While often comorbid with obesity and insulin resistance, this disease can also be found in lean individuals, suggesting specific metabolic dysfunction2. NAFLD has become one of the most prevalent liver diseases in adults worldwide, but its incidence in both children and adolescents has also markedly increased in developed nations4. Progression of this disease into nonalcoholic steatohepatitis (NASH), cirrhosis, liver failure, and hepatocellular carcinoma in combination with its widespread incidence thus makes NAFLD and its related pathologies a significant public health concern. Here, we review our understanding of the roles of dietary carbohydrates  and the gut microbiota, which provides essential carbon sources for hepatic fat synthesis during the development of NAFLD.Imbalance between fat production and consumption causes various metabolic disorders. Nonalcoholic fatty liver disease (NAFLD), one such pathology, is characterized by abnormally increased fat synthesis and subsequent fat accumulation in hepatocytes Evidence is emerging of a role for interactions between gut microbes and dietary carbohydrates in non-alcoholic fatty liver disease (NAFLD), one of the most prevalent liver disorders of adults worldwide, with children and adolescents also increasingly affected. NAFLD can progress into even more serious conditions such as steatohepatitis, cirrhosis, liver cancer and liver failure. Researchers led by Cholsoon Jang at the University of California Irvine review understanding of the role of dietary glucose, fructose, fiber and gut microbial metabolism in contributing to NAFLD. The involvement of gut microbes in hepatic assimilation of carbohydrates into fatty acids that accumulate in NAFLD indicates possible treatment strategies based on interfering with microbial metabolism. New understanding of the interactions between carbohydrate metabolism and gut microbes will also help reveal why some people develop NAFLD and suggest appropriate preventive interventions. On the other hand, dietary carbohydrates absorbed by the small intestine go directly to the liver. When the carbohydrate level reaches a certain threshold, diverse metabolic pathways in hepatocytes are triggered to clear away excess carbohydrates. Glycogen synthesis and DNL are such pathways. The resulting newly synthesized fatty acids are stored in hepatocytes as lipid droplets, released into the bloodstream as lipoprotein particles  to feed other organs, or oxidized in the liver when other energy sources are scarce . Thus, DNL is a key mechanism that contributes to the metabolic flux of dietary carbohydrates into lipids, especially in the postprandial state and under disease conditions7.Carbohydrate and lipid metabolism is highly intertwined in the liver. De novo lipogenesis (DNL), a process that converts dietary carbohydrates into fat, is one such metabolic link. Dietary carbohydrates and lipids follow distinct paths from the gut lumen to other parts of the body. Due to their hydrophobicity, dietary fats, mostly composed of long-chain fatty acids, are packaged as triglycerides (TGs) into chylomicrons in intestinal enterocytes and distributed to nonhepatic tissues  through the lymphatic system, thereby bypassing metabolism in the liver8. This generation of cytosolic acetyl-CoA by ACLY is the key step in lipogenesis, as mitochondrial acetyl-CoA cannot directly participate in DNL. Notably, in obese individuals, a small fraction of short-chain fatty acids can be transported from the mitochondria to feed DNL, but the biological significance of this pathway needs more investigation9. Acetyl-CoA carboxylase (ACC) then generates malonyl-CoA from cytosolic acetyl-CoA, the commitment step in DNL. This process is allosterically promoted by cytosolic citrate. Importantly, malonyl-CoA serves as a potent allosteric inhibitor of fatty acid β-oxidation. This mechanism is thought to act as a metabolic safeguard against wasteful energy expenditure from simultaneous fatty acid synthesis and oxidation10. The final step of DNL is the conversion of malonyl-CoA into palmitate (16:0) by fatty acid synthase (FAS). Other processes, such as fatty acid elongation and desaturation, also occur after palmitate is synthesized10.DNL is controlled by allosteric regulation of diverse lipogenic enzymes. Dietary carbohydrates (mainly glucose) first enter glycolysis in the cytosol and become pyruvate, the terminal product of glycolysis Fig. . Pyruvat14. In mice, the loss of Srebp-1c resulted in failure to induce DNL enzymes, including FAS and ACC, in response to postprandial increases in insulin levels, and this lack of response was only partially compensated by SREBP-1a/SREBP-215. Therefore, SREBP-1c is the primary isoform responsible for insulin-induced DNL in hepatocytes. The other SREBP isoforms have been linked to separate metabolic pathways; SREBP-2 is associated with cholesterol synthesis (another important feature of lipid metabolism), while SREBP-1a is mainly expressed in the spleen17.Another crucial layer of DNL regulation occurs at the transcriptional level, which is in part hormonally controlled. Under postprandial conditions, the release of insulin stimulates the expression of lipogenic genes in the liver to promote DNL. Sterol regulatory element-binding protein (SREBP) is a well-known transcription factor that simultaneously induces many lipogenic genes. While there are several SREBP isomers, SREBP-1c is particularly insulin-sensitive compared to the other SREBP isoforms SREBP-1a and SREBP-218. While glucose itself has not been found to modulate transcriptional DNL activity, one glycolytic metabolite, glucose-6-phosphate (G6P), has been linked to increased FAS and ACC expression20. This result was further substantiated by in vitro analysis of Fas expression and glucokinase activity, the enzyme that converts glucose into G6P. Both glucose and glucokinase were found to be required to induce Fas expression in response to insulin20. Glucose-induced DNL proceeds through the upregulation of both glucokinase and SREBP-1c in response to insulin21. Dentin et al. further expanded on this model and implicated carbohydrate responsive element-binding protein (ChREBP) as another important transcription factor regulated by G6P to trigger DNL22. However, this link between ChREBP and dietary carbohydrates is not exclusive to glucose; hepatic ChREBP is particularly sensitive to fructose (see more details in the section below)23. Indeed, G6P may not be the only metabolite that stimulates ChREBP activity, and various other metabolites  have also been suggested to have this effect25.In addition to insulin, full activation of the hepatic lipogenic program requires glucoseChREBP knockdown decreased the effects of glucose on both lipogenic and glycolytic gene induction in response to insulin24. It is thus evident that ChREBP mediates insulin-glucose synergy26. One mechanism of action involves glucose-mediated increases in ChREBP expression, nuclear entry, and binding to the carbohydrate response element (ChoRE), which in turn induces not only lipogenic genes but also SREBP-1c28. ChREBP and SREBP thus rely on each other and work synergistically as follows: 1) ChREBP relies on activation by glycolytic intermediates, which, in turn, are generated by genes controlled by SREBP-1c22. 2) Meanwhile, the deletion of hepatic Chrebp reduces SREBP-1c levels29. 3) Furthermore, overexpression of hepatic Chrebp does not increase postprandial lipogenesis when Srebp-1c is lacking29.Importantly, 18.Collectively, maximum activation of DNL requires concurrent upregulation of lipogenic and glycolytic gene expression, for which glucose and insulin are necessary to stimulate lipogenic transcription factors. In this regard, for insulin-resistant individuals with NAFLD, increases in both circulating glucose and insulin can be synergistic drivers of continuously elevated DNL, the hallmark of this disease6H12O6), they are metabolized by organs quite differently and have divergent effects on hepatic DNL30. For example, fructose can serve as a preferential substrate of DNL due to the faster rate of fructolysis compared to that of glycolysis33. During the primary steps of glycolysis, hexokinase and phosphofructokinase are allosterically regulated, suppressing excessive glycolysis. In contrast, ketohexokinase-c (KHK-C), the primary fructolytic enzyme, has constitutively high activity and thus bypasses these rate-limiting glycolytic steps. This results in rapid fructose catabolism, which triggers ATP depletion and carbon entry into the TCA cycle as well as DNL34. However, most in vitro hepatocyte studies used supraphysiological millimolar concentrations of fructose in the culture medium. Isotope tracing studies did not carefully differentiate between the direct and indirect contributions of fructose to the hepatic DNL pathway.In addition to glucose, fructose is another major dietary carbohydrate in modern society that is closely linked to NAFLD/NASH development. While fructose and glucose have the same molecular formula  and cancer were found to be the leading causes of mortality two decades after initial diagnosis58. It is possible that this proclivity for CVD over cirrhosis and end-stage liver disease is a result of systemic metabolic disorder and dysfunction, as NAFLD is often also co-diagnosed with insulin resistance, obesity, and hyperlipidemia60. Thus, the lethality of NAFLD may be due to hepatic manifestation of a systemic metabolic syndrome rather than the direct result of damage by steatosis.Mild cases of NAFLD can be asymptomatic or benign. However, progression into NASH is characterized by inflammation, hepatocyte apoptosis, and steatosis. One longitudinal study found that individuals with NASH, but not those with only NAFLD, showed increased mortality and the increased occurrence of end-stage liver disease than the general population61. A body of longitudinal human studies also suggests that steatosis contributes to the development of insulin resistance. Ekstedt et al. found that 78% of NAFLD patients developed impaired glucose tolerance or type 2 diabetes after their initial diagnosis57. Cohort studies of East Asian men corroborated these results and established NAFLD as a risk factor for type 2 diabetes, even in nonobese individuals63. Accumulated hepatic lipids, formed either by DNL or from free fatty acids, have been linked to the onset of hepatic insulin resistance. A positive correlation between high hepatic/visceral fat and hepatic insulin resistance was found in NAFLD patients65. In rats, 2,4-dinitrophenol (DNP), a drug that promotes β-oxidation in hepatocytes, reduced the hepatic lipid content and improved insulin sensitivity66. Conversely, systemic insulin resistance can cause hepatic lipid accumulation in NAFLD. Insulin-resistant adipocytes have higher rates of lipolysis, releasing excessive free fatty acids into the circulation that are incorporated into liver lipids. Another insulin-mediated mechanism is DNL upregulation. Glucose uptake in skeletal muscle and adipose tissue is dependent on insulin, while in hepatocytes, glucose uptake is largely insulin-independent67. Thus, this excessive influx of glucose into hepatocytes can cause steatosis via DNL68.Systemic insulin resistance is a key component in NAFLD, as patients have decreased insulin sensitivity across adipocytes, hepatocytes, and skeletal muscle69. This finding indicates that the quality of accumulated lipids rather than their quantity matters, as further