Obesity is an established risk factor for metabolic disease. This study explores the functional complementation of anti-adipogenic phytonutrients for obesity prevention and management. Nine phytonutrients were selected based on their ability to affect the expression of one or more selected adipogenic biomarker proteins. The phytonutrients include berberine, luteolin, resveratrol, fisetin, quercetin, fucoidan, epigallocatechin gallate, hesperidin, and curcumin. The selected adipogenic biomarker proteins include PPARɣ, SREBP1c, FASN, PLIN1, FABP4, and β-catenin. Individually, phytonutrients had variable effects on the expression level of selected adipogenic biomarker proteins. Collectively, the functional complementation of nine phytonutrients suppressed de novo fatty acid biosynthesis via the negative regulation of PPARɣ, FASN, PLIN1, and FABP4 expression; activated glycolysis via the positive regulation of SREBP1c expression; and preserved cell–cell adhesion via the inhibition of β-catenin degradation. In primary human subcutaneous preadipocytes, the composition of nine phytonutrients had more potent and longer lasting anti-adipogenic effects compared to individual phytonutrients. In a diet-induced obesity murine model, the composition of nine phytonutrients improved glucose tolerance and reduced weight gain, liver steatosis, visceral adiposity, circulating triglycerides, low-density lipoprotein cholesterol, and inflammatory cytokines and chemokines. The functional complementation of anti-adipogenic phytonutrients provides an effective approach toward engineering novel therapeutics for the prevention and management of obesity and metabolic syndrome. The current obesity epidemic presents a major challenge to global health management. In the United States, the prevalence of obesity in adults was 41.9% in 2017 . Weight Obesity is characterized by increased adipose tissue mass via hypertrophy, which describes an increase in the size of existing fat cells or adipocytes, or hyperplasia, which describes the formation of new adipocytes from precursor cells or preadipocytes . AdipogeThis study explores the functional complementation of anti-adipogenic phytonutrients for weight management. Automated and multiplexed proteomic methods are used to screen phytonutrients for their anti-adipogenicity by affecting the expression of one or more adipogenic proteins in primary human subcutaneous preadipocytes ,31,32,33Primary human subcutaneous preadipocytes were isolated from the thigh white adipose tissues of a healthy Caucasian female who was forty-three years old and had a BMI < 24.9 . Fat cell differentiation was induced using a previously published protocol . BrieflyThree cell cultures were generated for the screening of each phytonutrient: a cell culture of preadipocytes on d0, a cell culture of differentiating adipocytes on d6 post-differentiation, and a cell culture of differentiating adipocytes on d6 post-differentiation in the presence of phytonutrients. Phytonutrients were added to the complete differentiation media on d0 and d3 post-differentiation. No phytonutrient was present in the maintenance media from d7 to d14. The sources of phytonutrients and their purities are listed in Approximately one million cells were mixed with 60 µL of lysis buffer  and incubated on ice for 10 min; then, they were sonicated four times at 5 s duration, mixed by rotation for 2 h at 4 °C, and centrifuged at 12,000 rpm in an Eppendorf 5430R microfuge for 20 min at 4 °C. The total protein concentration in the supernatant cell lysate was determined with a Bradford protein assay and adjusted to a final concentration of 0.3 µg/µL with separation gradients  for charge-based cIEF immunoassays or to 0.4 µg/µL with denaturing buffers  for size-based Western immunoassays.Cell lysates were denatured at 95 °C for 5 min and then transferred to assay plates  preloaded with blocking reagents, wash buffer, primary and secondary antibodies, and chemiluminescent substrates. The default protocols of the Jess system (ProteinSimple) were used for sized-based protein separation and detection in capillaries. β-Actin and HSP60 were used as loading controls. Triplicate measurements for each protein and duplicate experiments were performed for each treatment condition to produce six repeated measurements per protein analyte. The expression levels of PPARγ, SREBP1c, FASN, PLIN1, and β-catenin were detected with capillary Western immunoassays.Cell lysates were loaded into 384-well assay plates  preloaded with primary and secondary antibodies and chemiluminescent substrates. The default protocols of the NanoPro 1000 system (ProteinSimple) were used for charge-based protein separation and detection in individual capillaries. Hsp70 was used as the loading control. Triplicate measurements for each protein and duplicate experiments were performed for each treatment condition to produce six repeated measurements per protein analyte. The expression level of FABP4 was detected with capillary isoelectric focusing immunoassays.The primary antibodies used for this study were: PPARɣ , SREBP1c , FASN , PLIN1 , FABP4 , β-catenin , β-actin , HSP60 , and HSP70 . The secondary antibodies used for this study were: anti-rabbit HRP , anti-mouse HRP , anti-rabbit NIR , and anti-mouse NIR .C57BL/6J mice  were divided into three groups: a group fed with a lean diet , a group fed with a high-fat diet , and a group fed with a high-fat diet supplemented with F1 . The lean diet  comprised proteins (25.2% by weight), carbohydrates (39.5% by weight), fats (4.4% by weight), and others . The lean diet has 3 kcal/g, with 34% of kcal from proteins, 53% of kcal from carbohydrates, and 13% of kcal from fats. The high-fat diet  comprised proteins (17.3% by weight), carbohydrates (48.5% by weight), fats (21.2% by weight), and others . The high-fat diet has 4.5 g/kcal, with 15.2% of kcal from proteins, 42.7% of kcal from carbohydrates, and 42% of kcal from fats. F1 was supplemented at 0.1% by weight, leading to an approximate daily dose of 200 mg/kg for mice, or approximately 16 mg/kg of a human equivalent dose. The mice groups were placed on their respective diets for seventeen weeks. Glucose tolerance tests were performed at week sixteen. Terminal tissue and blood samples collection was performed at week seventeen. The histology of the liver and visceral adipose tissues was performed by IHC WORLD . Blood triglycerides, cholesterol, HDL, and LDL were measured by IDEXX Analytics . The Proteome Profiler Mouse Cytokine Array Kits  were used to measure inflammatory cytokines in collected blood samples. All animal studies were performed in accordance with the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and with the approval of the Animal Care and Use Committee at Roseman University of Health Sciences.The mice were housed in cages that did not have bedding that could serve as alternative food sources. The mice were fasted overnight for 16 h, with access to water. The morning following fasting, the mice were anesthetized with 1.5–4% isoflurane at a flow rate of 1 L/min. The tail tips were clipped with a pair of scissors and wiped off with alcohol gauzes. Blood was collected at 0 min for measurement with a glucometer. Next, the mice were injected with 2 g dextrose per kg body mass using 27G needles. The volume of injected dextrose was calculated using the following formula: 50% dextrose (µL) = 4 × body weight (g). Blood was collected at 30, 60, 90, and 120 min after injection for the determination of glucose levels. Between each of these time points, the mice were returned to their cages and monitored continuously. At the end of the glucose tolerance tests, the mice were returned to their previous housing and feeding conditions. Primary human subcutaneous preadipocytes were cultured on uncoated 55 mm glass-bottom dishes for confocal fluorescence imaging . The cells were fixed with 5% formaldehyde and stained for the nucleus with Hoechst 33,258 , F-actin with Alexa Fluor 488 phalloidin , tubulin with paclitaxel Bodipy 564/570 conjugate , and neutral lipids with HCS LipidTOX™ Deep Red . A Nikon A1R inverted confocal microscope with a 60X oil-immersion objective and a numerical aperture of 1.49 was used for imaging. Solid-state lasers at 405, 488, 561, and 638 nm were used for excitation, and fluorescence emissions were collected using 425–475, 500–550, 570–620, and 662–737 nm filters.p-value ≤ 0.01 versus the control.Statistical significance was calculated using Excel software  and Student’s paired t-test with two-tailed distribution and a threshold at The effect of phytonutrients on the expression of adipogenic marker proteins was examined in primary human subcutaneous preadipocytes undergoing fat cell differentiation. Briefly, post-confluent preadipocytes were induced to undergo adipogenesis via the addition of complete differentiation media for six days in the absence or presence of phytonutrients. On d6, total cell extracts were collected, and the expression levels of six adipogenic protein biomarkers were analyzed using capillary Western immunoassays or capillary isoelectric focusing immunoassays. The expression level of the PPARɣ transcription factor was undetectable in preadipocytes on d0 and highly expressed in differentiating preadipocytes on d6. Individually, phytonutrients exhibited