiology of depression is complicated and includes genetic factors, stress, aging, and special physical status (pregnancy, metabolic syndrome, and trauma). Numerous animal and human studies have demonstrated that n-3 polyunsaturated fatty acids (n-3 PUFAs) are highly correlated to cognition and depression. These nutritional antidepressants, including EPA and DHA, have a range of neurobiological activities contributing to their potential antidepressant effects. Our preclinical and clinical studies have indicated that n-3 PUFA supplementation in addition to standard antidepressant medications may provide synergistic neuroprotective and antioxidant/inflammatory effects. To translate our preliminary findings into clinical application, this paper reviews the existing evidence on the antidepressant effects of n-3 PUFAs and the potential underlying mechanisms, which include modulation of chronic lowgrade inflammation and the corresponding changes in peripheral blood immune biomarkers.

Treatment of depression can be roughly categorized into three modalities: medications antidepressants and other medications that enhance the efficacy of antidepressants , psychotherapy psychodynamic therapy, cognitive-behavioral therapy CBT , and interpersonal psychotherapy IPT 10,11 , and somatic nonpharmacological treatments electroconvulsive therapy ECT and the more moderate repetitive transcranial magnetic stimulation rTMS 12 .
To date, there is a lack of clinically efficient predictors of depression and markers of the efficacy of therapies against MDD 13 . The neurotransmitter theory of depression describes that the depressive symptoms result from the impaired serotonin-kynurenine pathway, which decreases the serotonin concentration in the central nervous system CNS 14 . Most antidepressants, including selective serotonin reuptake inhibitors SSRIs , are designed based on this theory and increase the serotonin concentration in the CNS. However, because of the heterogeneity of depression, over 50 of patients with depression do not respond adequately to these antidepressants, and efficient clinical biomarkers for identifying such individuals are lacking 15 . The structure and constitution of human brains are extremely complicated, which hinders the determination of the exact pathomechanisms of depression. However, a large database of characteristics of patients with depression may help in this regard 16 . Accumulating evidence indicates that patients with depression have increased inflammation. Chronic low-grade inflammation, especially in the CNS, plays a pivotal role in the pathology of depression 17 .

n-3 Polyunsaturated Fatty Acids
Polyunsaturated fatty acids PUFAs are classified into different subsets based on the carbon number and double bonds of the fatty acid chain. n-3 PUFA is one of the major fatty acid subgroups and contains eicosapentaenoic acid EPA, 20:5 and docosahexaenoic acid DHA, 22:6 ; these have been found to exist in substantial concentrations in marine algae and animals. Other n-3 PUFAs include α-linolenic acid ALA, 18:3 and conjugated linolenic acid CLNA . ALA, which is found mostly in plants, had beneficial effects on reducing liver cholesterol levels via increasing the acetyl-CoA oxidase-associated proteins and suppressing the mechanisms of PPAR-α 18 . The dietary oil extracted from Perilla frutescens seed upregulated the BDNF expression in prefrontal cortex PFC and improved the depressive-like behaviors as well 19 . Conjugated linolenic acid CLNA , which is formed via the usage of microorganisms, has been proved that exert the anti-inflammatory properties and lipid/energy metabolism modulatory functions in some cell culture and animal studies 20,21 . EPA is an anti-inflammatory fatty acid that can be metabolized to n-3 series eicosanoids. For example, EPA-derived prostaglandin PGE -3 is metabolized by cyclooxygenase 2 COX2 , and the E-series-resolvins are metabolized by 5-lipoxygenase LOX 22 Fig. 1 . Over 20 of the net dry weight of the human brain is composed of lipids, mainly arachidonic acid AA, 20:4 and DHA. Such marine-derived n-3 PUFAs are a structural constituent of membranes, specifically in the CNS. DHA is crucial for brain function, neuronal cell growth and differentiation, and neuronal signaling 23 . Fur- Fig. 1 Transformation of PUFA into lipid oxylipins. Arachidonic acid AA , eicosapentaenoic acid EPA and docosahexaenoic acid DHA , hydrolyzed and released from membrane can be converted into several different kinds of mediators via cyclooxygenase COX or lipoxygenase LOX . The AA-related metabolites are pro-inflammatory whereas DHA-and EPA-derivatives are anti-inflammatory. Lower n-6/n-3 ratio can efficiently inhibit the production of n-6 metabolites and improve the inflammation status. LT: leukotriene, PG: prostaglandin. TX: thromboxane, Lx: lipoxin, RvEs: E-series resolvin, RvDs: D-series resolvin, NPD1: neuroprotectin D1, MaR1: maresin 1, EFOX: eletrophilic oxo-derivatives.