discussed below.However, it is noteworthy that hepatic lipid accumulation does not always share a causal relationship with insulin resistance. Overexpression of diacylglycerol acyltransferase 2 (DGAT2), an important enzyme in the final production of TG synthesis, raised hepatic TG levels and caused steatosis but did not result in hepatic or systemic insulin resistance70. In this model, the accumulation of TGs is thought to dysregulate lipid flux throughout the liver in this “first hit”. However, Listenberger et al. suggested that TG formation may be a form of protection against lipotoxicity from free fatty acids71. The results from a similar study with obese Dgat2-knockout mice with NASH also support the findings of Listenberger et al., as a decrease in hepatic TG stores failed to dampen oxidative stress signals or NASH progression72.As previously mentioned, not every case of NAFLD leads to chronic inflammation, hepatocyte apoptosis, and cirrhosis. As such, there must be a metabolic distinction between individuals with steatosis and those with progressive degeneration. The development of NASH and cirrhosis from NAFLD has been described by the “multiple-hit” hypothesis. The “first hit” of insulin resistance leads to lipid accumulation in the liver, which makes the organ more susceptible to the secondary “multiple hits” of oxidative stress and inflammation74. The conversion of lipotoxic SFAs to MUFAs is mediated by stearoyl-CoA desaturase-1 (SCD1). Free fatty acid influx upregulates SCD1 expression, resulting in MUFA formation, TG storage, and ultimately, liver adaptation with no progression beyond steatosis. However, SCD1 downregulation triggers mitochondria-mediated apoptosis through death receptor signaling, ER stress, and the release of proteases in the cytoplasm from increased lysosomal permeability74. Identification of key lipotoxic lipid species  and associated metabolic and signaling pathways is thus crucial to prevent disease progression from NAFLD to NASH.Even more than the level of fatty acid influx to the liver, the nature of these fatty acids may contribute to lipotoxicity. A higher ratio of saturated fatty acids (SFAs) to monosaturated fatty acids (MUFAs) has been connected to NASH development58. In obese individuals with type 2 diabetes, regulation of adipocyte lipolysis is largely absent, which promotes the release of fatty acids into the circulation. An increase in fat deposits, particularly an increase in the mass of visceral adipose tissue, further contributes to the influx of free fatty acids into the liver75. Yamaguchi et al. found that the suppression of TG production failed to stop liver damage or fibrosis in obese mouse models, suggesting that increased adipocyte lipolysis and the resultant influx of fatty acids into the liver make a large contribution to NASH pathologies72. Quantitatively, the distribution of visceral fat in the body is marginal compared to the abundance of subcutaneous fat. However, large visceral deposits in obese women have been linked to suppression of the antilipolytic effect of insulin, and increased levels of free fatty acids have been found in the hepatic portal in individuals with upper body obesity76. Although the mechanism by which visceral adiposity selectively contributes to NAFLD remains contentious, the close proximity of visceral fat to the portal circulation may facilitate the delivery of free fatty acids and inflammatory factors  to the liver77.While some NAFLD patients are lean, obesity is still prevalent in individuals with NAFLDWhile there are many enzymes in the DNL pathway, only a few of them are altered in NAFLD patients and are potentially targetable for NAFLD treatment without detrimental side effects. Here, among these enzymes, we focus on two crucial lipogenic enzymes that have been extensively studied for therapeutic intervention.79. ACLY silencing, inhibition, and genetic deletion were found to suppress cell proliferation and tumor growth in several preclinical models85. One recent study on atherosclerosis also found that Acly deletion in macrophages stabilized atherosclerotic plaques—a necessary preventative treatment for stroke—and attenuated the ACLY-mediated inflammatory response in the macrophages. These phenotypes were also linked to dysregulated cholesterol and fatty acid synthesis86.ACLY is a critical enzyme in DNL that converts cytosolic citrate into acetyl-CoA to provide carbon sources for DNL Fig. . Acetyl-ACLY has been linked to NAFLD in both humans and mice. Dietary carbohydrates (both glucose and fructose) increase the mRNA expression of Acly in mice and rats87. In NAFLD patients, this diet-mediated ACLY induction occurs mainly through SREBP-1c88, but a recent mouse study showed that ChREBP is also responsible for fructose-induced expression of ACLY and other glycolytic, fructolytic, and lipogenic genes89. Furthermore, ACLY is an essential contributor to global histone acetylation. Under nutrient-rich conditions, ACLY is the primary enzyme that mediates the production of glucose-derived nuclear acetyl-CoA, the source of histone acetyl groups80. ACLY-dependent histone acetylation has also been linked to selective control of key genes involved in glucose usage in adipocytes, including phosphofructokinase (PFK) and glucose transporter 4 (GLUT4)90.Abnormally elevated expression of ACLY is transcriptionally upregulated in NAFLD, posttranslational regulation of ACLY may also be an important contributing factor to disease pathology. ACLY nuclear localization is activated by phosphorylation at residue S455 by protein kinase B (AKT)92. ACLY is also phosphorylated at this site by protein kinase A (PKA), which significantly increases ACLY enzyme activity (6-fold increase in Vmax compared to that of nonphosphorylated ACLY)93. Recently, BDK, a kinase of branched chain ketoacid dehydrogenase (BCKDH), was shown to phosphorylate ACLY at the same serine residue independent of AKT and is also upregulated by ChREBP94. Small molecule-mediated inhibition of BDK improved hepatic steatosis and the insulin response in insulin-resistant Zucker fatty rats. Furthermore, overexpression of BDK increased ACLY phosphorylation and hepatic DNL.While it is evident that 96. Both mice and humans with NAFLD showed increased levels of not only total ACLY but also acetylated ACLY. Furthermore, while Acly knockdown suppressed steatosis in mice fed a high-fat, high-sugar diet, high levels of ACLY 3KQ (mimicking the constitutively acetylated form) induced steatosis more strongly than overexpression of ACLY wild-type96. Mechanistically, ACLY acetylation increases enzyme stability by antagonizing its ubiquitylation96. At least two responsible E3 ligases have been identified to negatively regulate ACLY98. Overexpression of one of these E3 ligases, HMG-CoA reductase degradation protein (Hrd1), decreased both acetyl-CoA and ACLY-mediated DNL in the hepatocytes of db/db and ob/ob mice97. This effect ameliorated hepatic lipid droplet accumulation and decreased hepatocyte inflammation in vivo. Hrd1 expression was also negatively correlated with NAFLD in mice, indicating that failure to activate the ubiquitin pathway and the subsequent accumulation of ACLY contribute to NAFLD development. More broadly, the proteosome-mediated pathway seems to prevent inflammation-dependent proteotoxicity in NAFLD99.In addition to its phosphorylation, the acetylation of ACLY seems to play important roles in NAFLD. In hepatocytes, acetylation at three lysine residues has been shown to increase ACLY enzyme stability, resulting in constitutive activation of DNL100. In one rodent model, BemA relieved atherosclerotic symptoms and lowered LDL cholesterol and triglyceride levels in ApoE-knockout and ApoE/AMPK β-1 double-knockout mice101. These results suggest that BemA regulates lipid metabolism and protects against LDL cholesterol and inflammation regardless of AMPK activation in the liver. As a prodrug, BemA also requires bioactivation by very long-chain acyl-CoA synthetase-1 (ACSVL1), an enzyme primarily found in the liver102. This mediation by hepatic ACSVL1 may thus circumvent muscle effects from statin usage. Current data for BemA usage in humans are primarily directed toward the treatment of atherosclerosis over NAFLD; nevertheless, phase III clinical trials have suggested positive outcomes in lowering LDL cholesterol levels when BemA is administered in conjunction with other therapeutics103.Statins, a class of HMG-CoA reductase inhibitors, have traditionally been used to attenuate atherosclerosis by reducing low-density lipoprotein (LDL) cholesterol levels. However, the emergence of small-molecule therapeutics targeting ACLY has presented new treatment options for individuals particularly sensitive to statin-related muscle symptoms or those who require a more robust reduction in LDL cholesterol. Bempedoic acid (BemA/ETC-1002), a recently FDA-approved LDL cholesterol-lowering drug, is one such therapeutic that has been singled out for ACLY inhibition; BemA was found to reduce lipogenic intermediates such as acetyl-CoA, malonyl-CoA, and HMG-CoA while subsequently increasing citrate in rat hepatocytesAcly-knockout mouse cell lines80. Importantly, acetate was found to be a requirement for maintaining viability in Acly-knockout cells by supporting cellular acetyl-CoA levels and DNL80. Mice heterozygous for Acly also did not show fluctuations in lipid concentration or synthesis, suggesting that the lipogenic program proceeds normally at half ACLY protein levels104. ACSS2 is one enzyme that may explain this rescue of lipogenic capacity via its involvement in an alternative acetyl-CoA-generating pathway  are required. Most studies that support the causal link between the microbiota and NAFLD have reported the results of fecal transplantation from donors fed NAFLD-inducing diets138, which are summarized in Table Given that the liver is directly linked to the intestine via the portal blood and that the gut microbiome is a major source of acetate, it is not surprising that the gut microbiome plays important roles in NAFLD. Sequencing studies indicated specific changes in microbiota composition in NAFLD subjects. Healthy versus obese/NAFLD individuals showed marked population differences in members of the phyla Bacteroidetes and Firmicutes138. Increases in cecal SCFA concentrations have been found in obese mice with a concurrent similar increase in the Firmicutes:Bacteroidetes ratio, supporting the clinical findings140. While studies often report inconsistencies in the Firmicutes to Bacteroidetes ratios in obese and NAFLD patients, increased SCFA levels are consistently reported141. Another potential factor is microbiome diversity. One comparative study of 154 monozygotic and dizygotic twins that were incongruous for obesity found that the twin with obesity exhibited higher Firmicutes levels and an overall decrease in gut flora diversity142. It is thus possible that this diminished microbiome diversity, rather than specific microbial populations, contributes to NAFLD pathogenesis.How do fructose-mediated microbial population shifts affect the onset of NAFLD? One clinical study found a link between higher SCFA concentrations and an increased Firmicutes:Bacteroidetes ratio in obese and overweight individuals143. Another study found that infusion with SCFAs lowered free fatty acids while increasing serum TG and cholesterol levels144. Isotopic tracing of cecum-infused SCFAs confirmed a significant increase in the incorporation