differential effects on the expression of adipogenic protein biomarkers. The phytonutrients berberine, luteolin, fisetin, quercetin, EGCG, hesperidin, and curcumin suppressed PPARɣ expression in differentiating preadipocytes C,F. CollThe expression level of the SREBP1c transcription factor was undetectable in preadipocytes on d0 and highly expressed in differentiating preadipocytes on d6. The phytonutrients luteolin and resveratrol enhanced SREBP1c expression in differentiating preadipocytes B,C. The The expression level of FASN was undetectable in preadipocytes on d0 and highly expressed in differentiating preadipocytes on d6. Eight phytonutrients, including berberine, luteolin, resveratrol, fisetin, quercetin, EGCG, hesperidin, and curcumin, suppressed FASN expression in differentiating preadipocytes A–E,G–I. The expression level of PLIN1 was undetectable in preadipocytes on d0 and highly expressed in differentiating preadipocytes on d6. All selected phytonutrients suppressed PLIN1 expression in differentiating preadipocytes A–I. ConsThe expression level of FABP4 was undetectable in preadipocytes on d0 and highly expressed in differentiating preadipocytes on d6. Six phytonutrients, including berberine, resveratrol, quercetin, EGCG, hesperidin, and curcumin, did not suppress the expression of FABP4 but reduced its distribution as a function of isoelectric points A,C,E,G–IThe expression level of β-catenin was high in preadipocytes on d0 and strongly repressed in differentiating preadipocytes on d6. Three phytonutrients, including berberine, fucoidan, and hesperidin, did not interfere with β-catenin degradation in differentiating preadipocytes A,F,H. SiAll selected phytonutrients in this study were anti-adipogenic phytonutrients. Their anti-adipogenicity was most prominent when they were present in differentiating media from d0 to d6 . Most inThe high-resolution imaging of intracellular lipid droplets by independent methods further confirmed the anti-adipogenic effects of the F1 composition of phytonutrients. Following hematoxylin, eosin, and Oil Red O staining, brightfield microscopy revealed the minor Oil Red O staining of intracellular lipid droplets on d14 in differentiating adipocytes treated with the F1 composition of phytonutrients versus untreated control A. FurtheThe anti-adipogenic effects of F1 composition were further evaluated in a diet-induced obesity murine model A–C. MiceGlucose tolerance tests were performed for all mice after 16 h of overnight fasting A. The faBlood and tissue samples were terminally collected from all mice at the end of this study. The average blood triglyceride levels were 76 mg/dL, 123 mg/dL, and 93 mg/dL for the LD, HFD, and HFD + F1 mice, respectively A. The avTerminally collected liver and visceral adipose tissues were used for hematoxylin and eosin (H&E) histology A,B. H&E Terminally collected blood samples were used to measure inflammatory chemokines and cytokines A. SubstaFat cell differentiation is a complex and highly regulated process . The actHerein, nine anti-adipogenic phytonutrients were selected based on their effects on the expression of six adipogenic biomarker proteins: PPARɣ, SREBP1c, FASN, FABP4, PLIN1, and β-catenin. The phytonutrients include berberine, luteolin, resveratrol, fisetin, quercetin, fucoidan, EGCG, hesperidin, and curcumin. The anti-adipogenicity of these phytonutrients had been reported in the literature and was further confirmed by this study ,40,41,42Combinations of phytonutrients improved both the potency and duration of anti-adipogenicity. Notably, the F1 composition of all nine phytonutrients exhibited highly potent and lasting anti-adipogenicity. In primary human subcutaneous preadipocytes, F1 composition suppressed lipid metabolism, fatty acid transport and biosynthesis, and lipid droplet formation via the negative regulation of PPARɣ, FABP4, FASN, and PLIN1. F1 composition activated glycolysis via the positive regulation of SREBP1c expression. In addition, F1 composition preserved cell–cell adhesion via the inhibition of β-catenin degradation. Most interestingly, F1 composition continued to inhibit intracellular lipid droplet accumulation up to seven days following its removal from the culturing media. The functional complementarity of phytonutrients in F1 composition plausibly exerted multi-targeted effects, suppressed the expression of multiple adipogenic proteins, and delayed the resumption of cytoplasmic lipid droplet accumulation. In a diet-induced obesity murine model, dietary supplement with F1 composition improved glucose tolerance and reduced weight gain, liver steatosis, visceral adiposity, circulating triglycerides, LDL cholesterol, and inflammatory cytokines and chemokines. Obesity is associated with visceral adiposity, liver steatosis, hyperlipidemia, hypercholesterolemia, systemic low-grade chronic inflammation, and increased risks for the development of metabolic disease ,50. DietThe bioavailability assessment of F1 composition will be technically challenging due to the presence of many phytonutrients. Fortunately, high-throughput in vitro absorption, distribution, metabolism, and excretion (HT-ADME) screening and in vivo drug metabolism pharmacokinetics (DMPK) have become an essential part of the drug discovery process of synthetic molecules over the last two decades ,52. The Targeting adipogenesis is a rational therapeutic approach for obesity prevention and management ,54. NumeThe functional complementation of anti-adipogenic phytonutrients increased the potency and duration of anti-adipogenicity in primary human subcutaneous preadipocytes. Dietary supplement with the F1 composition of nine anti-adipogenic phytonutrients—berberine, luteolin, resveratrol, fisetin, quercetin, fucoidan, EGCG, hesperidin, and curcumin—was highly effective at reducing weight gain, liver steatosis, visceral adiposity, circulating triglycerides, LDL cholesterol, and inflammatory cytokines and chemokines in a diet-induced obesity murine model. The functional complementation of anti-adipogenic phytonutrients provides an effective approach toward engineering novel therapeutics for the prevention and management of obesity and metabolic syndrome.The authors are co-inventors of a U.S. patent application regarding a composition of phytonutrients for the prevention of obesity .


Calcitriol mediates autocrine (intracrine) and paracrine signalling in immune cells, which provides broader, protective immune functions crucial to overcoming infections. The synthesis of 1,25(OH)2D  in peripheral target cells is dependent on diffusion and endocytosis of D3 and 25(OH)D from the circulation into them, which requires maintenance of serum 25(OH)D concentration above 50 ng/mL. Therefore, in acute infections such as sepsis and respiratory infections like COVID-19, it is necessary to rapidly provide its precursors, D3 and 25(OH)D, through the circulation to generate adequate intracellular calcitriol. Immune defence is one of the crucial non-hormonal functions of vitamin D. A single oral (bolus) dose or divided upfront loading doses between 100,000 and 500,000 IU, using 50,000 IU vitamin D3 increase the serum 25(OH)D concentrations to a therapeutic level of above 50 ng/mL that lasts between two to three months. This takes three to five days to raise serum 25(OH)D. In contrast, a single oral dose of calcifediol (0.014 mg/kg body weight) can generate the needed 25(OH)D concentration within four hours. Considering both D3 and 25(OH)D enter immune cells for generating calcitriol, using the combination of D3 (medium-term) and calcifediol (immediate) is cost-effective and leads to the best clinical outcome. To maximise protection against infections, particularly to reduce COVID-19-associated complications and deaths, healthcare workers should advise patients on safe sun exposure, adequate vitamin D supplementation and balanced diets containing zinc, magnesium, and other micronutrients to support the immune system. Meanwhile, governments, the World Health Organisation, the Centers for Disease Control, and governments should consider similar recommendations to physicians and the public, change the outdated vitamin D and other micronutrient recommendations directed to their population, and organise targetted food fortification programs for the vulnerable groups. This article discusses a rational approach to maintaining a sustained serum 25(OH)D concentration above 50 ng/mL, necessary to attain a robust immune system for overcoming infections. Such would cost-effectively improve the population’s health and reduce healthcare costs. It also describes three cost-effective, straightforward protocols for achieving and sustaining therapeutic serum 25(OH)D concentrations above 50 ng/mL (>125 nmol/L) to keep the population healthy, reduce absenteeism, improve productivity, and lower healthcare costs.Vitamin D deficiency is a global public health problem, a pandemic that commonly affects the elderly and those with comorbidities such as obesity, diabetes, hypertension, respiratory disorders, recurrent infections, immune deficiency, and malignancies, as well as ethnic minorities living in temperate countries. The same groups were worst affected by COVID-19. Since vitamin D deficiency weakens the immune system, it increases the risk of infections, complications, and deaths, such