thermore, DHA-derived lipids play a vital role in maintaining the integrity of the blood-brain barrier BBB , which prevents the entry of harmful materials into the brain environment 24 . n-3 PUFAs, especially EPA and DHA, exerted antidepressive efficacy in many preclinical and clinical trials 25 30 . Moderate intake of n-3 PUFAs approximately 0.5-1 g/day was significantly associated with a lower prevalence of depression 31 . EPA may exert anti-inflammatory benefits in women with perinatal depression 32 . The treatment efficacy of n-3 PUFA supplementation depends on the proportion and dose of EPA and DHA 33 . The mechanisms underlying the antidepressive effects of n-3 PUFAs, however, remain unclear. Several studies have indicated that DHA has no efficacy for depression except in postnatal depression , and their findings highlight that EPA might be the main n-3 PUFA component with antidepression activity 33,34 . Some meta-analyses have proposed that n-3 PUFA supplements with high concentrations of EPA 50 , 60 , or 80 have significantly greater efficacy than those with high concentrations of DHA 35 37 . Furthermore, the anti-inflammatory capability of EPA, which can compete with n-6 PUFAs and lead to a decrease in the production of proinflammatory cytokines, may be its potential mechanism of action against depression.

CNS Inflammation
The immune system involves complex communication among various types of immune cells, including antigenpresenting cells APCs , T-helper cells, T-regulator cells, macrophages, and monocytes. The complete immune response can be roughly divided into four steps: 1 recognition of infection or damage, 2 initiation of immune responses to infection or damage, 3 modulation of the duration and scale of immune responses to avoid self-harm, and 4 induction of memory to enhance future responses to the same infectious agent or damage 38 . The immune system aims to maintain homeostasis by eliminating pathologic agents and repairing injuries, but its dysregulation leads to diseases, including psychiatric diseases such as depression 7 . Thus, the role of immune dysregulation in depression is being increasingly explored.
Neurons in the CNS are sensitive and fragile, and the immune cells in the CNS quickly respond to the introduction of any harmful materials. The BBB is composed of capillary endothelial cells, astrocytes end-feet ensheathing the capillary, and pericytes embedded in the capillary basement membrane. This barrier resists the entry of pathogens; it allows the entry of some small molecules by simple diffusion and of ions and micronutrients through selective and active transport. Penetration of the BBB by pathogens or peripheral immune cells causes immune dysregulation, aberrant inflammation, and neuronal cell dysfunction. Such possible mechanisms have shown in Fig. 2.
Damage-associated molecular patterns DAMPs and pathogen-associated molecular patterns PAMPs trigger the receptors e.g., toll-like receptor 4 TLR4 on innate immune cells and activate them to immediately produce proinflammatory cytokines to attract more monocytes/ macrophages. DAMPs include host molecules associated with cellular damage e.g., heat shock protein, ATP, and HMGB1 , whereas PAMPs are infectious pathogen-related substances 39 . After the innate immune system responds to PAMPs or DAMPs, the adaptive immune system, which is a specific immune system and the second-line protection in mammals, is activated by specific antigens via APCs e.g., dendritic cells . Upon antigen recognition, the T and B cells differentiate to become functional lymphocytes.

Special Immune System in the CNS
Microglia are specialized immune cells in the CNS; they are one of the neuroglial cells and are characterized by a small and thin cell body. They account for 5 -10 of total brain cells and exert macrophage functions in the CNS. Their major functions include maintaining CNS homeostasis, eliminating infection, and repairing damage. They are extremely sensitive to even the smallest of stimuli and immediately respond to any alteration in the CNS environment 9 by transforming from the resting ramified form to the reactive form. Microglia are associated with synapse pruning and neurogenesis and are activated to their reactive form in many neurodegenerative and psychiatric diseases, thereby promoting neuroinflammation 40 42 .