of SCFAs into glucose, cholesterol, and lipids145. Acetate, the most abundant SCFA, is preferentially utilized by hepatocytes via ACSS2 to fuel DNL. Zhao et al. found a twofold increase in acetate after acute fructose feeding in portal blood but not systemic blood, indicating that acetate is efficiently cleared by the liver37. The authors further found that depleting the microbiome using antibiotics or silencing hepatic ACSS2 markedly blocked fructose conversion into hepatic acetyl-CoA and fatty acids. Intriguingly, fructose-induced lipogenic gene expression was intact, consistent with the notion that proximal steps of fructolysis provide a signal to transcriptionally activate the DNL program. While involved in a range of various metabolic pathways, SCFAs also have an established role as signaling molecules via G-protein coupled receptors (GPCRs)147. In the liver, RGS5, a protein that suppresses these GPCRs, suppresses NAFLD/NASH pathology via downregulation of lipogenic genes and inflammatory cytokines in vivo148. Thus, targeted GPCR therapeutics may represent an alternative to chronic antibiotic usage in the treatment of NAFLD/NASH.One interesting development in the field is the idea that excessive microbiome-derived SCFAs feed hepatic DNL, thereby triggering NAFLD. However, different SCFAs likely have different metabolic fates, with primarily acetate feeding DNL and propionate and butyrate feeding gluconeogenesis. In one clinical study, healthy fasted individuals who received acetate and propionate did not show increases in serum free fatty acids, presumably due to hepatic glucose production rather than DNL to compensate for fasting-induced hypoglycemia150. Additionally, in contrast to what it does in the liver, acetate in the gut-brain axis has been shown to suppress appetite by upregulating γ-aminobutyric acid (GABA) activity in the hypothalamus and was thus flagged as a possible therapeutic for obesity151. The exact roles of propionate and butyrate in the liver are also under contention. Butyrate has been shown to robustly promote the mevalonate and fatty acid synthesis pathways by yielding intermediates such as HMG-CoA153. Propionate, on the other hand, inhibits the incorporation of acetyl-CoA into fatty acids without reducing cytosolic citrate levels, suggesting that it may compete with acetate for CoA and thereby suppress DNL and drive odd-chain fatty acid synthesis155. To make matters more complex, Weitkunat et al. found that the addition of both propionate and high amounts acetate in a long-term high-fat diet decreased the liver TG content and DNL gene expression, suggesting that SCFAs may play a protective role against steatosis156. Therefore, the role of SCFAs in NAFLD is likely context-dependent, and more systematic investigation is required.In contrast, some studies proposed the beneficial effects of SCFAs in reducing adiposity and restoring insulin sensitivity in obese mice157. Bacterial endotoxins induce adipocyte lipolysis via Toll-like receptor 4 (TLR4) signaling and increase serum-free fatty acids158. Elevated free fatty acid levels have been linked to impaired insulin signaling159, and serum fatty acids that enter the liver can be incorporated into TGs, contributing to steatosis and insulin resistance161. A recent study further supported the notion that fructose-fed mice developed NAFLD/NASH phenotypes as a result of intestinal barrier deterioration and TLR signaling162. Antibiotic treatment blocked intestinal tight junction degeneration and lowered inflammatory hepatic cytokines and chemokines. Interestingly, antibiotics also suppressed DNL induction. Gut-mediated inflammation and endotoxemia may also explain NASH development. The progression of NAFLD into NASH has been described in the previous sections by the ‘multiple hit’ hypothesis, in which the first hits of lipid accumulation and insulin resistance are exacerbated by inflammation, leading to further hepatic injury. It is thus possible that fructose-induced gut permeability increases the liver’s susceptibility to these ‘second hits’ of systemic inflammation from serum endotoxins.In addition to supplying lipogenic substrates via the microbiome, fructose itself causes intestinal damage to induce NAFLD/NASH. Fructose feeding has been shown to elevate plasma endotoxin levels, presumably due to increased oxidative stress and decreased intestinal junction integrity126. It is possible that the concentration of fructose administered in animal studies is beyond the concentrations received by typical human consumption, causing chronic and systemic changes. Conversely, these phenotypes may also be seen in humans if high fructose consumption proceeds long enough. Therefore, while understanding of the relationship between the microbiome, fructose, and hepatotoxicity in animal models has increased, more human data are necessary to draw the same conclusions regarding human NAFLD pathologies.However, the human relevance of fructose-induced gut leakiness and microbial dysbiosis is still controversial. A recent clinical study showed that obese participants given a daily dose of 75 g of fructose over a course of a few weeks displayed no gut permeability or endotoxemia164. Some dietary fibers are resistant to digestive enzymes and therefore neither hydrolyzed nor absorbed until they reach the colon. Depending on their physicochemical characteristics, dietary fibers are subdivided into insoluble and soluble dietary fibers165. Insoluble fibers, such as cellulose and lignin, are barely fermented by the gut microbiota and thus have a fecal bulking effect166. In contrast, soluble fibers, such as inulin, β-glucan, pectins, and maltodextrins, are readily fermented to produce SCFAs167.As described above, acetate is the preferential substrate of hepatic DNL. Even in the absence of fructose, copious acetate is produced by the microbial fermentation of dietary fibers. Dietary fibers, carbohydrate polymers with three or more carbohydrate units (primarily glucose or fructose), are naturally found in fruits, vegetables, and grains168. In general, a diet containing more soluble fiber induces a higher concentration of acetate in fecal samples169, and this acetate readily feeds hepatic lipogenesis171. The roles of fiber-derived propionate and butyrate are less understood, but their presence in excessive amounts can induce insulin resistance172 or global gene alterations in colonocytes or hepatocytes, as butyrate can inhibit histone deacetylases (HDACs)177. Butyrate may thus provide a mode of epigenetic control. In colonocytes, the accumulation of butyrate has been shown to have a therapeutic effect as an HDAC inhibitor and suppresses colon tumorigenesis179. However, whether fiber-derived butyrate can reach millimolar concentrations to have any effects in hepatocytes is unclear.The concentration of SCFAs produced by the gut microbiota is largely influenced by the composition and type of dietary fibers in food181. In animal studies with a fructose-rich diet, supplementation with inulin significantly reduced blood TG and cholesterol levels183. Similar effects were also observed in clinical studies. In a dietary intervention study, increasing inulin intake reduced blood TG levels and fat mass in obese children184. Twelve healthy participants who consumed cereal containing high amounts of inulin for four weeks showed significantly lower plasma TG and cholesterol levels than the control group185.Despite the potentially detrimental effect of dietary fibers through increased hepatic lipogenesis and perturbation of signaling pathways, certain types of dietary fibers reduce the risk of metabolic diseases186. On the other hand, in type 2 diabetic rats, acetate supplementation reduced hepatic TG accumulation and improved glucose tolerance by decreasing SREBP-1187. SCFA supplementation has also been implicated in the prevention of CVD. Acetate significantly reduced blood pressures, cardiac fibrosis, and left ventricular hypertrophy in a mouse model of CVD induced with deoxycorticosterone, and a high-fiber diet generated consistent improvement188. This protective action of dietary fibers and acetate likely involves downregulation of CVD-associated genes, such as early growth response protein 1 (Egr1).To determine the causal relationship between fiber fermentation and improved metabolism, several studies have focused on the impact of an SCFA-enriched diet. Supplementation with propionate and butyrate improved glucose homeostasis in both clinical and animal studies189. A high-inulin diet was also shown to worsen liver cancer. Feeding TLR5-deficient mice a 7.5% inulin diet caused cholestasis, hepatic inflammation, and hepatocellular carcinoma190. Germ-free or antibiotic-treated mice did not show these phenotypes, indicating that microbiota dysbiosis was involved. This intriguing idea of context-dependent fiber effects on lipogenesis and liver pathology merits further investigation.However, other recent studies have indicated that dietary fibers can be detrimental. In a randomized clinical study, Chambers et al. compared the effect of inulin or inulin-propionate ester consumption in NAFLD patients. In contrast to the authors’ prediction, after 42 days of 20 g/day inulin supplementation, the subjects showed significantly increased intrahepatocellular lipid accumulation. The provision of inulin-propionate ester did not have this effect. Given that NAFLD patients exhibit high levels of hepatic DNL, the authors speculated that acetate derived from the high-inulin diet provided excessive carbon for increased DNL, while propionate supplementation suppressed it by competing with acetate for CoATo conclude, despite the widespread belief in the beneficial effects of dietary fibers, the composition of dietary fibers and health status of the individual  are likely critical factors that determine the impact of dietary fibers on metabolic health. This notion supports the increasingly popular notion of personalized medicine and diet. Future studies using isotope tracing and metabolomics to define the metabolic fates of various dietary fibers and their byproducts in diverse pathophysiological contexts will expand our understanding of the link between fibers, their fermentation products, and NAFLD onset.A recent alarming increase in NAFLD patients and patients with associated detrimental metabolic diseases has sparked extensive research in the field. Numerous dietary and genetic animal models have been prepared to recapitulate the human pathology, and the use of these models has identified several new metabolic pathways involved in the disease process. Among them, increased hepatic lipogenesis driven by a high-carbohydrate diet and microbial metabolism/dysbiosis have been repeatedly reported in NAFLD. Accordingly, targeting hepatic lipogenesis via inhibition of a specific lipogenic enzyme or limiting the supply of lipogenic carbon substrates has been performed in clinical trials and achieved some promising results. In addition to the further development of therapeutic strategies, a more detailed understanding of the complex mechanisms of NAFLD pathogenesis in various genetic and dietary backgrounds  will explain why some people are more susceptible to NAFLD than others. This will also expedite the development of personalized dietary guidelines and pharmaceutical interventions. Finally, identifying accurate and early diagnostic markers of NAFLD in the blood and/or fecal samples is another crucial future research avenue.