as from sepsis and COVID-19. Deficiency can be remedied cost-effectively through targeted food fortification, supplementation, and/or daily safe sun exposure. Its endocrine functions are limited to mineral metabolism, musculoskeletal systems, specific cell membrane interactions, and parathyroid gland functions. Except for the rapid, endocrine, and cell membrane-based non-genomic functions, all other biological and physiological activities of vitamin D depend on the adequate intracellular synthesis of 1,25(OH) Most vitamin D-dependent peripheral target cell functions, including intracrine/autocrine and paracrine signalling, are determined by the adequate intracellularly generation of calcitriol from 25(OH)D via its 1α-hydroxylation.The endocrine functions of vitamin D encompass the musculoskeletal system, membrane—vitamin D- interactions mediations actions, and vitamin D−ionised calcium-dependent parathyroid gland functions, calcium and phosphate metabolism including skeletal mineralisation and gastrointestinal and renal tubular calcium absorption. In contrast, peripheral target cell signalling and functions depend on the diffusion of sufficient quantities of precursors of calcitriol—vitamin D3 and 25(OH)D hinder their entry into immune cells and, thus, generation of calcitriol. Therefore, vitamin D deficiency D in the circulation) impairs the beneficial effects of vitamin D from peripheral target cells, such as immune cell functions. Consequently, hypovitaminosis D increases the vulnerability to infections and worsens chronic diseases and the severity of illnesses, leading to higher complications and premature deaths. Despite vitamin D supplements being widely available and economical to use and the availability of sunlight worldwide, vitamin D inadequacy is highly prevalent. Chronic vitamin D deficiency significantly increases ill-health, reduces productivity, and escalates healthcare costs  impairs the beneficial clinical outcomes of vitamin D, despite having normal circulatory concentrations of calcitriol. Unlike D2D. This is due to issues such as gastrointestinal malabsorption, increased catabolism, and impaired renal 1α-hydroxylation. The latter is partly due to hyperphosphatemia and elevated fibroblast growth factor-23 (FGF-23) levels, negatively controlling calcitriol production in renal tubular cells  entering target immune cells lead to failure to generate intracellular calcitriol. It causes hypovitaminosis-associated generalised hyper-inflammation and oxidation and cytokine storms , the European Food Safety Authority (EFSA), the UK Scientific Advisory Committee for Nutrition , National Institute of Health & Care Excellence , and certain European countries, and their recommendations to maintain the minimum circulatory 25(OH)D concentration above 20 ng/mL (50 nmol/L) .3 repletion, as mentioned in While laboratory facilities to measure serum 25(OH)D concentrations available in most industrialised countries, they are expensive. In most other countries, measurements of 25(OH)D are not available; thus, relying on shipping samples abroad add to the cost. However, as illustrated in While there are protocols where the needed vitamin D doses could be calculated based on body weight , none isFor average, non-obese healthy persons, weight-based doses are calculated as 70 to 90 IU/mL/kg/day. For individuals with obesity, 90 to 130 IU/kg/day, and for morbid obesity, 140 to 180 IU/kg/day. In extreme obesity, it can be increased up to 200 IU/kg/day. Information provided in 3 described in Using the upfront loading doses of vitamin D3 could be administered safely as a single dose without any adverse effects  into peripheral target cells [Many other peripheral target cells do not have the megalin–cubilin system. Therefore, they are dependent on a concentration-dependent gradient for diffusions of vitamin D and 25(OH)D  and, in some instances, endocytosis [circulating Det cells . Based oet cells . MoreoveTherefore, more vitamin D than 25(OH)D expect to enter target cells and become 25-hydroxylated and then 1α-hydroxylated to form calcitriol. If this is the case, the current gold standard, the measured serum concentration of 25(OH)D alone, may not provide the appropriate information or an accurate picture of vitamin D status. Consequently, serum 25(OH)D concentration alone is unlikely to provide complete information on vitamin D adequacy and the requirements for extra-skeletal target cell functions . This hiFor robust innate and adaptive immune functions, immune cells need to be stimulated by intracellularly generated calcitriol. This initiates autocrine/intracrine and paracrine signalling processes crucial to suppressing inflammatory cytokines and excessive tissue oxidative processes ,147,148 Several recent systematic reviews conclude that, as with parental vitamin D, early administration of calcifediol reported a significant reduction of complications, intensive care unit admissions, and mortality from COVID-19 ,150. A s3 can be substituted, as illustrated in the last column in If calcifediol is unavailable, cumulative doses of vitamin D3 [3, where patients have to wait for a few days, calcifediol increases serum 25(OH)D concentration within hours [Oral calcifediol is readily absorbed in the upper gastrointestinal tract and is one-third better absorbed than parental D3 ,102. Wit3 , 25-hydrin hours ,63,151.3 [3. For example, when administered as 25 µg/day, an equivalent of 1 µg of D3 increased serum 25(OH)D by 1.5 ± 0.9 nmol/L, while one µg of calcifediol increased 25(OH)D by 4.8 ± 1.2 nmol/L [When the potency of calcifediol is calculated on a weight-to-weight basis for raising serum 25(OH)D concentrations , it is between 3.2 and 4 times more effective than D3 . To simp2 nmol/L .There are no meaningful physiological or pharmacological differences between calcifediol and its extended-release analogues. For example, while ordinary calcifediol raises serum 25(OH)D concentrations in four hours, extended-release formulations do the same in three hours ,152. WitPharmacodynamic studies reported that calcifediol’s circulatory half-life is between 12 and 21 days . Therefo3 and calcitriol , not calcifediol or its analogues.Most synthetic vitamin D analogues do not subject to typical physiological feedback control as original vitamin D compounds. Consequently, they have a significantly higher risk of adverse effects . Based oFor many vitamin D deficient persons with sepsis  or COVID3 and 25(OH)D into immune cells for generating calcitriol. The latter required maintaining a serum 25(OH)D concentration of over 50 ng/mL. Therefore, to successfully manage and overcome an infectious epidemic or a pandemic, it is crucial to maintain the population’s serum 25(OH)D concentration above the mentioned therapeutic level.A robust immune system is essential to overcome infections without complications. It depends on the adequate entry of vitamin D3 and 25(OH)D concentrations quickly is paramount and life-saving. In these urgent situations, 0.5 to 1.0 mg of calcifediol can raise serum 25(OH)D concentrations above the minimum therapeutic levels of 50 ng/mL in four hours and boosts the immune system within a day that facilitates to overcome infections.In acutely ill persons, especially those with vitamin D deficiency having infections, raising serum D3 may take a week to increase serum 25(OH)D concentration. Therefore, it is unhelpful in emergencies like SARS-CoV-2 infections.While calcifediol raises serum 25(OH)D within hours, the oral administration of even high doses of vitamin D takes three to five days to raise serum 25(OH)D concentrations. This delay is due to its less efficient absorption than calcifediol and the need for vitamin D to undergo 25-hydroxylation in the liver, a rate-limiting step. In acutely ill patients, as in those in the ICU, administering even high doses of oral D3, administered as loading or bolus, will maintain serum 25(OH)D concentrations between two to three months. Although the circulatory half-life of D3 is short, due to the larger initial doses, it maintains a higher circulatory concentration of both D3 and 25(OH)D for several weeks—partly because of the release from the storage in fat and muscle tissues.With a weight-based, single dose of calcifediol, as described in 3. This can be done using 50,000 IU vitamin D capsules in outpatients’ setups and emergencies, as illustrated in 3 and its longer duration of physiological actions described above, the combination of D3 and calcifediol provides better clinical outcomes than either alone. Therefore, administering the proper doses of D3 and calcifediol is recommended for patients with infections as an adjunct therapy at the first outpatient or inpatient encounter.Therefore, with calcifediol, one should administer a suitable higher dose of vitamin DMultiple observational and RCTs have demonstrated that serum 25(OH)D concentrations (pre-infection or on admission) inversely correlated with the incidence, severity, and rates of death from COVID-19 ,56,153. 