Normally, peripheral cells cannot cross the BBB. However, in some special conditions involving BBB disruption, infectious agents and peripheral immune cells e.g., macrophages and T cells can cross the BBB and enter the brain parenchyma. This results in damage to the brain tissue and may contribute to the pathology of psychiatric or neurodegenerative diseases 43 . Activated T cells entering the CNS upregulate adhesion molecules i.e., vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 , chemokines, and integrins 7,44 . Although pathogenic T cells have been associated with autoimmune and psychiatric diseases, peripheral immune cells in the CNS are not always detrimental 45 .
Cytokines have various proinflammatory or anti-inflammatory capabilities. Interleukin IL -6 IL-6 , IL-1β, and interferon-γ IFN-γ are predominantly produced by immune cells, microglia, and astrocytes. In the CNS, the suitable production of cytokines by neuroglial cells is crucial to maintaining neuroplasticity and normal neuroprotective functions 46 . However, sustained physical or physiologic stress might result in persistent cytokine production, which accounts for the chronic inflammation status in the brain. Chronic neuroinflammation status results in perturbed multiple neuronal functions, including impairment of the synthesis, reuptake, and release of neurotransmitters 47 . One of the hypotheses implicated is that increased and persistent cytokine activity in the brain might lead to psychiatric diseases, including depression 48 .
It also remains unclear why the same cytokines exhibit different functions in different contexts. Peripheral cytokines are the most influential factors contributing to neurobehaviors, whereas central cytokines also increase neuroinflammation. Maintenance of BBB integrity is sufficient to prevent the entry of peripheral cytokines and improve the symptoms of depression 48,49 . Peripheral immune cells and cytokines can enter the CNS through the disrupted BBB directly due to its increased leakiness 50 or by being attracted to the chemokines secreted by the local immune cells.

Dysregulation of Cytokines in the Brain
Several meta-analyses have concluded that patients with MDD have upregulated proinflammatory cytokine levels and acute phase proteins 51 55 . Inflammation has been positively associated with depression even in younger age groups 51 . Antidepressants also decrease the levels of inflammatory biomarkers; patients with depression who are refractory to treatment tend to have higher baseline inflammation 52 . The peripheral blood IL-6, TNF-α, and C-reactive protein CRP levels are higher in patients with MDD than in healthy controls, indicating that the overactive immune system can be a therapeutic target in patients with MDD 56 .
Studies have always focused on measuring IL-6, IL-1β, TNF-α or CRP levels in patients with MDD. Recently, with advancements in measurement technology, more types of cytokines and chemokines have been evaluated in association with MDD. A 2017 meta-analysis including 82 studies and 3212 patients with MDD revealed upregulation of IL-6, TNF, IL-10, sIL-2, C-C motif ligand CCL 2, IL-13, IL-18, IL-12, IL-1RA, and soluble TNF receptor 2 sTNFR2 53 , thus providing new insights into unknown potential pathways of inflammation in patients with MDD. Another metaanalysis including 69 studies focused on CNS cytokine levels by analyzing cerebrospinal fluid CSF , positron emission tomography PET images, or postmortem brain tissues in patients with MDD compared with healthy controls 55 . The results revealed that IL-6 and TNF-α levels were higher in the CSF of patients with MDD. The PET marker of CNS inflammation was elevated in some brain regions; however, no correlation was observed between inflammatory markers and peripheral marker levels. Postmortem data indicated that certain brain tissues had high TNF-α levels and a significantly smaller number of astrocytes, which may account for the compromised integrity of BBB, leading to increased peripheral monocyte infiltration 55 .
Because neuroinflammation is significantly related to depression, many questions must be considered: 1 whether the adequate response to antidepressants is associated with improved inflammatory status and 2 whether conventional anti-inflammatory treatments are effective in treating depression, especially in patients with MDD with increased inflammation 7 . Many conventional antidepressants exert some degree of anti-inflammatory effects, implying that reducing inflammation might be one of the mechanisms underlying their antidepressant efficacy 57 . SSRIs were reported to decrease IL-6, IL-1β, and TNF-α levels 58 , whereas some studies have stated that SNRIs increase IL-6 and TNF-α production 52,59 . Another meta-analysis including 45 trials and 1517 patients with MDD demonstrated that antidepressant medication significantly decreases the peripheral levels of IL-6, IL-10, TNF-α, and CCL2, but this decrease did not correlate with treatment response 57 . Intriguingly, higher baseline IL-17A levels were associated with better treatment efficacy after treatment with the combination of bupropion and an SSRI 60 . Higher CRP levels have also been associated with better treatment outcomes with SSRI or SNRI 61 . Thus, the anti-inflammatory effects of antidepressants remain inconsistent; however, antidepressants may exert some degree of anti-inflammatory activity.