Genomic integrity is constantly insulted by solar ultraviolet (UV) radiation. Adaptative cellular mechanisms called DNA damage responses comprising DNA repair, cell cycle checkpoint, and apoptosis, are believed to be evolved to limit genomic instability according to the photoperiod during a day. As seen in many other key cellular metabolisms, genome surveillance mechanisms against genotoxic UV radiation are under the control of circadian clock systems, thereby exhibiting daily oscillations in their catalytic activities. Indeed, it has been demonstrated that nucleotide excision repair (NER), the sole DNA repair mechanism correcting UV-induced DNA photolesions, and ataxia–telangiectasia-mutated and Rad3-related (ATR)-mediated cell cycle checkpoint kinase are subjected to the robust control of the circadian clock. The molecular foundation for the circadian rhythm of UV-induced DNA damage responses in mammalian cells will be discussed. Everlasting day-night cycles and the accompanying environmental oscillations have placed evolutionary pressure on most organisms on Earth. To accommodate these recurring environmental changes, organisms have been equipped with an internal oscillating system to keep track of light-dark cycles, the “circadian clock system.” The evolutionarily conserved feature of the circadian clock across most living organisms strongly suggests that it must confer selective advantages .Rhythmic oscillations in physiology and behavior with a period close to 24 h are called circadian rhythms, which are generated by the cell-autonomous molecular clock operating virtually in every single cell . This trUltraviolet (UV) radiation from sunlight consists of three different wavelengths termed UVA (315–400 nm), UVB 280–315 nm), and UVC 100–280 nm). The one with shorter wavelengths generates more DNA lesions efficiently. Indeed, UVB and UVC readily respond to DNA to produce genotoxic photoproducts such as cyclobutane pyrimidine dimers (CPD) and pyrimidine–pyrimidone 6-4) photoproducts (6-4PP) -4 photop,8. Indee0–315 nm,0–280 nm.Nucleotide excision repair (NER) is the sole and error-free DNA repair mechanism capable of correcting UV-induced DNA photolesions in placental mammals . Based oNER is exceptional among the DNA repair systems in its ability to eliminate the widest class of structurally unrelated DNA lesions, including UV-photolesions, chemical-DNA bulky adducts, reactive oxygen species induced base alterations, and intrastrand crosslinks . The NERIn addition to activating the NER mechanism, UV-photolesions trigger the ataxia–telangiectasia-mutated and Rad3-related (ATR) checkpoint pathway to transiently arrest the cell cycle to allow sufficient time for NER . To stimConsequently, the NER and the ATR pathways are expected to be interconnected intimately to each other to cope with UV-photolesions on genomic DNA. The recent findings on the circadian rhythm of NER and the ATR pathways and possible crosstalk between the two systems will be discussed to shed light on the role of the circadian clock system in UV-evoked DNA damage responses and ultimately to translate it into clinical application for human health.Circadian oscillations of metabolic processes are pervasive and play key roles in ensuring homeostatic balance with the environment to coordinate virtually every aspect of the cellular event, physiology, and behavior . LikewisThe efficiency of NER is essential for the maintenance of genome integrity against UV irradiation in placental mammals, including humans, because it represents the sole system capable of neutralizing the two major UV-photolesions (CPD and 6-4PP) on genomic DNA. Mutations in genes associated with the NER mechanism, consequently, cause a wide range of cutaneous symptoms, from mild solar sensitivity to severe skin cancers .XPA is an essential component of both subpathways for NER, and its transcriptional and post-transcriptional regulation may have significant effects on cellular repair and survival following exposure to UV and UV-mimetic agents . FunctioA decade ago, the circadian rhythm of XPA and NER activity was observed in mammalian tissues. To gain insight into the mechanistic foundation underlying this phenomenon, Kang et al. analyzed the levels of key components consisting of the NER system and revealed that only the level of XPA was governed by the circadian clock, showing a daily oscillation with maximum and minimum levels closely correlating with the NER activity . By demoIn line with this report, a study by Gaddameedhi et al., in which a mouse skin was analyzed for assessment of circadian rhythmicity of NER, also confirmed that the transcription of the XPA gene is regulated positively by the CLOCK-BMAL1 complex and negatively by CRY-PER complex . The peaWith few exceptions for a protein to demonstrate robust circadian oscillation, the gene encoding it must first be transcribed with circadian rhythmicity, and secondly, the protein must have a relatively short lifetime; even if a gene is transcribed with circadian rhythm, it would not show high-amplitude oscillation if it is stable . HERC2, HERC2 is initially reported to function as a scaffold to recruit a RING finger E3 ligase 8 (RNF8) to the sites of double-strand breaks to aid in double-strand break repair . In the ATR has been reported as a binding partner of XPA, and this interaction can substantially increase the NER activity and cell viability in response to UV damage . To thisMeanwhile, it has been reported that XPA is acetylated at lysines 63 and 67 by an unknown acetyltransferase, and this acetylation significantly reduced XPA function in NER by interfering with the XPA-RPA interaction . It is aApart from the UV-photolesion repair, the removal of a chemotherapeutic agent cisplatin-induced intrastrand crosslink is strongly dependent on the circadian clock due to the circadian oscillation of XPA expression . CisplatDNA damage-induced cell cycle checkpoints are signal transduction pathways activated following DNA damage and serve as a genome surveillance mechanism to coordinate multiple cellular pathways for ensuring genomic integrity . The ATRApart from their canonical clock function, the mammalian clock factors have also been implicated in diverse noncanonical functions such as the DNA damage response (DDR), which includes cell cycle checkpoint ,61 and DDKO) are arrhythmic under constant darkness [The clock repressor cryptochromes (CRYs) are indispensable for molecular clockwork  and robudarkness . IntriguDKO cells retained substantial ATR activity compared with clock-proficient wild-type cells, although the CRY1-modulated rhythmic ATR activity was abolished in CRYDKO cells. The molecular foundation of this phenomenon is driven by the temporal interaction of CRY1 and TIM in the nucleus due to the cyclical expression of CRY1 protein. Depending on the level of nuclear CRY1, the differential ATR activity was observed, eventually generating a circadian oscillation of ATR activity. And when CRY1 was ectopically expressed in CRYDKO cells, the rhythmic ATR activity was recurred. Importantly, the significantly altered ATR activity in mouse liver that was intraperitoneally injected with cisplatin at different circadian times was detected, which consequently affected the removal rate of cisplatin-DNA adducts from genomic DNA in the liver. Collectively, this study demonstrates the intimate interaction between the circadian clock and the ATR pathway during genotoxic stress in clock-ticking cells. In line with the story, CRY1 has recently been characterized as a protumorigenic factor that promotes most DNA repair mechanisms  and cell survival through temporal transcriptional regulation, nominating CRY1 as a new therapeutic target [The ATR activity measured by the level of the phosphorylation status of ATR substrates such as CHK1 and MCM2 exhibits circadian rhythm. Interestingly, clock-deficient CRYc target .Several studies have already demonstrated that UV-induced ATR activation is dependent on NER in the quiescent cells ,74. The The melanocortin 1 receptor (MC1R) regulates pigmentation, adaptive tanning, and melanoma resistance by activating adenylyl cyclase which accumulates intracellular cyclic AMP (cAMP) levels . Upon UVImportantly, a defective NER activity exclusively during the S phase of a majority of human melanoma cell lines is speculated as a result of decreased ATR signaling, which may constitute an unrecognized determinant in melanoma pathogenesis .In the last 20 years, most of the core factors dedicated to the NER mechanism have been elaborately described, shifting attention to the mechanisms that facilitate NER in a spatiotemporal context and to cooperative interactions between NER and other signaling pathways. While increased NER activity protects the genome against the accumulation of DNA lesions, thereby maintaining genome integrity, it might be beneficial to reduce the NER capacity in patients with cancer who are undergoing chemotherapy, because doing so might help ensure the efficient action of DNA damage-inducing drugs such as cisplatin. In that regard, we expect that the transient suppression of NER through the pharmacological manipulation of NER core factors or ATR pathways will synergize with DNA-damaging agents to optimize chemotherapeutic outcomes. A wide range of transcriptional and post-translational regulatory mechanisms of NER factors has been revealed recently, providing attractive targets to adjust the NER threshold . DetaileChronotherapeutics aim at treating illnesses according to the endogenous biological rhythms, which moderate pharmacokinetics and pharmacodynamics of a certain drug . Given t


Findings suggest that telehealth coaching procedures implemented by community-based EI providers resulted in improvements in caregiver identified goals for young children.Coaching has been identified as a best practice for early intervention (EI) services provided through the Individuals with Disabilities Education Act (IDEA) Part C. The current study describes the establishment and progress of a research-relationship partnership to deliver coaching via telehealth during the COVID-19 pandemic. Community-based EI providers implemented 9-weeks of telehealth coaching and evaluated the extent to which child and caregiver outcomes differed between families that had previously received in-person services versus telehealth only. Four EI providers completed the intervention with n=17 families of children aged 6-34 months during the pandemic (April-August 2020). We used the Canadian Occupational Performance Measure (COPM) and Goal Attainment Scaling (GAS) to collect outcomes on caregiver identified goals; we used Wilcoxon Signed Rank Tests to examine pre- to post-intervention data. Results showed significant improvements in parent satisfaction, child performance, and goal attainment (all  Due to the COVID-19 pandemic, telehealth rapidly expanded as a service delivery model across state early intervention (EI) systems. Telehealth is the use of information and communication technology (ICT) to support access to care . While ICT may encompass both synchronous and asynchronous methods to deliver care, we refer to the use of synchronous videoconferencing methods as ‘telehealth’ within this article. Extant evidence shows that EI positively impacts children's developmental trajectories e.g.,  and famiEarly childhood coaching  has beenResearch shows that telehealth may be an efficacious model to serve families in EI ; 2011, iThe COVID-19 pandemic presented the opportunity to research the efficacy of a rapid expansion of telehealth practice from a community-based perspective. To date, the number of effective interventions for children with disabilities far outweighs the number of interventions actually used in practice, and it often takes an average of 17 years before evidence-based practices are used in practice . To reduOur team, comprised of two researchers and two community-based EI providers, engaged in collaborative efforts to increase the use of early childhood coaching in telehealth. Prior to the COVID-19 pandemic, an EI organization, part of a midwestern state-wide EI network, became interested in expanding their telehealth practice. A collaborative partnership was developed to train an EI occupational therapy practitioner (first author) on the coaching via telehealth model described above. As part of the training, this EI provider was trained to use telehealth to provide EI services for a research study led by the last author, who provided ongoing feedback, including fidelity checks. This EI provider began training other providers in the EI organization after completing the research study. When the COVID-19 pandemic occurred, all EI providers in the state completed coaching via telehealth training provided by the third and fourth authors and received a certificate of completion