3 maintenance dose is 5000 IU/(0.125 µg) day or 50,000 IU (1.25 mg)/week (or every ten days). Nevertheless, this regimen takes a few months to reach the desired serum 25(OH)D concentration above 50 ng/mL. It can be expedited by ingesting vitamin D, 10,000 IU/day (250 µg/day) for 8 to 10 weeks and reverting to the daily dose of 5000 IU.The described schedules in the three tables are highly cost-effective ways to raise serum 25(OH)D concentrations and maintain it to keep the immune system on high alert. Consequently, it prevents and/or reduces infections and complications from COVID-19 and other infections. For non-obese 70 kg adults, the recommended longer-term vitamin DRectifying vitamin D deficiency costs less than 0.1% of the costs related to evaluating and treating comorbidities and complications associated with vitamin D deficiency . For exa3 supplements exceeds 1 in 20,000. Despite these data, no country is yet to recommend vitamin D (or has published proper guidelines with the right doses) for disease prevention or recommended it as an adjunct therapy to prevent complications and deaths from infections or other diseases. This report provides rationale, justifications, straightforward guidance, and practical tables that provide regimens for use in clinical practice for achieving and maintaining the serum 25(OH)D concentrations needed to ensure a robust immune system that helps to overcome infections, including SARS-CoV-2.Considering the described significant benefits associated with disease prevention, reduced illness severity, reduced absenteeism, complications and deaths, improved well-being and higher productivity, the calculated overall cost-benefit ratio for administered vitamin D


A reductive, high-affinity Cu uptake system in marine microalgae is identified which enables cells to directly utilize organically complexed Cu, highlighting the importance of cell surface Cu reduction in the marine Cu cycle. This review provides new insights into Cu uptake models that may update the existing knowledge of Cu availability in the ocean.Copper is a critical metal nutrient required by marine microalgae but may be toxic when supplied in excess. Maintaining an optimal intracellular Cu content is thus fundamentally necessary for microalgae and relies on cellular regulatory metabolisms and the process of Cu uptake that buffers the variation in environmental Cu availability. In this article the current progress in understanding the Cu requirements and acquisition mechanisms of marine microalgae is reviewed. Cu requirement by microalgae is primarily determined by the amount of Cu-dependent enzymes involved in cellular metabolisms and can be adjusted by Cu-sparing pathways. Decrease in metabolic Cu quotas caused a decline in the abundance of cuproenzymes and the dependent cellular metabolisms, and an induction of Cu acquisition pathways. Conventional models of Cu uptake describe the dependence of Cu uptake rate on free Cu Marine microalgae require both macronutrients  and micronutrients  to grow. The trace metals mainly function in metabolic pathways as cofactors of enzymes, and their availability is a crucial environmental factor regulating algal growth ,2. Coppe2) and biological uptake  above 10−12 M) and eukaryotic algae more tolerant to Cu overload, assessed by growth performance at high Cu concentrations [Copper toxicity occasionally occurs in some Cu-enriched environments, such as coastal regions that may receive a large quantity of atmospheric aerosols containing high Cu concentrations and is thought to alter the abundance of particular microalgae ,50,51,52trations ,54,55. Ttrations ,56. Sometrations . Eukaryotrations . Moreovetrations .Metabolic Cu quotas are the amount of cellular Cu required by Cu-dependent metabolisms. The quotas are usually maintained within narrow limits by tightly regulated cellular processes even as environmental concentrations vary by orders of magnitude ,60. At lCu quotas also vary among species by a factor of 10  because The rise of dissolved oxygen produced by oxygenic photosynthesis changed the redox state of the paleoocean, greatly increasing the solubility of dissolved Cu and decreasing the bioavailability of iron . Since tc oxidase and multicopper oxidase [6/f complex to photosystem I [c oxidase catalyzes the final step of respiration by reducing oxygen to water [The three most abundant cuproenzymes in eukaryotic microalgae are plastocyanin, cytochrome  oxidase ,74. Plassystem I . Cytochrto water . Multicoto water ,65. Otheto water ,78,79,80to water ,82.Chlamydomonas [c oxidase and multicopper oxidase [Chlamydomonas can be restored by up-regulation of a Fe-containing protein, cytochrome c6 [c6 [Under Cu deficiency, the cellular concentration of cuproenzymes declines with the decrease in the amount of cellular Cu . Plastocydomonas ,85. Down oxidase  and the hrome c6 . The rephrome c6  and a CRhrome c6 ,88 undere c6 [c6 , then a e c6 [c6 ,89. Beca2+  in marine cyanobacteria and diatoms [+) via specific cuprous transporters [Chlamydomonas, the high-affinity system is induced by low Cu and increases the uptake rate by 20-fold in Cu-limited compared to Cu-replete cells [2+ (pCu = 13.2 to 14.2), more than 100-fold lower than the Km of low-affinity uptake systems [Copper uptake is of great importance in maintaining cellular Cu homeostasis, because it controls the amount of Cu that is transported into cells. Uptake proceeds by a two-step reaction involving binding of the metal to a specific transport ligand on the cell surface followed by internalization . In mari diatoms ,93,94 ansporters ,96. In tte cells . Half-sa+ transportation and consist of two molecular components, high-affinity CTR-type Cu transporters (CTR) and cupric reductases [12-GxxxG in transmembrane domains is characterized as the signature motif found in all CTR proteins [Thalassiosira oceanica, by functional complementation of a CTR-defect yeast mutant [T. oceanica in the yeast mutant successfully restored growth in Cu-deficient medium and the expressed proteins selectively transported Cu(I). A closely related coastal isolate, T. pseudonana, also contained candidate CTR genes, but the expressed proteins failed to restore growth of the yeast mutant [High-affinity Cu uptake systems feature high specificity for Cuductases . CTRs weductases  and lateductases ,97. The proteins . The N-tproteins , and Cu proteins . CTRs arproteins ,101,102.t mutant . Expresst mutant . The post mutant . For exat mutant ,60,74 ant mutant ,105.2+, Mn2+, Co2+, Zn2+, Cd2+, Cu2+, Ni2+ and Pb2+ [T. pseudonana, contains an NRAMP homologue protein (TpNRAMP) in its genome and its expression increases by 2-fold in Cu-limited cells [Arabidopsis [Arabidopsis, suggested that ZIP may also transport Cu [Chlamydomonas [Low-affinity uptake proceeds by some non-specific divalent metal ion transporters. One type of this assimilatory Cu transporter is the NRAMP  family of divalent metal transporters. NRAMP was named for its first detection in membranes of macrophages and its function in the digestion of intracellular parasites . The humand Pb2+ ,108. Theand Pb2+ . The mared cells . Very sibidopsis ,112,113 bidopsis . Inhibitsport Cu . Expressydomonas , but theThe first description of Cu reduction in marine algae showed that Cu(II) bound to inorganic and organic complexes was reduced to Cu(I) extracellularly ,116. Extphox, of human NADPH oxidases [TpFRE1,2 of T. pseudonana, PtFRE1,2 of Phaeodactylum tricornutum, ToFRE1,2 of T. oceanica) share conserved amino acid motifs and protein domains with yeast FREs and NADPH oxidases [The molecular mechanism of cell surface Cu reduction in marine species is poorly understood, but there is some evidence that it is associated with nitrate reductase activity ,116. In oxidases . NADPH ooxidases ,125. Putoxidases ,127,128.Saccharomyces cerevisiae [2−) [Biochemical function of cupric reductases was originally investigated in revisiae ,120. A srevisiae ,110. Cuprevisiae . Some stiae [2−) ,129, so iae [2−) ,117. Giviae [2−) ,130, and2+ but not organically complexed Cu [2+, Cu2+, Fe3+, Mn2+, Zn2+) [2+ or inorganic Cu′ species cannot fulfill the Cu requirements for uptake at growth limiting concentrations of Cu because of its extremely low concentration [The initial work on the relationship between Cu availability and algae growth showed that the toxicity of Cu in freshwater and marine species was related to the concentration of free Culexed Cu ,19,53. T+, Zn2+) ,132,133.ntration ,134,135.ntration , so othentration . Similarntration ,138. It ntration ,140. Altntration . Acquisintration . So far,ntration ,23.2+) are so low that uptake of Cu′ via low-affinity transporters contributes only a negligible amount to the bulk uptake of Cu from the environment (The possession of both high-affinity (reductive) and low-affinity (nonreductive) Cu uptake systems allows marine diatoms to adjust strategies to acquire Cu from different environments . For example, at growth-limiting, low Cu environments, concentrations of Cu′  is reduced to Cu(I) prior to Cu internalization. Biological and photochemical reactions that catalyze reactions of Cu(II) reduction are thus fundamental to Cu uptake by marine microalgae in the ocean. Current views recognize that extracellular Cu(II) reduction is required for Cu(I) uptake by Cu(I) transporters, but pay little attention to the reduction step ,93,94. A