The Anti-inflammatory Ability of Fish Oil
Many studies have explored the depression response following treatment with immune-modulating antidepressants. Some trials have tried to treat depression using antiinflammatory medicine, including nonsteroid antiinflammatory drugs NSAIDs or cytokine inhibitors. However, the add-on therapy was not appropriate for most patients. Therefore, natural anti-inflammatory as dietary components or supplements, including polyphenols, vitamin D, and fish oil, have been discussed extensively 33,62,63 . A large-scale trial was conducted to measure the anti-inflammatory efficacy of n-3 PUFA, especially marine fish oil. As an effective anti-inflammatory supplement, fish oil was recommended to improve the inflammatory conditions in cancer and heart disease 64 . One randomized controlled trial RCT in 2017 demonstrated that daily intake of EPA and DHA total 1.8 g of n-3 PUFA for 30 days to newly diagnosed breast cancer patients can reduce the level of inflammaion 65 . A 2019 meta-analysis including 13 RCTs revealed that marine n-3 PUFA supplementation lowered inflammation, myocardial infarction, and CVD risks 66 . In summary, n-3 PUFAs seem to exert strong anti-inflammatory effects and may be ideal adjuvants for treating inflammation-associated diseases.

Possible Cellular Mechanisms Underlying the Antiinflammatory Effects of n-3 PUFA
The mechanisms of n-3 PUFAs crossing the BBB have been fully demonstrated by several articles. Three pathways dominated the entry of n-3 PUFAs including simple diffusion, lipoprotein transcytosis and transport via transmembrane proteins. In situ perfusion study demonstrated that the rates of simple diffusion of EPA and DHA were no significant difference 67 . Additional study also confirmed that diffusion of plasma non-esterified DHA is important for supplying brain 68 . Transcytosis showed that lipoprotein receptor e.g. LDL receptor can bind the lipoprotein and transport them into brain via clathrin-or caveolae-dependent endocytosis 69,70 . Last, some transporters on the surface of endothelium cells involved in the transporting PUFAs including FA transport protein-1 FATP-1 , FATP-4 and FA translocase/CD36. Mfsd2a was also presented in the endothelium of the brain, which is particularly important for maintaining DHA levels in brain 71 . In addition, FAs esterified to a glycerol backbone including lysophosphatidylcholine lysoPC has been shown crossing the BBB easily 71 .
Some cross-sectional studies have indicated a significant association between n-3 PUFA and depressive symptoms 72,73 . The French Supplementation en Vitamines et Mineraux Antioxydants SUVIMAX study with a 2-year follow-up indicated that participants consuming fish containing full n-3 PUFA or taking n-3 PUFA at amounts higher than 0.1 of energy intake had a significantly lower risk of depressive disorders 74 . Although the Nurse s Health Study did not conclude a significant association between n-3 PUFA intake and depression occurrence over a 10-year follow-up period in 54,000 women aged 50-77 years in the United States, the findings indicated that lower linoleic acid intake decreases the depression risk, implying that the n-6/n-3 fatty acid ratio should be considered in the diet 75 .
The mechanisms underlying n-3 PUFAs preventive and therapeutic effects on depression remain unclear, and various hypotheses have been proposed. As mentioned earlier, neurotransmitter dysregulation is a well-known hypothesis, and SSRIs are the first-line medications for patients with MDD. However, chronic inflammation may also play a critical role in inducing depression. Because of the limitation and difficulty in obtaining human brain tissue samples, studies on possible molecular mechanisms have mostly involved animal models. Chronic stress is a major risk factor for depression. The chronic mild stress CMS animal model is an effective depression animal model 30, 76 78 . This model overcomes the limitations of single-stimuli response adaptation, and various mental stresses are administered to induce depression in rodents 30 . It also induces a neuroinflammatory response by activating the microglia in the CNS 79 . The neuroinflammation ensures the release of cytokines and impaired the neurotrophin system, synaptic plasticity, and neurogenesis of the hippocampus 80 . n-3 PUFAs exert their anti-inflammatory effect by inhibiting the production of n-6 series eicosanoids. Eicosanoids are biologically active lipid mediators produced from PUFAs, which modulate inflammation and regulate immune activity 81 . The plasma fatty acid composition of patients with depression had a higher n-6/n-3 ratio or percentage of n-3 fatty acids than in healthy controls 82 ; similar results were reported in a rodent study 30 . Whether higher plasma n-3 PUFA concentrations reduce the prevalence of depressive symptoms should be explored.