prior to providing EI services through telehealth in April 2020. At the same time, additional EI providers from this partnering community organization were trained in Goal Attainment Scaling GAS;  and the The current study extends previous studies of coaching and telehealth in numerous ways. First, previous studies used strictly trained interventionists whereas the current intervention was delivered by community-based EI providers that attended a 2.5 hour coaching training and a 1.5 hour outcome measure training, and received one-on-one support from the trained fidelity occupational therapy practitioner. Second, previous studies of coaching have used primarily homogenous samples based on diagnosis e.g., ; the curThe current study was initiated by a community-based EI agency at the onset of telehealth use during the pandemic. Using a permission form developed by the community agency, EI providers asked families if they would like to participate in a data tracking project on parent and child outcomes resulting from participation in telehealth delivered EI services during the pandemic. The community agency then partnered with the research team for training in outcome measures. The research team then used a retrospective data review of families enrolled in telehealth EI services at the onset of the pandemic . The data used for analyses were de-identified and the study was approved by a University Institutional Review Board as a quality improvement project.After participating in training opportunities, four EI providers delivered the coaching intervention, including one physical therapist, two occupational therapists, and one speech-language pathologist. Coaching trainings were rooted in Early Childhood Coaching , DivisioFamilies (n=17) were enrolled in the state EI program; children ranged from ages 6-34 months . Seven (42%) of families were Hispanic/Latino, nine (52.9%) families were White, non-Hispanic and one (5.9%) family preferred to not report race/ethnicity. Eight families received services through Medicaid (47.1%); primary languages included English , Spanish , and other languages . Families that spoke Spanish and other languages used a translator for EI service delivery. Child diagnoses included developmental delay, genetic disorders, and expressive/receptive language delay. Seven families were enrolled in EI during the pandemic and thus only experienced EI services via telehealth, whereas 10 families had received in-person EI services prior to transitioning to telehealth services.EI providers invited families to create specific, measurable short-term goals based on Individualized Family Service Plan (IFSP) outcomes using GAS  and the Demographic measures were collected at baseline, and all interventionists had access to families’ IFSP reports. The below assessments were administered pre- and 9 weeks post-intervention.Goal Attainment Scaling (GAS) is a method to track progress toward goals created by the individual, or in this case the family. Goals are placed on a 4-point scale with a scale from −2 (less than expected) to +1 (much more than expected). When a family identifies a goal for their child, the interventionist asks the family to describe specific child behaviors that would fall on a 4-point scale . GAS demonstrates sound psychometric properties in pediatric focused studies .Canadian Occupational Performance Measure , a criterion referenced measure, assesses client-centered outcomes over time  and the collapsed scores across all goals. The Mann Whitney U tests were used to examine differences between those that had previously received in-person EI services (n=10) versus those that had only experienced telehealth (n=7) across the change scores of COPM child performance, COPM parent satisfaction, and GAS ratings.Descriptive analyses showed that goals included a variety of family centered goals across disciplines see .p<.01) and COPM parent satisfaction scores (p<.01). Across all goals on the COPM, parents reported a mean increase in child performance of 2.97  and a mean increase in parent satisfaction of 2.42 . There was also a significant increase in GAS scores overall (p<.001). Refer to With regard to child participation outcomes see , particiMann Whitney U results showed a lack of significant differences between families that had previously experienced in-person EI services versus those that only experienced telehealth EI services for child performance Z=−1.03, p=.315, parent satisfaction Z=−1.075, p=.282, and GAS scores Z=−1.235, p=.230.Findings suggested that a 9-week coaching intervention delivered through telehealth by trained, community-based EI providers may be effective in promoting child goals and parent reported satisfaction with child gains. Short term coaching interventions have previously shown effectiveness in increasing child participation . In a prAnother novel finding from the current study suggests that there were no significant differences found among families that had received in-home services previously to telehealth and those that had not. These findings coincide with other studies reporting high satisfaction with the use of telehealth by families and therapists ; howeverLastly, data showed that some goals overlapped among disciplines . In EI, providers use a team-based approach and collaboration. Multiple providers may work toward a similar family goal through the lens of their individual discipline. For example, if a family's goal relates to mealtime participation, an occupational therapy practitioner may focus on collaborating with the caregiver on increasing children's self-feeding skills whereas the speech-language pathologist may focus on increasing oral-motor skills. The commonalities demonstrated among goals by EI provider serve as evidence of the collaborative approach using their discipline-specific expertise to achieving family identified goals; the coaching approach likely increases parent capacity to reach goals.First, limitations of the current study include a lack of fidelity measure to track EI providers’ use of the coaching model. Second, each EI provider also had varying knowledge related to their professional training which was integrated within their telehealth sessions; therefore, intervention content varied by provider and by session. Third, we cannot account for any additional services the families were receiving during the intervention window. Fourth, the measures included in this study relied on parent report measures. Lastly, we did not include potential moderating factors in the statistical models such as child age, diagnosis, and potential influence of primary and secondary effects of COVID-19 on family functioning and well-being.Future directions for research would include a larger sample size in order to assess outcomes among a large variety of families. This also includes the examination of factors related to state-wide EI adoption, sustainability, and large-scale measurement of how the coaching model is used when services are provided through telehealth. In order to facilitate large scale adoption of coaching via telehealth in practice, EI networks must consider factors associated with access to technology to ensure widespread access for all families needing services. Further, future research should use a systematic approach from an implementation science framework to better understand the uptake and adoption of evidence-based coaching intervention via telehealth in state-wide EI networks.


Hip osteoarthritis (OA) is characterized by chronic pain, but there remains a mismatch between symptoms and radiological findings. Recently, brain connectivity has been implicated in the modulation of chronic peripheral pain, however its association with perceived pain in hip OA is not understood. We used resting-state functional magnetic resonance imaging (fMRI) to examine functional connectivity associated with pain in hip OA patients. Thirty participants with hip OA and 10 non-OA controls were recruited. Using the visual analogue scale (VAS), pain scores were obtained before and after performing a painful hip activity. All participants underwent 3.0 T resting-state fMRI, and functional connectivity of brain regions associated with pain was determined and compared between participants, and before and after hip activity. Relative to controls, functional connectivity between the secondary somatosensory cortex and left posterior insula was increased, and functional connectivity between the bilateral posterior insula and motor cortices was significantly decreased in hip OA participants. In response to painful hip activity, functional connectivity increased between the thalamus, periaqueductal grey matter and brainstem. Functional connections between brain regions associated with pain are altered in hip OA patients, and several connections are modulated by performing painful activity. Unique lateralization of left posterior insula and linked brain functional connectivity patterns allows assessment of pain perception in hip OA providing an unbiased method to evaluate pain perception and pain modulation strategies. This places limits on how we effectively treat the perceived pain associated with chronic hip OA. Whereas the hallmark signs of joint pathology—degradation and inflammation—are in the periphery, it is unclear why joint pain progresses from local, sharp and episodic, to widespread, dull and persistent. Several studies suggest that pathological changes, evident within the joint afferent neurons6, spinal cord7, and brain circuits9, are distinct features of long-standing OA. Thus, there is increasing recognition that mechanisms within the central nervous system (CNS) play a significant role in the modulation and perception of pain10. Indeed, central sensitization (typified by CNS neuroinflammation and neuroplasticity) characterizes neuropathic-like pain in OA patients16. In one study, pain was reduced and recovery was improved after total knee arthroplasty when patients were treated with the CNS-acting drug, duloxetine, a serotonin–norepinephrine reuptake inhibitor17. Hence, there is a need to better understand the impact of OA on the CNS such that patient-specific therapy can be tailored.The lack of association between radiological indicators of hip osteoarthritis (OA) and self-reported pain by individual patients18.These studies using fMRI report increased activity within the thalamus, posterior and anterior insula, secondary somatosensory cortex, anterior cingulate cortex and periacqueductal grey matter. Wager called this a pain signature within the brain19. Other investigations using fMRI in patients with chronic pain have shown multiple changes, i.e., distinct signatures, in a number of brain regions20. In patients with hand OA, increased activation of the thalamus, anterior cingulate, frontal and somatosensory cortices was observed, relative to controls, when subjects were asked to perform a hand movement task21. Specific changes within the brain circuitry of knee OA patients have been reported to involve the periaqueductal grey matter, ventral tegmental area, and bilateral medial orbital prefrontal cortex22. Most recently, a resting-state fMRI study of knee OA patients following arthroplasty demonstrated changes in the temporal synchrony between the rostal anterior cingulate cortex and rostral ventromedial medulla23 .Functional magnetic resonance imaging (fMRI) has demonstrated changes within brain circuits involved in the perception of pain11. Interestingly, a volumetric brain MRI study of hip OA patients demonstrated evidence of reversal of thalamic atrophy following total joint arthroplasty24, suggesting that CNS changes may be reversible following the removal of the  stimulus of chronic joint pain.With regards to hip OA patients, alterations in periacqeductal gray matter activity in response to skin stimulation have been reportedCollectively, these studies demonstrate a clear involvement of the CNS in OA; however, the pain signatures vary depending on the condition and joint itself. Hence, there is a need to investigate the association between hip OA pain and CNS brain circuity, to determine the potential of CNS-targeted therapies to improve the quality of life of hip OA patients. The present study used resting-state fMRI to explore differences in functional connectivity between hip OA patients and control subjects. Further, changes in connectivity were examined in hip OA patients following an acute painful hip activity.This study was approved by the Conjoint Health Research Ethics Board of the University of Calgary (REB14-2375), and written Informed consent was obtained from all participants. All methods were performed in accordance with the relevant guidelines and regulations. Thirty patients with hip OA requiring total hip replacement were recruited from the Alberta Hip and Knee Clinic, in Calgary, Alberta . As determined by SF-36 questionnaire, collection of a Harris Hip Score and interview, patients were recruited for the study if they had no history of other chronic pain issues , diabetes, neurological or psychiatric disorders, or were using antidepressants or marijuna. None of the patients used regularly prescribed medications for analgesia. Many had intermittently tried nonsteroidal anti-inflammatory agents and acetaminophen with no sustained benefit. The patients were symptomatic for greater than 1 year, many for multiple years before qualifying for total joint arthroplasty.Ten healthy participants  were also recruited and similarly assessed.29 ranging from 0 to 10, where 0 indicates no pain and 10 indicates severe pain. All participants underwent MRI using a 3.0 T GE Discovery MR750 scanner equipped with a 12-channel receive-only phased array head coil . The MRI session consisted of resting-state fMRI using a gradient-recalled echo, echo planar imaging sequence , during which participants were instructed to keep their eyes open and focussed on a fixation cross at the center of a projection screen inside the MRI scanner bore. Anatomical images for registration of the resting-state data were collected using a three-dimensional magnetization-prepared rapid gradient echo sequence . Hip OA participants and controls were removed from the MRI scanner and completed 10 min of physical activity engaging the hip  and were re-assessed for pain using the VAS and for blood pressure, heart rate and oxygen saturation levels. The participants were immediately placed back inside the MRI scanner and underwent a repeated resting-state fMRI scan. VAS scores were compared between baseline and post painful activity by a Wilcoxon Signed-Rank test.For both OA patients and controls, blood pressure, heart rate and oxygen saturation levels were acquired prior to the MRI session. Baseline levels of perceived pain was assessed for hip OA patients using a visual analogue scale (VAS) for painhttp://www.fmrib.ox.ac.uk/fsl/). Using MELODIC (part of the FSL software library), Independent component analysis (ICA) was used to identify time-varying signals arising from artifact due to non-physiological noise, residual non-corrected head motion, and cardiac and respiratory pulsatility, which were subsequently removed by linear regression30. The total number of ICA components reviewed were automatically estimated using MELODIC  of which 4-6 components are identified as artifact. To permit group comparisons, resting-state fMRI images were registered to anatomical images and subsequently to the Montreal Neurological Institute’s (MNI) standard brain template, using a 12 DOF linear registration model. All images were visually inspected to ensure alignment with the brain template was successful using well-established anatomical landmarks .Resting-state fMRI images underwent pre-processing, including skull stripping, motion correction using a six degrees-of-freedom (DOF) rigid body trasnsformation, intensity normalization of the entire 4D volume, slice-timing correction using sinc interpolatiom to account for timing difference of each slice within the TR, high-pass temporal filtering using a 0.001 Hz cutoff, and 6-mm Gaussian kernel spatial smoothing. These steps were performed using FSL image analysis software . Comparisons of functional connectivity of each ROI between hip OA and control subjects as well as between baseline and after painful hip activity were determined using a mixed-effects analysis in FEAT. Results were presented as brain maps of the Z-score of the difference in connectivity, corrected for multiple comparisons using a false-discovery rate (FDR) threshold of 0.05, corresponding to a cluster volume of greater than 322 voxels, as determined by AlphaSim 31, which uses simulations of a null distribution of Z-scores given the spatial smoothness of the images. Other than the MR data, all results are presented as mean ± SD. Comparison between groups was performed using Student’s t-test, with significance level set at p < 0.05.A region-of-interest (ROI) approach was used to analyse the resting-state fMRI data. Specific ROIs were chosen as ‘seed’ regions, to determine their functional connectivity with the rest of the brain and how connectivity differed between subject groups and after physical activity involving the hip. The ROIs included the secondary somatosensory cortex (S2), anterior/posterior insulae and thalamus, as these regions have been shown to be associated with acute onset of painp < 0.001), confirming that the painful hip activity intervention was successful in reproducing the hip pain. Participants’ vital signs were monitored and the changes in the vital sign post exercise are noted in Table The VAS scores of pain for hip OA patients at baseline and after physical activity are shown in Fig. Functional connectivity based on seed regions and their activation comparing OA patients and controls is summarized in Table The connectivity changes in the study patients before and after activity are shown in Table Functional connectivity between the secondary somatosensory cortex S2) and the left posterior insula was significantly greater in hip OA patients than in control subjects . The majority, 88% of right handed people are left hemispheric dominant and 78% of left handed people are also left hemisphere dominant. It would be interesting to speculate if localization of pain to the left posterior insula would hold true for patients who are right hemisphere dominant, i.e. only ~ 7% of left handed people41.The present study demonstrates a difference in functional connectivity of controls and patients with severe osteoarthritis. We observed a unique signature of increased connectivity between the secondary somatosensory cortex and the left posterior insula. The posterior insula and its relationship to nociception has been well describedPost-surgical fMRI studies could also lead to a fuller understanding of the potential reversibility of the observed imaging characteristics, particularly the circuitry involved in central sensitization associated with OA disease. Despite the cost related to MRI studies, the fMRI could be a worthwhile consideration as a tool for patients with chronic OA pain, including persistent post-surgical pain. The present study revealed that resting-state fMRI defines an unusual signature of brain connectivity characteristic of chronic OA hip pain at baseline.42. Our findings are similar to what has been reported for brain activation following acute pain and include nociceptive, emotional and motivational circuitry59. When pain is acute and severe, brain regions involved in emotion, cognition and motivation are predominantly activated60.Following acute pain brought on by painful hip activities, we observed patterns of altered interconnectivity of multiple brain regions sometimes referred to as the pain matrix49. As the same VAS was used repeatedly on each patient, before and after painful hip activities, each patient served as their own comparator for this measure of clinical hip pain. The documentation of brain connectivity afforded by the resting state fMRI analyses used here is based on an unbiased statistical methodology. The choice of predetermined ROIs permitted an hypothesis-based analysis; however, there may be other brain regions involved that are not revealed using our approach.The connectivity results presented here need to be considered in two phases: (a) connectivity differences between OA and control patients, and (b) connectivity differences in OA patients before and after provocative hip tests. Presumably, connectivity differences at baseline between OA and controls reflect neuroplastic changes that occur in response to chronic OA hip pain, which typically takes months or years to develop, conceivably in a timeframe that corresponds in some way with development of hip OA pathology. In contrast, changes from baseline connectivity detected in OA patients after provocative hip tests infers activation of pre-existing neural connections in the brain rather than structural neuroplastic changes. This later type of brain activation is analogous (but not homologous) to brain activation in BOLD fMRI images in response to a noxious stimulus . While these two connectivity signals are not mechanistically congruent nor directly comparable, they represent valid and intriguing brain signals corresponding to hip OA pain. As such, this resting-state fMRI approach would seem to be advantageous for evaluating both spontaneous (baseline) and evoked pain that in turn assess two important aspects of pain-circuit connectivity: development and activation.While we present novel observation, the study does rely on a few assumptions and limitations. An important limitation is that all but one patients were right handed. In addition, the VAS methodology is based on subjective self-reporting by patients. While known to be variable, this methodology is widely used and has been shown to have validity for studies of OA pain61. Future work should examine this component of change as well.One final limitation that can be recognized is that this assessment is only measuring functional connectivity and has not assessed structural changes in the brain. Baliki and coauthors have shown that different clinical conditions have different patterns of structural change in the brain, for example Chronic low back pain showed reduced whole-brain gray matter volume, whereas 2 other chronic pain conditions did not, Chronic Regional Pain Syndrome and Knee OAIn summary, the resting-state fMRI of patients with hip OA relative to controls demonstrated greater connectivity between secondary somatosensory cortex and the left posterior insula. Connectivity was reduced between the posterior insula and the hip area of the primary motor and sensory cortices. We believe that this is a resting-state fMRI signature for OA of the hip with chronic pain, and indeed may hold an important clue in understanding pain pathways in patients with chronic OA of the hip. Similar to other reports, we also demonstrated connectivity changes after painful hip activity.


Nasopharyngeal carcinoma (NPC) represents a molecularly paradigmatic tumor given the complex diversity of environmental as well as host dependent factors that are closely implicated in tissue transformation and carcinogenesis. Epstein Barr Virus (EBV) plays a key role in tissue invasion, hyperplasia and malignant transformation. Therefore, EBV related oncoviral proteins such as Latent Membrane Protein family , Epstein Barr Nuclear Antigen 1 (EBNA1) and EBV related glycoprotein B (gB) are responsible for inducing intracellular signalling aberrations leading to sustained proliferation and further acquisition of NPC related invasive nature and metastatic potential.Dysregulation of proteasome signaling seems to be centrally implicated in oncoviral protein stabilization as well as in modulating tumor microenvironment. Different studies in vitro and in vivo suggest a potential role of proteasome inhibitors in the therapeutic setting of NPC. Furthermore, alterations affecting proteasome signalling in NPC have been associated to tumor growth and invasion, distant metastasis, immune exclusion and resistance as well as to clinical poor prognosis. So on, recent studies have shown the efficacy of immunotherapy as a suitable therapeutic approach to NPC. Nevertheless, novel strategies seem to look for combinatorial regimens aiming to potentiate immune recognition as well as to restore both primary and acquired immune resistance.In this work, our goal is to thoroughly review the molecular implications of proteasome dysregulation in the molecular pathogenesis of NPC, together with their direct relationship with EBV related oncoviral proteins and their role in promoting immune evasion and resistance. We also aim to hypothesize about the feasibility of the use of proteasome inhibitors as part of immunotherapy-including combinatorial regimens for their potential role in reversing immune resistance and favouring tumor recognition and eventual tumor death. Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated malignancy arising from the nasopharynx. It displays remarkable characteristics regarding ethnic and geographic distribution, including higher prevalence in Southeast Asia, South China, Northern Africa, Greenland and Alaska –3.Malignant transformation and tumor progression of NPCs are directly related to environmental factors, lifestyle, dietary habits, latent EBV infection, local inflammation, host immunity, and genetic and epigenetic alterations –10 as brA combined modality of concurrent chemo-radiotherapy based on platinum regimen is the main approach for locally advanced NPC, showing an improvement in overall survival, although there is a relatively high chance of recurrence in non-metastatic NPC –16. GiveEBV is a human gamma-herpes virus which establishes lifelong asymptomatic infections in more than 90% of individuals worldwide –22. ThisFour major types of EBV latent infections have been identified. NPC exhibits frequently the type II latency infection characterized by expressing several non-coding RNAs such as EBERs, LMP1, LMP2, BARTs and EBNA viral proteins –30. On tProteasomes are responsible for the degradation of ubiquitinated proteins, and its role has been extensively documented in EBV-positive NPCs. Ubiquitin system is responsible for protein processing and degradation. In this sense, targeted proteins are primarily poly-ubiquitinated and eventually degraded by the 26S subunit. This process is mediated by consecutive activation of several enzymes, namely ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3), which may exert protein poly-ubiquitination, enabling further proteasome recognition and eventual proteolysis –36.Cancer cells are highly dependent on proteasome-mediated degradation because of their intrinsically accelerated tumor turn-over. Thus, therapeutic efforts have been directed so as to promote direct proteasome inhibition which may in turn facilitate intracellular protein accumulation. An excess of intracellular misfolded protein-burden leads to further endoplasmic reticulum stress and cellular death. EBV induced proteasome truncation seems to be centrally implicated in viral replication as well as in EBV-induced carcinogenesis . ConsequGiven the importance of EBV related oncogenesis in NPC as well as the extensively acknowledged role of proteasome associated tumor transformation with further implications in neoplastic proliferation and perpetuation, our aim with this review is to thoroughly assess the molecular basis and oncologic implications of the ubiquitin-proteasome system in NPC as well as its therapeutic significance so far. This may further broaden the clinical and molecular perspective of the rationale beyond the suitability of targeting the ubiquitin-proteasome pathway, both as single therapy and as part of novel combinatorial strategies in the management of NPC.LMP1 (latent membrane protein 1), LMP2A (latent membrane protein 2A), LMP2B (latent membrane protein 2B), EBNA1 (Epstein Barr nuclear antigen 1), BARF1 (Epstein–Barr virus BamHI-A rightward frame 1), as well as two small nuclear RNA namely EBERs , and the heterogeneously spliced group of BART RNAs [NPC is molecularly characterized by EBV mediated type II latent infection. In this scenario, the main genetic expression related to the presence of latent EBV infection is composed by ART RNAs .LMP1 is a transmembrane protein which belongs to the tumor necrosis factor receptor (TNFR) family and is constitutively activated in EBV infected cells leading to further activation of multiple downstream signaling pathways in a ligand-independent manner. Even if variable levels of LMP1 protein may be detected in NPC, incidence of tissue LMP1 mRNA detection has been reported in up to 90% of all cases published so far, which supports its central role in the pathomolecular development of NPC . HoweverOn the other hand, overexpression of TP53, which is widely known to be responsible for cell cycle regulation, is often found in NPC, even though established mutations of TP53 are infrequent in this tumor. Unlike other double-stranded DNA oncoviruses, EBV does not inactivate TP53, however different studies have demonstrated the role of LMP1 which contributes to consecutive activation of NF-kB and AP-1 signaling which leads to further p53 accumulation , 37, 55.In addition to the aforementioned, overexpression of the protooncogene MDM2 has also been widely reported in NPC samples, and its presence has been associated to both EBV infection as well as to tumor metastasis. Besides MDM2 self-ubiquitination, LMP1 has been also described to further increase MDM2 expression by directly regulating the MDM2 ubiquitination. Such phenomenon could be explained as MDM2 ubiquitination occurs in a different linkage manner which shows different functions rather than single degradative signaling via proteasome pathway, which indicated its crucial role in the oncogenic signaling transduction . As a coin vivo [Another EBV related oncoviral protein is Latent Membrane protein 2 A (LMP2A) which has been commonly detected in EBV-positive NPC cells in vivo . In vitrin vivo , 60. In in vivo , and itsin vivo .As mentioned above, EBNA1 (Epstein Barr nuclear antigen 1) represents another EBV-associated key protein which has been shown to be responsible for the regulation of viral DNA synthesis as well as for episome segregation during mitosis . TriptolOn the other hand, EBV related glycoprotein B (gB) is expressed in almost all herpesvirus. This protein is a core component of the viral fusion machinery and plays an important role in viral entry intro epithelial cells by promoting cell-to-cell fusion. This envelope protein is modified by the addition of high-mannose-linked N-glycans, and it has been shown that F-box only protein 2 (FBXO2), an SKP1/Cul1/F-box protein glycan-dependent E3 ubiquitin ligase substrate adaptor attenuate EBV infectivity by targeting N-glycosylated gB via ubiquitin-proteasome pathway . Therefocontinuum in which there is a viral-mediated basal cellular functioning that accounts for different activity rates of intracellular signaling pathways modulated by oncoviral proteins which in turn leads to further instability of physiologic proteasome functioning. Despite the fact that proteasome inhibition might play a role in further stabilization of oncoviral proteins and, therefore, in perpetuating cellular transformation, an established continuum remarks that proteasome does not display a basal physiologic role in tumor cells. Far from that, there is an unbalanced activity of proteasome which further favors oncoviral protein stabilization and activity. Therefore, whole inhibition of ubiquitine-proteasome machinery by proteasome inhibitors may lead to cell destruction by promoting further aggregation of stabilized oncoviral proteins which may in turn perpetuate the activation of intracellular stress cascades and, consequently, it may lead to the eventual activation of proapoptotic signaling and tumor cell death.So on, the role of proteasome in the setting of cellular stability and proteic machinery seems to establish a Despite the high cure rates of NPC, local recurrence as well as development of distant metastasis are still the main causes of treatment failure and represent the main prognostic markers of this disease. In this sense, strong efforts are being developed so as to identify potential molecular markers implicated in the molecular pathogenesis of NPC-related metastasis and tumor recurrence.S100 calcium-binding protein A14 (S100A14) is a member of the multi-functional S100 protein family. Constitutive expression of this protein has been found to be deregulated in several cancer types. To this matter, Meng et al.  showed tOn the other hand, cyclin-dependent kinase inhibitor p27 is widely known to be primarily regulated by the ubiquitin-proteasome pathway . Pan et Pharmacologic approach in tumor cell lines has shown different natural compounds and natural product derivatives with pharmacologic activity as suitable proteasome inhibitors . Thus, cAnother pharmacologic approach studied in the setting of NPC is the use of molecules targeting the Inhibitor of Apoptosis Protein 2 (c-IAP2), as high concentrations of c-IAP2 have been identified in NPC cell lines, supporting the hypothesis that c-IAP2 may play a critical role in the molecular pathogenesis of NPC. To this matter, pharmacologic inhibition of c-IAP2 demonstrated a dramatic decrease of c-IAP2 levels within NPC cells. Further proteomics revealed markedly elevated levels of ubiquitinated c-IAP2 in NPC cell lines previously treated with c-IAP2 inhibitors. Actually, pretreatment of NPC cells with the proteasome inhibitor MG132 or epoxomicin showed to prevent c-IAP2 decrease after c-IAP2 inhibitor treatment. Thus, the authors conclude that pretreatment of NPC cells with c-IAP2 inhibitors may induce proteasome-dependent degradation of c-IAP2 by directly enhancing polyubiquitination of c-IAP2 substrate , 71, 72.In addition to EBV infection and environmental exposures, there are other factors which are centrally involved in the pathogenesis of NPC , 75.BeyoNFKBIA, CYLD, and TNFAIP3. Functional studies also confirmed that inhibition of NFKBIA has a significant impact on NF-kB activity and NPC cell growth. Moreover, an APOBEC mediated signature was described. On the other hand, CYLD (cylindromatosis lysine 63 deubiquitinase), a NF-kB inhibitor, has been described as one of the most frequently mutated genes in NPC. Functional assays indicate that CYLD suppresses NPC tumorogenicity and metastasis by negatively regulating the NF-kB signaling pathway [Among different strategies assessing novel biomarkers in NPC, missregulation of the NF-κB pathway, either by mutation or epigenetic mechanisms, is also involved in cancer pathogenesis. Zheng et al. ,characte pathway . Consequ pathway .ENO1), which has been shown to be overexpressed in NPC [In the line with the above mentioned, NESG1  has been described as a potential tumor suppressor in NPC. NESG1 is specifically expressed in the epithelial cells of nasopharynx and its reduced expression is an unfavorable factor which is related with further promotion of NPC progression and poor prognosis . Interesd in NPC  samples,d in NPC . In thisd in NPC . Thus, eIn addition, the vacuolar protein sorting 33B (VPS33B) is rarely reported in malignant tumors. However, it has been shown that overexpression of VPS33B inhibits proliferation and bypasses chemoresistance to 5-fluorouracil in NPC. Moreover, VPS33B interacts with NESG1 by colocalizing in the cytoplasm. Literature so far has shown that knockdown of NESG1 is able to reverse the inhibitory effect of the overexpression of VPS33B in NPC cells by downregulating the PI3K/AKT/c-JUN-mediated transcription repression. Curiously, VPS33B is downregulated by viral LMP-1 and nicotine, and thus suppresses the proliferation of NPC cells by disrupting its interaction with NESG1 in NPC cells .On the other hand, O6- methylguanine–DNA methyltransferase (MGMT) has been well-characterized to be a therapeutic determinant of O6-alkylguanine alkylating drugs, and it is related to cisplatin resistant in NPC models. Thus, it has been shown that platinum levels after cisplatin exposure is significantly lower in MGMT-proficient cells than in MGMT-deficient ones, and that MGMT protein directly binds to cisplatin-induced DNA damage. Proposed mechanisms suggest that cisplatin-bound MGMT protein became ubiquitinated and eventualy degraded through the proteasome-ubiquitin system. As a consequence, patients who received cisplatin treatment who did show higher MGMT expression level, did also exhibit shorter progression free survival and overall survival compared with patients with lower MGMT expression . TherefoAnother interesting fact is that the expression of ANXA1 and EphA2 is significantly higher in NPC tissues than in normal nasopharyngeal epithelium. Basally, ANXA1 competes with Cbl for binding to EphA2, which in turn increases its stability by inhibiting Cbl-mediated EphA2 ubiquitination and degradation in NPC . Feng etAdditionally, TMEM8B-a protein, which represents an isoform of the NGX6 gene product (Transmembrane protein 8B) has been described as a metastasis suppressor in NPC. TMEM8B-a is rapidly degraded via proteasome pathway which is mediated by Ezrin in many NPC and lung cancer cell lines. However, TMEM8B-a is not ubiquitinated. To this matter, Wang et al. demonstrated that cell lines with stable TMEM8B-a protein expression treated with MG-132 or bortezomib could prevent TMEM8B-a degradation .Following other factors involved in NPC carcinogenesis, the expression of CULLIN 3 is increased in both NPC tissues and cell lines, and correlate with Ki-67 based proliferation index in NPC samples. Moreover, in patients with NPC, the overexpression of this protein is associated with local relapse and distant metastasis. In vitro experiments show that knockdown of CULLIN 3 is related to a reduced proliferation rate, inhibition of tumor cell formation as well as to reduced migratory and invasive capacity of NPC cells. In addition, Zeng et al. also suggested that the CULLIN 3 based ubiquitin proteasome targeting may be a useful and promising therapeutic target in NPC .As a matter of fact, polymorphisms in the genes of histocompatibility locus antigens (HLA) are reported in NPC. These polymorphism might play an important role given the resultant lack of proper immune functions against EBV infection and, eventually, for NPC perpetuation in endemic populations. Chin et al. describe a strong association of