Health-related quality of life (HrQoL) is a major concern for patients with differentiated thyroid carcinoma (DTC). We aimed to systematically review the literature comparing HrQol following total thyroidectomy (TT) and hemithyroidectomy (HT) in DTC patients. A systematic review of publications indexed in Medline, Embase, and EBM reviews—Cochrane Central Register of Controlled Trials, which evaluated HrQoL following thyroid surgery for DTC, was conducted. Of 2507 identified records, 25 fulfilled the inclusion criteria. Our results suggest that patients undergoing TT may suffer more impairment in physical and social HrQoL than patients undergoing HT. Psychological-related HrQoL and long-term global HrQoL are, however, equivalent in both groups, which highlights the multidimensional nature of HrQoL and the importance of a multitude of factors aside from treatment modalities and related morbidities, such as the experience of receiving a cancer diagnosis, the fear of cancer recurrence, and other psychosocial factors. Addressing postoperative HrQoL when discussing therapeutic options with patients is an integral part of patient-centered care and informed shared decision-making, and should be approached in a holistic manner, accounting for its physical, psychological, and social aspects. This review supplies evidence regarding HrQoL following thyroid surgery, which can be employed in such decisions. Over the last 50 years, the incidence of thyroid cancer has more than tripled in the United States, with mortality rates remaining largely stable over the last three decades ,2. The iA systematic review of the literature was performed to identify relevant studies reporting on patients’ HrQoL following TT or HT. The PRISMA  framework was used to guide the search and the reporting of the review . The seaReferences were assessed for eligibility according to the criteria outlined below  in a twoWe included empirical studies conducted in human patients with a histopathologically confirmed diagnosis of well-differentiated thyroid cancer undergoing any thyroid surgery, with or without subsequent RAI ablation. For empirical studies using quantitative methodology, we included any studies comparing preoperative HrQoL with postoperative HrQoL, postoperative HrQoL with healthy controls, postoperative HrQoL with active surveillance, or postoperative HrQoL following TT vs. HT. For empirical studies using qualitative methodology, we included any studies providing information related to patients’ experiences with thyroid surgery, opinions related to the intervention, and life after the procedure such as satisfaction, preferences, and feelings. All included studies had to provide results according to surgery extent, and study groups had to be at least 95% homogenous in terms of surgical procedure performed (TT or HT). Due to limited resources for the translation of studies, only articles written in English or French were included.Nonempirical studies were excluded . Studies including patients with medullary or anaplastic thyroid cancer were excluded. Studies reporting results only in the context of thyroid-hormone withdrawal or recombinant human TSH stimulation were also excluded.Two researchers independently assessed the risk of bias using the Joanna Briggs Institute (JBI) critical-appraisal checklists, which allows for the assessment of quantitative and qualitative studies .For the purposes of this review, any surgical intervention restricted to one lobe of the thyroid, with or without isthmusectomy, was classified as a HT (lobectomy). Any bilateral resection, including total and near-total thyroidectomy, was classified as a total thyroidectomy. HrQoL is a multidimensional concept including physical and psychological symptoms, social wellbeing, and symptoms associated with illness or treatment . MultiplThe initial search resulted in a total of 2507 records that were imported into the Covidence systematic review software Version v2625  for screening and eligibility assessment . Four ren = 6), the United States (n = 5), the Netherlands (n = 3), and South Korea (n = 2). The remaining studies were from Canada (n = 1), Austria (n = 1), Sweden (n = 1), Finland (n = 1), Italy (n = 1), Greece (n = 1), Japan (n = 1), Australia (n = 1), and Egypt (n = 1). Twenty studies of quantitative design were included; two studies of qualitative and three studies of mixed methods were included. Nineteen studies were cross-sectional in nature, while six studies were transversal. Among transversal studies, four focused on early postoperative HrQoL (≤1 year following surgery), while two studies provided a long-term longitudinal assessment of HrQoL (≥2 years postoperatively and 4 years postoperatively). The postoperative time to HrQoL assessment varied greatly across studies, ranging from 1 week to 44 years. The majority of studies focused on TT patients (n = 14), while only one study focused exclusively on HT patients, and ten studies directly compared TT and HT patients. All studies evaluated DTC patients, with six studies including papillary thyroid microcarcinoma (PTMC) patients  employed in nine different studies. It is a general questionnaire comprised of 36 items measuring eight different scales ,26. EachThe 12-Item Short-Form Health Survey (SF-12) is an abbreviated version of its predecessor, the SF-36 . It evalThe Short-Form Six Dimensions (SF-6D) is an 11-item general HrQoL instrument frequently used in economic evaluations . It evalThe 5-Level EQ-5D (EQ-5D-5L) is a questionnaire comprised of 25 items and a visual analog scale (VAS) used in economic evaluations . Each itThe Health Utilities Indexes 2 and 3 (HUI2 and HUI3) are 40-item health-status classification systems used in economic evaluations . HUI2 scThe 15D instrument is a general HrQoL questionnaire assessing 15 different dimensions that can be used both as a profile and as a single index instrument . 15D indThe Fear of Progression questionnaire (FoP) is a 43-item instrument used to assess anxiety and fear related to disease progression . Items aThe Short Form of the Fear of Progression Questionnaire (FoP-Q-SF) is a 12-item version of its predecessor (FoP) . Each itThe Assessment of Survivor’s Concerns (ASC) questionnaire includes six items evaluating cancer-related worries in cancer survivors . Items aThe European Organisation for Research and Treatment of Cancer’s Core Quality of Life Questionnaire (EORTC QLQ-C30) is a 30-item instrument designed to evaluate HrQoL in cancer patients . All iteThe EORTC Quality of Life Questionnaire’s supplementary module for thyroid cancer (EORTC QLQ-THY34) is a 34-item instrument meant to be employed in conjunction with the EORTC QLQ-C30 in thyroid cancer patients . A scoreThe THYCA-QoL is a 24-item questionnaire that was designed specifically to assess HrQoL in thyroid cancer survivors. Each item is ranked on a scale of 1–4 . HoweverThe Korean Thyroid QoL questionnaire (KT-QOL) is the validated Korean translation of the QOL-Thyroid Scale, first developed by Dow et al. in the United States . It is aThe ThyPRO questionnaire is an 84-item instrument designed to assess HrQoL in patients with benign thyroid disorders . Scores The Billewicz Score is a clinical 14-item scale used to detect hypothyroidism. Its scores range from −47 to +67, with higher values denoting decreased thyroid function .The Multidimensional Fatigue Inventory (MFI-20) is a 20-item questionnaire measuring five different dimensions of fatigue . Each suThe Pittsburgh Sleep Quality Index (PSQI) is a 19-item instrument evaluating seven different components of sleep . Global The Hospital Anxiety and Depression Scale (HADS) is a 14-item questionnaire, with seven items assessing depression and seven items assessing anxiety . Each itThe somatoform disorders questionnaire (SDW) assesses for the presence of somatoform disorders, as described in the third edition of the Diagnostic and Statistical Manual of Mental Disorders . Scores The State-Trait Anxiety Inventory (STAI) is a 40-item questionnaire used to assess an anxiety state at a given time in relation to basal anxiety levels . Each itThe Voice Handicap Index (VHI) is a 30-item instrument designed to assess the psychosocial impact of voice disorders. Scores range from 0 to 120, with higher scores denoting a greater handicap .A visual analog scale (VAS) was used by Teliti et al. to assess complaints related to medical therapy . Scores The JBI critical appraisal checklists were used to perform the quality assessment of included studies  22]. Mo. Mo22]. n = 1), vitality (n = 2), physical functioning (n = 3), speech (n = 1), general or physical fatigue (n = 1), and sleep (n = 1) [n = 1) or discomfort and symptoms (n = 1) following TT compared with the reference population [n = 1), cognitive complaints (n = 1), and sleep efficiency (n = 1) [In this review, we identified 20 studies reporting on physical HrQoL following TT or HT ,68,70,73 (n = 1) ,55,58,61pulation ,58. Teli (n = 1) . One stuThree studies compared patients’ preoperative and postoperative physical-related HrQoL following TT ,70,73. On = 1), voice symptoms (n = 1), trouble with concentration (n = 2), neuromuscular symptoms (n = 1), throat or mouth symptoms (n = 2), feeling chilly (n = 1), or problems with scar (n = 2) [n = 1), weight gain (n = 1), problems with scar (n = 1), and vocal symptoms (n = 1) [Three studies assessed postoperative physical-related HrQoL in comparison with patients undergoing active surveillance (AS) ,60,67. A (n = 2) ,60,67. F (n = 1) .n = 1), voice symptoms (n = 1), sensory symptoms (n = 1), neuromuscular symptoms (n = 1), vitality (n = 1), and problems with scar (n = 1) [n = 1) and cognitive functioning (n = 1) [Finally, nine studies directly compared physical-related HrQoL for patients undergoing TT versus HT using six different questionnaires  ,64,65,67 (n = 1) ,59,64,67 (n = 1) ,60,62. T (n = 1) ,57.n = 2), mental fatigue (n = 2), reduced motivation (n = 1), distress (n = 1), anxiety (n = 1), depression (n = 1), somatization (n = 1) [In this review, 19 studies reporting on psychological-related HrQoL were identified ,67,68,73 (n = 1) ,53,58,61 (n = 1) ,55,68. NOnly two studies compared preoperative and postoperative psychological-related HrQoL ,73. ThesThree studies compared psychological-related HrQoL for patients undergoing surgery versus AS ,60,67. Tn = 1), emotional functioning (n = 1), state anxiety (n = 1), and mental health (n = 1) for patients undergoing TT versus HT [Finally, ten studies directly compared psychological-related HrQoL in TT and HT patients ,64,65,67ersus HT ,59,62,63ersus HT .n = 4), role limitations due to emotional or physical health (n = 5), and reduced activity (n = 1) [In this review, 16 studies reporting on social functioning through 10 different instruments  were identified ,65,67,68 (n = 1) ,55,58,61 (n = 1) . No studOne study compared patients’ preoperative and postoperative social-related HrQoL following TT, while no study evaluated this aspect in HT patients . This stn = 4), social functioning (n = 1), and role limitations due to physical or emotional health (n = 1) [Two studies compared the social-related HrQoL of patients undergoing surgery versus AS and found a significant decrease in the social subscale ( (n = 1) ,67. Of nn = 2) and role limitations due to physical (n = 3) or emotional health (n = 2) [Finally, seven studies directly compared social-related HrQoL in TT and HT patients ,62,65,67 (n = 2) ,57,59. O (n = 2) . One stuFinally, 16 studies evaluated the global postoperative HrQoL in DTC patients using ten different instruments  ,67,68,73Three studies compared preoperative versus postoperative global HrQoL following TT ,68,73. OTwo studies assessed patients’ global HrQoL following surgery or during AS ,67. One Seven studies directly compared HT and TT patients in terms of global HrQoL ,62,65,67Qualitative studies are valuable in HrQoL research, as they allow for the consideration of individuals’ perceptions and interpretations; the understanding of their experiences of wellbeing; and the discovery of new issues related to HrQoL .n = 2) to content analysis (n = 2) and thematic analysis (n = 1) [In this review, two different studies of qualitative and three studies of mixed methodology were identified, among which four focused on TT patients, and one focused on both TT and HT patients ,71,72,73 (n = 1) ,71,72,73n = 4), psychological symptoms (n = 4), social functioning (n = 1), regret or dissatisfaction with treatment (n = 2), satisfaction with care provided (n = 1), and adaptative strategies (n = 2) [Included qualitative studies identified themes related to physical symptoms ( (n = 2) ,71,72,73Physical symptoms such as numbness and tingling, impaired communication, xerostomia, xeropthalmia, and dysgeusia were frequently reported (18–57%) following DTC treatment with TT ,72,73. VPsychological symptoms following TT such as distress, anxiety, and fear were described in three studies, although the incidence of such symptoms was not reported ,72,73. Tn = 1) or “impact on work” (n = 1), which was more frequently reported by HT patients than TT patients [Themes relevant to social-related HrQoL were only described in two studies as “feeling supported”  may be associated with better HrQoL due to the lower risk of surgical and post-operative complications, thus leading to better postoperative physical health ,16,17,18n = 3). While it has frequently been assumed that the decrease in HrQoL of patients with DTC was mostly attributed to treatment modalities and their associated complications, current evidence remains unclear on this matter. There is currently insufficient evidence to conclude that DTC patients’ postoperative HrQoL is worse than their preoperative HrQoL, which calls into question other possible contributive factors, such as the experience of receiving a cancer diagnosis, the fear of recurrence, and other psychosocial factors.We found that evidence regarding HrQoL following HT versus TT remains mitigated, with a tendency towards worse physical-related HrQoL in patients undergoing TT, which may be explained by the higher complication rates observed in the latter ,76,77. GThe psychological impacts of cancer diagnoses are significant clinical issues to consider ,80,81. ICurrent evidence seems to indicate that there are no significant differences in psychological-related HrQoL for patients undergoing HT versus TT, although the latter may be associated with less fear of recurrence or cancer-related worry. This relationship between fear of recurrence and surgery extent has seldom been explored in DTC but is extensively described in breast cancer by the trade-off hypothesis, i.e., the founded or unfounded rationale that an organ can be sacrificed or “traded” through radical surgery in exchange for a lower risk of recurrence . CurrentDirectly related to the physical and psychological components of HrQoL is the social functioning component; that is, the ability to fulfill social roles and maintain social relationships, interactions, and societal integration . AdequatCurrent evidence suggests that there is no difference in long-term HrQoL between patients undergoing HT versus TT, although a greater impairment may be observed in TT patients in the first year following surgery. Current research is nuanced concerning a potential decrease in global HrQoL in TT patients compared with the general population. If this difference does exist, it may be partly due to the diagnosis of cancer, rather than only treatments and associated complications. This hypothesis is supported by the fact that no differences are observed between preoperative and postoperative global HrQoL in patients undergoing TT. Additionally, there is currently insufficient evidence to conclude to the existence of a difference in global HrQoL for patients undergoing surgery versus AS.Current research highlights that, whether patients undergo TT or HT, the decision of undergoing thyroid surgery has far-reaching consequences that can affect long-term HrQoL. However, there has been limited research focused on developing interventions aimed at improving HrQoL in DTC patients. Nuria Javaloyes et al. presented a psycho-oncological intervention based on counseling, which successfully decreased anxiety and depression and increased psychological general well-being in DTC patients . Wu et aSeveral limitations limit the conclusions that can be drawn from included studies. First, the important heterogeneity of included studies, both in terms of QoL instruments used, and postoperative times of assessment limited comparison across studies and did not allow for a meta-analysis to be performed. Second, the vast majority of included articles were cross-sectional, which limits the establishment of causal relationships. Third, there was a paucity of studies directly comparing HT and TT patients’ HrQoL or focusing on HT patients’ HrQoL in general. Fourth, the scope of our review did not allow us to assess the impact of thyroid surgery on the HrQoL of patients who are ultimately found to have benign diseases. These patients are expected to experience an entirely different set of challenges postoperatively, whether it is regarding the fear of cancer recurrence, the necessity for medical appointments, RAI ablation or external beam radiation therapy, or attitudes towards potential postoperative complications. To date, there has been limited research on postoperative HrQoL of this group of patients, although they represent a significant proportion of patients who undergo thyroid surgery ,92,93,94HrQoL is increasingly being recognized as a significant clinical issue in thyroid cancer. This study provides an up-to-date synthesis of HrQoL following thyroid surgery that can help clinicians better counsel their patients while allowing for a more personalized approach, congruent with patients’ preferences and priorities. Our findings highlight the multidimensional nature of HrQoL and the importance of a multitude of factors aside from treatment choice and related morbidities, such as the experience of receiving a cancer diagnosis, the fear of cancer recurrence, and other psychosocial factors. Qualitative research furthermore emphasizes therapeutic alliances and relationships with healthcare providers as decisive factors in DTC patients’ HrQoL. Addressing postoperative HrQoL when discussing therapeutic options with patients is an integral part of patient-centered care and informed shared decision making, and should be approached in a holistic manner, accounting for its physical, psychological, social, and global aspects. As thyroid cancers are detected in greater numbers and in younger patients, HrQoL is an issue that will continue to gain importance over the next few years. More comprehensive, longitudinal, homogenous studies, including larger sample sizes, are needed in order to fully elucidate the impacts of TT and HT on DTC patients’ HrQoL.