The antidepressant effect of n-3 PUFAs is likely exerted by modulating inflammatory cytokine production in microglial and other neuronal cells. Some studies have indicated that n-3 PUFA treatment can influence neuronal differentiation 7,83,84 . A study derived neuronal stem cells iNSCs from patients with MDD and treated with EPA, DHA, and stearic acid SA indicated that n-3 PUFAs, but not SA, facilitate astrocyte differentiation dependence on the cAMP-response element binding protein CREB pathway and may exert some antidepressant effects by increasing the production of neurotrophins such as brain-derived neurotrophic factor BDNF and glial cell-derived neurotrophic factor GDNF 83 . n-3 PUFAs can increase the expression of CREB and phosphorylated CREB pCREB 85 . Clinical and preclinical studies have also supported the relevance of neurotrophins in depression, implying that MDD is associated with reduced BDNF or GDNF levels, which can be alleviated by n-3 PUFA supplementation.
n-3 PUFAs also substantially influence the microglia to modulate neuroinflammation by altering their morphology reactive inflammatory or ramified resolving . Understanding how n-3 PUFAs modulate microglial phenotypes and functions can aid the development of innovative therapies with positive effects on physiology and behavior 86 89 . In the BV-2 cell culture model, n-3 PUFAs, especially EPA, inhibited the activation of the NLR family pyrin domain containing 3 NLRP3 inflammasome and decreased IL-1β and IL-18 levels and neurological deficit score via the GPR 40 pathway 87 . Another BV-2 cell culture study revealed that EPA dose-dependently inhibited the expression of two inflammation-related enzymes, inducible NO synthase iNOS and COX2, as well as the subsequent production of NO and PGE 2 . n-3 PUFAs can significantly and dose-dependently reduce the expression and activity of matrix metallopeptidase 9 MMP9 under lipopolysaccharide LPS stimulation in microglial cells. In vitro, n-3 PUFAs inhibit the production and activity of iNOS and COX2 and the production of NO and ROS, which is correlated with the alteration of microglia from anti-inflammatory form to proinflammatory form.
Additionally, n-3 PUFAs modulate the phagocytic capacity and migration capacity of the microglia. DHA dose-dependently inhibited LPS-induced microglial migration; however, it did not exert the same function under normal physical conditions 90 . Autophagy is essential for immune cell homeostasis; it accounts for the dampening of inflammatory processes, highlighting a new pathway to modulate the microglial inflammatory response. EPA and DHA treatment significantly activated the SIRT1 pathway and autophagy in MG6 microglial cells, which decreased the inflammatory responses of LPS stimulation 91 . Studies have indicated that n-3 PUFAs can serve as various ligands for several nuclear receptors, including peroxisome proliferator-activated receptors PPARs and demonstrated that DHA and EPA activate PPARγ and lowered the presence of proinflammatory cytokines in microglial cells 92,93 Fig. 3 .
Many studies have reported a close relationship between microglial function and n-3 PUFAs. A CMS study demonstrated that EPA and DHA can improve aberrant corticos-terone and IL-1β levels as well as hippocampal noradrenaline and 5-hydroxytryptamine concentrations. The researchers also reported that n-3 PUFAs can ameliorate microglial and astrocyte functions suppressed by CMS stimuli and improve depressive symptoms of rodent animals 88 . Notably, studies on models of brain development have indicated that n-3 PUFA supplementation reduces microglial activation or cause phenotype alteration. In a mouse study, lower dietary consumption of n-3 PUFA precursors over the whole perinatal period influenced the microglia phenotype and its morphologic dynamic in the postnatal developing brain 94 . Another study reported the similar result that low maternal intake of n-3 PUFAs during the perinatal period led to increased microglia-mediated phagocytosis of synaptic elements in the developing hippocampus, altered neuronal morphology, and impaired the cognitive performance of the offspring through 12/15-LOX/12-HETE signaling 95 . Impairment of DHA synthesis of mice alters synaptic plasticity, BDNF expression, learning, and memory formation and results in brain inflammation, including higher levels of TNF-α, IL-1β, and iNOS 96 . Overall, n-3 PUFAs are crucial for CNS development and reduce CNS inflammation by modulating various neuronal glial cells.