HLA-A with NPC susceptibility in a GWAS based study of Malaysian Chinese patients . FurtherIn summary, NPC carcinogenesis is a diverse and molecularly complex process in which, aside for EBV driven molecular alterations, many other EBV-independent environmental as well as molecular factors take part. Untangling these processes seems crucial for their potential implications in different clinical fields, both in prevention, diagnosis and tumor prognosis as well as in the therapeutic setting of NPC.Tumor microenvironment in NPC is highly defined by its immune-tolerant nature which leads to further avoidance of tumor cell recognition and, thus, to immune cell-mediated tumor excytotoxicity. Immunotherapy aims to promote cell to cell recognition by either inhibiting tumor mediated immunologic silencing or by enhancing immunologic recognition of tumoral cells. In this way, since immunotherapy strongly irrupted in Oncology, it has demonstrated promising results in different tumors and, specifically, in NPC , 95–97.RIG-I) as well as melanoma differentiation associated protein 5 (MDA5) are both cytosolic receptors implicated in the type 1 interferon (IFN-I) induced primary immune response which is responsible for viral recognition and destruction. Thus, RIG-I activation has been proposed as a feasible therapeutic strategy in diverse neoplasms [The central implication of EBV in natural pathogenesis of NPC plays a crucial role in terms of immunity and carcinogenesis. To this matter, innate immunity is important for viral recognition and primary host defense. To this matter, Retinoic acid-inducible gene I  expression in diverse tumors, which is known to be a major contributor of tumor-induced immune exclusion  and, theAs mentioned above, immunotherapy (IO) has become an essential among the oncologic therapeutic armamentarium proving to be effective in different tumor types among which NPC needs to be remarked. NPC is characterized by high expression of Programmed Death Ligand 1 (PD-L1) as well as by the abundant presence of Tumor Infiltrating Lymphocytes (TILs) which highlights the immunogenic nature of NPC which makes this disease highly suitable for therapies targeting the immune checkpoint blockade –113. MorAltogether, given the immunogenic nature of NPC as well as its complex pathomolecular characteristics involving EBV infection and EBV-derived cellular alterations, which include proteasome alterations, combination of proteasome inhibitors together with IO may represent a promising therapeutic modality in NPC. Therefore, future studies are granted to elucidate the role of IO-based strategies as well as to study potential therapeutic combinations aimed to look for multi-targeting modalities that may favor tumor response by blocking tumor proliferation at different pathomolecular levels.The use of proteasome inhibitors (PI) is an extended practice in different hematologic tumors and, to this date, several drugs have been developed and approved. The major clinical use of these inhibitors has been linked to multiple myeloma where the firstly approved peptide bortezomib boronate (Velcade®) had a great positive efficacy as a first-in-class agent for this disease. Also, second generation PI have been developed such as carfilzomib , marizomib, ixazomib , oprozomib and delanzomib with promising clinical outcomes. Their toxicity can vary from mild nausea and diarrhea up to a non-reversible peripheral neuropathy leading to drug withdrawal. Furthermore, the study of PI has been developed in other malignancies such as leukemia, lymphomas and solid tumors; even in the setting of GVHD (Graft-Versus-Host-Disease). For example, marizomib is currently being investigated in glioblastoma (NCT03345095), bortezomib plus vandetanib in solid tumors with special focus on medullary thyroid cancer (NCT00923247), bortezomib plus herceptin for advanced breast cancer with HER2 overexpression (NCT00199212) and orIxazomib and selinexor for sarcoma (NCT03880123) among others. A recent phase I trial evaluated the maximum tolerated dose, safety and preliminary efficacy of lapatinib plus bortezomib, in adult patients with refractory solid tumors . AlthougThe novel VLX1570 small molecule which induces inhibition of deubiquitinases has been tested in multiple myeloma, but due to pulmonary toxicity, the study was discontinued . HoweverAs previously highlighted, Pis have been satisfactorily explored among different hematologic malignancies. However, the role of PIs in solid tumors remains under active research. In fact, therapeutic strategies exploring the role of PIs in solid tumors seem of highest interest given the conceptual application of these drugs among solid tumors. Up to now, lung cancer has been the most extensively studied tumor regarding the combinatorial use of PIs. For instance, preliminary clinical approaches using bortezomib as a single therapy in both platinum-refractory small cell lung cancer (SCLC) as well as in non-small cell lung cancer (NSCLC) failed to prove their efficacy. So on, the phase II trial studying the role of bortezomib as a single-agent based therapy in refractory SCLC failed to prove efficacy In this subset of patients with a mean PFS and OS of 1 month and 3 months respectively . MoreoveConclusively, even if proteasome inhibition as single therapy has modestly proven to have no activity among certain solid tumors as in the case of lung cancer, we find highly interesting that the addition of bortezomib to classic chemotherapy as combinatorial or maintenance regimen seems to enhance antitumoral activity over classic approaches. To this matter, we aim to emphasize our idea that combinatorial strategies multi-targeting tumor pathobiology may show promising results in further controlling neoplastic disease. As a result, addition of PIs together with novel therapies, if not extensively studied so far, might add valuable benefit to patients with solid tumors in which proteasome dysregulation may play a role.Following the previous point, and in regard of NPC, few clinical trials have tried to prove the efficacy of PIs in the clinical setting of recurrent or metastatic disease (RM-NPC). The phase II trial conducted by Gilbert J et al  evaluateIn this sense, even if the information on the efficacy and safety of PIs in NPC is far from being satisfactory, we think that, given the advances mentioned above on the evidence about the molecular implication of proteasome in NPC and benefit observed in different solid tumors, the use of PIs should be newly reconsidered due to different reasons. On the one hand, PIs might not be useful as single therapy based regimens, albeit, they could play a role in combination with other novel antitumor therapies such as immunotherapy given the immune molecular background associated with proteasome dysregulation and tumor immunephenotype. On the other hand, combinatorial strategies including different immunotherapy-based modalities together with classic chemotherapy could also represent suitable strategies in the setting of both locally advanced and metastatic NPC.In line with the above mentioned, since classic strategies that have endeavoured the use of PIs as single-agent based therapy have failed to prove its efficacy, the initial development and clinical success in some tumor types, such as multiple myeloma as well as for the promising results of bortezomib in combination with other therapies in solid tumors, different strategies are granted in further developing innovative targets against proteasome. Nevertheless, targeting proteasome as a therapeutic approach in cancer has currently yielded different barriers that need to be eventually overcome.For instance, resistance mechanisms and toxicity profiles of first generations PIs have casted doubts on their usefulness. Despite this fact, current diagnostic strategies together with omic profiling are increasingly playing a key role in developing more precise therapies. Since cancer cells may eventually develop acquired resistance to PIs by different mechanisms, two are the main lines in novel targeting of proteasome inhibition. To this matter, research focused on design and synthesis of novel compounds seems of high interest. Beside novel therapies, implementation of combinatorial strategies together with novel therapeutic approaches such as cancer immune-landscape targeting drugs seems of greatest interest.Regarding acquired resistance to proteasome inhibitors, multiple mechanisms have been reported so far including aberrant expression of ubiquitin proteasome system pathway components, induction of active extracellular drug efflux as well as activation of signaling cascades that avoid apoptosis, thus promoting further cell survival and tumor progression with central implication of eventual upregulation of the Unfolded Protein Response by direct induction of Endoplasmic Reticulum Stress –130. IntOther resistance mechanisms described so far have been recently documented involving both mitochondrial dysfunction  as well In the line with the aforementioned, and despite the lack of activity observed for single agent bortezomib-based clinical trials in solid tumors , 121, coreoviridae have yielded promising results in multiple myeloma which furthermore is associated with cytokine release and proinflammatory changes that may further favor more effective combinations with ICPis [In addition to the aforementioned, other therapeutic strategies targeting immune mediated tumor recognition are those derived from the administration of modified viruses with oncolytic activity which may further be combined with ICPis and from which it has been observed promising activity in the therapeutic strategy of solid tumors . In factth ICPis .Finally, another innovative strategy targeting different neoplasms is Adoptive Cell Therapy (ACT). Primary studies using cytotoxic T lymphocytes (CTLs) with specific affinity for EBV antigen recognition proved to be safe in terms of toxicity and to further induce LMP2 specific immune response . DespiteAltogether, futures perspectives in NPC are under active development. The role of PIs has proved to be limited in single-agent based strategies. However, since combinatorial strategies in solid tumors support the role of PIs for their potential capacity of further enhancing treatment effect, their use in this scenario seems a promising future strategy. So on, current novelties in clinical use of PIs include the development of new compounds with more specific ability of targeting proteasome and to eventually enable suitable bypass to tumor acquired resistance. In addition, recent advances in the clinical approach of NPC based on immunotherapy and immunotherapy based combinations, may grant future studies evaluating the efficacy of combinatorial regimens aiming to elucidate the role of PIs together with chemo and immunotherapy for their molecular implications whitin tumor immunephenotype and lymphocytic exclusion.Molecular pathogenesis of NPC is highly complex and biologically varied. EBV related latent infection plays a central role in tissue damage, cellular hyperplasia and malignant transformation. To this matter, proteasome dysregulation remains a poorly understood, albeit, crucial key pathogenic point of NPC which is responsible for direct modulation of intracellular signaling, thereby perpetuating cellular transformation and uncontrolled proliferation. Therefore, proteasome alterations may interact globally throughout different mechanisms affecting cellular proliferation, nuclear instability as well as immune exclusion.In this scenario, proteasome inhibitors represent a therapeutic strategy that despite its lack of activity among solid tumors in the case of single-therapy based approaches, combination with classic regimens of chemotherapy has yielded promising results as both induction or maintenance treatment. So on, even if literature so far assessing the use of PIs in solid tumors is scarce, mainly given their lack of activity as monotherapy, combinatorial strategies have yielded promising results in recent studies. Therefore, proteasome inhibitors may act as key deregulators of tumor-proteome balance which in combination with other therapies may result highly useful.To this matter, and given the efficacy of PIs in different hematologic tumors, future in clinics brings with it novel compounds aiming to further overcome tumor acquired secondary resistances to first generation PIs. But what is most interesting, given the central role of preoteasome inhibition in favoring immune exclusion and evasion, we hereby present novel perspectives in the therapeutic setting of nasopharyngeal carcinoma that may grant further research activity to elucidate the role of PIs in combination with novel immunotherapy-based modalities as well as the molecular rationale beyond it, that may further support such combinations.