The cellular functions are executed by biological macromolecular complexes in nonequilibrium dynamic processes, which exhibit a vast diversity of conformational states. Solving the conformational continuum of important biomolecular complexes at the atomic level is essential to understanding their functional mechanisms and guiding structure-based drug discovery. Here, we introduce a deep manifold learning framework, named AlphaCryo4D, which enables atomic-level cryogenic electron microscopy (cryo-EM) reconstructions that approximately visualize the conformational space of biomolecular complexes of interest. AlphaCryo4D integrates 3D deep residual learning with manifold embedding of pseudo-energy landscapes, which simultaneously improves 3D classification accuracy and reconstruction resolution via an energy-based particle-voting algorithm. In blind assessments using simulated heterogeneous datasets, AlphaCryo4D achieved 3D classification accuracy three times those of alternative methods and reconstructed continuous conformational changes of a 130-kDa protein at sub-3 Å resolution. By applying this approach to analyze several experimental datasets of the proteasome, ribosome and spliceosome, we demonstrate its potential generality in exploring hidden conformational space or transient states of macromolecular complexes that remain hitherto invisible. Integration of this approach with time-resolved cryo-EM further allows visualization of conformational continuum in a nonequilibrium regime at the atomic level, thus potentially enabling therapeutic discovery against highly dynamic biomolecular targets. Essential cellular functions are executed and regulated by macromolecular complexes comprising many subunits, such as the ribosome, proteasome, replisome, and spliceosome. Their structures in functional cycles are often compositionally heterogeneous and conformationally dynamic, involving many reversibly associated components in cells. Visualizing the conformational continuum of highly dynamic megadalton complexes at high resolution is crucial for understanding their functional mechanisms and for guiding structure-based therapeutic development. The current approaches in cryogenic electron microscopy (cryo-EM) structure determination allow atomic-level visualization of major conformational states of these dynamic complexes ,10,11,12The problem of structural heterogeneity in cryo-EM structure determination has been investigated with numerous computational approaches including maximum-likelihood-based classification and multivariate statistical analysis ,16,17,18The most widely used method so far to counteract structural heterogeneity in cryo-EM for resolution improvement is the hierarchical maximum-likelihood-based 3D classification due to its ease of application ,23,24. TIn addition to the approaches characterizing global structural heterogeneity, other methods were proposed to analyze local structural dynamics or to improve the local resolution of flexible regions in cryo-EM reconstructions. These include the multi-body or focused refinement ,28,29, nThe energy landscape is a statistical physical representation of the conformational space of a macromolecular complex and is the basis of the transition-state theory of chemical reaction dynamics ,38. The The combination of linear dimensionality reduction via 3D principal component analysis (PCA) and pseudo-energy landscape estimation has been demonstrated to be capable of capturing the overall conformational landscape ,42,43. TBreaking the resolution barrier in visualizing the conformational space of highly dynamic complexes represents a formidable technical challenge. Achieving this goal may further leverage cryo-EM applications in both basic biomedical science and drug discovery, where high-resolution features of highly dynamic components may provide crucial mechanistic insights and guide more accurate structure-based ligand design. This would require a substantial improvement in 3D classification techniques that are optimized for achieving higher resolution. We hypothesize that a high-quality estimation of the pseudo-energy landscape could be potentially used to improve the 3D classification of highly heterogeneous cryo-EM datasets. If different conformers or continuous conformational changes can be sufficiently mapped and differentiated on a pseudo-energy landscape, the energetic visualization of the conformational continuum could then be used to discover previously ‘invisible’ conformers and achieve higher resolution for lowly populated, transient, or nonequilibrium states. To explore these ideas in this study, we developed a novel machine learning framework named AlphaCryo4D that can break the existing limitations and enable 4D cryo-EM reconstruction of highly dynamic, lowly populated intermediates or transient states at the atomic level. We examined the general applicability of AlphaCryo4D in analyzing several large synthetic datasets over a wide range of signal-to-noise ratios (SNRs) and four experimental cryo-EM datasets of diverse sample behaviors in complex dynamics, including the human 26S proteasome , malariaM-fold particle shuffling and Bayesian clustering using a maximum-likelihood-based 3D (ML3D) classification algorithm implemented in RELION . . EnvCondrun_resnet.py in the folder DeepFeature, can be applied using the sample command like:As an example, given the properly configured environment, the Python program, such as python DeepFeature/run_resnet.py --batchsize 8 --validationsize 200 --regularization 0.001 --data data_dl.npy --gpu 0,1,2,3Three simulated large datasets with the SNRs of 0.05, 0.01 and 0.005, each including 2 million particle images, were employed to benchmark AlphaCryo4D and to compare its performance with alternative methods. For each synthetic dataset, the particles were computationally simulated by projecting the 20 3D density maps calculated from 20 hypothetical atomic models emulating continuous inter-domain rotation of the NLRP3 inflammasome protein. The 20 atomic models were interpolated between the inactive NLRP3 structure and its hypothetical active state, which was generated through homology modeling using the activated NLRC4 structure D 56]. T. T56]. TEach simulated heterogeneous NLRP3 dataset was analyzed separately by AlphaCryo4D and used to characterize the performance and robustness of AlphaCryo4D against the variation of SNRs. In the step of particle shuffling and volume resampling, 2,000,000 particles in the dataset of any given SNR were divided randomly into 10 sub-datasets for batch processing. The orientation of each particle was determined in the initial 3D consensus alignment in RELION, which did not change in the subsequent 3D classification. In this step, the maximum number of iterations of the 3D alignment was set up as 30, with the initial reference low-pass filtered to 60 Å. Three-fold particle shuffling (indicated as × 3 below) was conducted on each sub-dataset for volume resampling. The first round of maximum-likelihood 3D classification divided the input shuffled particle sub-dataset into five classes, each of these classes was then further classified into eight classes. This procedure was separately executed on all shuffled particle sub-datasets. The particle shuffling and volume resampling generated 1372, 1489, and 1587 volumes by the datasets with SNRs of 0.05, 0.01 and 0.005, respectively. These volume data were used as inputs for deep residual autoencoder to compute low-dimensional manifolds and pseudo-energy landscapes . After sUsing 3D classification precision as a benchmark indicator, the performance of AlphaCryo4D preprocessed by standardization or 5 Å low-pass filtering was compared with several other methods: (1) the conventional maximum-likelihood-based 3D (ML3D) classification in RELION v3.0   with andFor the tests using ML3D in RELION, we classified all particles directly into 20 classes and hierarchically into 4 × 5 classes, which first divided the dataset into four classes, with each class further classified into five sub-classes . To geneAlthough the computational cost of AlphaCryo4D is potentially higher than the conventional approach, it does not appear to increase drastically and likely falls in an affordable range, while reducing the average cost of computation per conformational state . In a nuA2. In this focused study, 147,108 particles of EA2 were utilized for computing the specific pseudo-energy landscape with 40 volumes. The alignment of these particles was firstly refined with a 19S mask. M = 3 particle shuffling was then conducted to resample the 3D volumes with a soft mask of 19S. By clustering on the pseudo-energy landscape and particle voting, two classes containing 99,043 and 47,389 particles were generated for high-resolution refinement using RELION v3.0, which were designated state EA2.1 and EA2.0, respectively. The density map of state EA2.0 exhibits a conformation identical to state EA2 in the previous report [The substrate-engaged human 26S proteasome dataset (EMPIAR-10669)  includess report .Pf80S ribosome dataset (EMPIAR-10028) contains 105,247 particle images. First, the alignment of all particles was refined with a global mask in RELION. To resample enough 3D volumes for the pseudo-energy landscape, we set M = 7 in the particle shuffling step, resulting in 79 volumes in total with a global mask and 15 Å resolution limit in the expectation step. Moreover, the regularization coefficient was set to 0.001 when training the 3D residual network with this small dataset. All volumes were then low-pass filtered at 5 Å prior to manifold embedding. Five clusters were obtained from the pseudo-energy landscape, which had 66,035, 21,482, 9922, 6424, and 657 particle images, respectively, after voting. All these clusters were refined independently using RELION v3.0.The M = 7 particle shuffling was utilized to resample 160 volumes with a soft global mask and 15 Å resolution limit in the expectation step. The regularization coefficient of 0.001 was set to train the 3D Autoencoder with this dataset. All volumes were then low-pass filtered at 5 Å prior to manifold embedding. Based on the pseudo-energy landscape, we obtained 14 classes with the particle number of 15,749, 17,703, 27,522, 13,820, 20,841, 14,989, 16,377, 22,047, 25,049, 21,327, 18,949, 6157, 9790 and 36,878 after voting. Each class was refined independently using RELION v3.0.The pre-catalytic spliceosome dataset (EMPIAR-10180) with the particle number of 327,490 shows high conformational dynamics. The consensus alignment of these particles in the original dataset was used in the first step. M = 7 particle shuffling was conducted to generate these volumes without any mask. The regularization coefficient of 0.001 was set when training the deep neural network. All volumes were then low-pass filtered at 5 Å prior to manifold embedding. Then the first pseudo-energy landscape resulted in nine clusters with the particle number of 766 (A), 15,129 (B), 1236, 322, 670, 22,037, 25,445 (D), 33,976 (E) and 25,115 (C), respectively. For the clusters of states B–E, four zoom-in pseudo-energy landscapes were then computed to discover more sub-states with 46, 56, 66 and 72 resampled volumes, respectively. In the zoom-in pseudo-energy landscape calculation, M = 7 was set in particle shuffling for resampling 3D volumes with a global mask and 20 Å resolution limit in the expectation step. Then the regularization coefficient of 0.001 was used to train the deep residual network. All volumes were then low-pass filtered at 5 Å prior to manifold embedding. After particle voting, the pseudo-energy landscape of state B resulted in three sub-clusters with particle numbers of 3494, 3600 and 7803. The state C pseudo-energy landscape resulted in five sub-clusters with the particle numbers of 2444, 7442, 711, 7633 and 5557. The state D pseudo-energy landscape resulted in five sub-clusters with the particle numbers of 6231, 8785, 2123, 3718 and 4092. Moreover, the pseudo-energy landscape of state E resulted in eight sub-clusters with the particle numbers of 4783, 1588, 5891, 3620, 7352, 5265, 357 and 2241. All these clusters were refined using RELION v3.0. The 131,899 particle images of the 50S ribosomal large subunit (EMPIAR-10076) are highly heterogeneous. We refined the alignment of all particles with a global mask of the 50S using RELION v3.0. In the first step, 119 resampled volumes were utilized to calculate the pseudo-energy landscape.