EPA or DHA?
n-3 PUFAs play a potent role in shaping the brain struc- Depressive Disorder, Lower Grade Inflammation and Fish Oil ture and maintaining homeostasis. Extensive research has been conducted to determine which n-3 PUFA, especially EPA or DHA in marine fish oil, is more efficacious in improving depressive symptoms. Notably, the fatty acid profile analysis of rodent brain tissues revealed that lipids extracted from the brain contain a part of DHA, whereas the levels of EPA are extremely scarce 30,89 . In the whole brain, DHA concentrations are approximately 250 times higher than EPA concentrations, but in the microglia, EPA concentrations are at least two-fold higher than DHA concentrations 97 . EPA may not cross the BBB, but EPA-only supplementation still has anti-inflammatory capability 89 Fig. 3 .
Although the EPA concentration is considerably lower than the DHA concentration in brain tissue, EPA is more neuroactive than DHA with respect to inhibition of neuroinflammation. A meta-analysis revealed that EPA might be more effective than DHA in treating depression 98 . An RCT demonstrated that EPA, but not DHA, can significantly reduce IFN-α-induced depression-like behaviors 99 . A rodent study also reported that EPA but not DHA lowered IL-6 and TNF-α levels and improved the suppressive expression of astrocyte markers and TrkB-BDNF signaling 88 . By contrast, a cell culture study suggested that EPA and DHA have equal potency and efficacy and that they both increased the expression of GFAP and BDNF but not SA 83 . A similar result revealed DHA and EPA both dose-dependently inhibited LPS-induced microglial migration by regulating the ligand of PPARγ receptors to ameliorate inflammation 92 .

Future Work
The relationship among n-3 PUFAs, depression, and neuroinflammation has been extensively examined in recent years, especially with the advancements in measurement technology. A wide variety of possible mechanisms of n-3 PUFAs in the CNS have been unveiled gradually, including shaping the brain structure, maintaining neuronal function, and modulating the immune response. Neuroinflammation, as the main reason for most psychiatric diseases, was relieved through the intake of n-3 PUFAs. However, some paradox still exists in the application of n-3 PUFAs for depressive symptoms even if hundreds of experiments are conducted to explore the clinical use of n-3 PUFA. Given that some meta-analyses have indicated that n-3 PUFAs are not effective for treating depression, the patients dietary habits and n-6/n-3 ratio may be critical factors. However, the most valuable problem might be the exact utilization of n-3 PUFAs in the CNS. Updated detection technology is lacking for measuring inflammation conditions and the entry/usage of one factor in people. However, the novel manufacturing approaches of n-3 PUFA supplements have increasingly attempted to increase the entry and utilization of n-3 PUFAs into the CNS Fig. 4 . For example, condensed fish oil with a higher percentage of n-3 PUFAs and lysophosphatidylcholine-EPA/DHA LPC-EPA/DHA form products were investigated in preclinical or clinical studies. These findings should inform the scope of future clinical trials on the efficacy of n-3 PUFAs. Finally, consolidated analysis could be completed being combined with the results via using the state-of-the-art detection technology-multiomics analysis, such as transcriptomic, proteomic, metabolomic and microbiome. In this context, the whole picture of anti-inflammatory ability of fish oil in CNS could be disclosured in the future.

Contributions
The author s responsibilities were as follows: Te-Hsuan Tung, Ngan Thi Kim Nguyen and Shih-Yi Huang conducted the conception of topic. Te-Hsuan Tung and Shih-Yi Huang collected the related articles. Ngan Thi Kim Nguyen, and Shih-Yi Huang assisted with the editing of the manuscript. Te-Hsuan Tung and Shih-Yi Huang prepared the initial draft and finalized the manuscript. All authors approved the final version of the manuscript.