The global coronavirus disease 2019 (COVID-19) pandemic has lasted for over 2 years now and has already caused millions of deaths. In COVID-19, leukocyte pyroptosis has been previously associated with both beneficial and detrimental effects, so its role in the development of this disease remains controversial. Using transcriptomic data (GSE157103) of blood leukocytes from 126 acute respiratory distress syndrome patients (ARDS) with or without COVID-19, we found that COVID-19 patients present with enhanced leukocyte pyroptosis. Based on unsupervised clustering, we divided 100 COVID-19 patients into two clusters (PYRcluster1 and PYRcluster2) according to the expression of 35 pyroptosis-related genes. The results revealed distinct pyroptotic patterns associated with different leukocytes in these PYRclusters. PYRcluster1 patients were in a hyperinflammatory state and had a worse prognosis than PYRcluster2 patients. The hyperinflammation of PYRcluster1 was validated by the results of gene set enrichment analysis (GSEA) of proteomic data (MSV000085703). These differences in pyroptosis between the two PYRclusters were confirmed by the PYRscore. To improve the clinical treatment of COVID-19 patients, we used least absolute shrinkage and selection operator (LASSO) regression to construct a prognostic model based on differentially expressed genes between PYRclusters (PYRsafescore), which can be applied as an effective prognosis tool. Lastly, we explored the upstream transcription factors of different pyroptotic patterns, thereby identifying 112 compounds with potential therapeutic value in public databases. The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed 6 million people until May 2022 –6. This A possible mechanism linking cytokine storm to organ damage is inflammatory cell death, namely pyroptosis and necroptosis. Pyroptosis has been intensely studied recently. Some patients with severe COVID-19 may develop a systemic cytokine storm because SARS-CoV-2 promotes cytokine storms by inducing pyroptosis in pro-inflammatory blood-born immune cells –19. HowePyroptosis is a mechanism of programmed cell death characterized by the inflow of sodium ions and water mediated by gasdermin proteins, resulting in cell membrane rupture, excessive cell swelling, and spontaneous release of cytosolic contents into the extracellular space . GasdermAlthough pyroptosis is crucial for innate immunity , 26, extIn COVID-19 patients, various cells undergo pyroptosis, including leukocytes  and other type of cells  , 35–39. Based on transcriptome data of patients with or without COVID-19 available in public databases (GSE157103), we found that the leukocytes of ARDS patients with COVID-19 have considerably higher pyroptotic markers than patients without COVID-19. Moreover, at least two different patterns of pyroptosis occur in patients with COVID-19, one correlated with a poor prognosis and the other with a benign prognosis. These two pyroptosis patterns may be regulated by different upstream transcription factor networks, which could prove therapeutically valuable for drug development.From the GEO dataset, we retrieved RNA-seq data (GSE157103) of 126 ARDS patients, namely 100 COVID-19 patients and 26 non-COVID-19 patients, in addition to their clinical data, including gender, age, underlying disease status (diabetes), coagulation , and hospital-free days post 45-day follow-up (HFD45), among other parameters. More specifically, HFD45 is defined as the number of days patients lived outside of a hospital from enrollment through death or the end of follow-up . The higThe label-free quantification (LFQ) intensities of 736 proteins of 126 ARDS patients were collected from Mass Spectrometry Interactive Virtual Environment (MassIVE) (MSV000085703). After calculating the logarithm of the FQL intensities, we used the R package “limma” to calculate the log2(Log2Fold of change) (log2FC) of 736 proteins between two PYRclusters. The 736 proteins were sorted from large to small by log2FC . Then, using the “org.Hs.eg.db” and “clusterProfiler” packages, gene set enrichment analysis (GSEA) was performed based on MSigDB gene sets C2, C5 and C7. Significant gene sets were identified when |Normalized Enrichment Score (NES)|>1 and False Discovery Rate (FDR) < 0.25.Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using the “org.Hs.eg.db” and “clusterProfiler” packages. GO terms and KEGG terms were identified as significantly enriched when p.adjust < 0.05.Single-sample gene set enrichment analysis (ssGSEA) and “Cibersort” were used to analyze the immune cell infiltration fractions. The former was based on the list of Pan-cancer Immune Metagenes , 46.AIM2, CASP1, CASP3, CASP4, CASP5, CASP6, CASP8, CASP9, ELANE, GPX4, GSDMA, GSDMB, GSDMC, GSDMD, GSDME, GZMA, GZMB, IL18, IL1B, IL6, NLRC4, NLRP1, NLRP2, NLRP3, NLRP6, NLRP7, NOD1, NOD2, PJVK, PLCG1, PRKACA, PYCARD, SCAF11, TIRAP, and TNF, we divided 100 COVID-19 patients into two clusters (PYRcluster) using the “nmf” package. We determine the k value based on the consensusMap function.Based on RNA-seq data of 35 pyroptosis-related genes including The differentially expressed genes (DEGs) of two PYRclusters were identified using the “limma” package. Using the median of HFD45 as the cutoff value, COVID-19 patients were divided into two groups. The DEGs in these two groups were calculated using the limma package and subsequently applied for PCA analysis. PC1 and PC2 were used to construct the PYRscore .i × Expression level of signaturei. Using the “caret” package, 100 patients were randomly divided into a training group and a test group with a ratio of 2:1. The model built with the training group data was validated in the test group. We used “ROCR” packages to plot receiver operating characteristic (ROC) curves and to calculate area under the curve (AUC) scores to evaluate model performance.Based on the log2(TPM) of the 570 DEGs between PYRclusters and the HDF45 of each patient, we used the “glmnet” package to build a PYRsafescore model by LASSO regression. We determine the signatures of the model by selecting the lambda value with the smallest mean-squared error by 20-fold-cross-validation. The coefficients of the final signatures were used to calculate the PYRsafescore as follows: protective score = ∑ CoefficientFirst, based on the transcription factor targets (TFT) and their gene sets in MSigDB , we usedWe then used the “CoRegNet” package to enrich the transcription factor co-regulatory network in COVID-19 patients from PYRcluster1 and PYRcluster2 based on the dataset of transcription factor targets  from Harmonizome , 50. LasWe used the transcription factors that we screened as keywords to search for the corresponding compounds on ChEMBL , thus scThe Wilcoxon sum-rank test and the t-test were used to compare different groups, and the Pearson’s product-moment correlation test was used for correlation analysis. All statistical tests were two-sided, and a significant difference was defined as a p-value of 0.05. Power calculations were performed using the following R packages: “pwr” and “rstatix” at sig.level=0.05.A GEO dataset provided RNA-seq data and clinical data from 126 samples of 100 patients with COVID-19 and 26 patients without COVID-19 (GSE157103) . InitialAIM2, CASP1, CASP3, CASP6, CASP8, CASP9, GSDMA, GSDMC, GZMB, IL6, NLRP3, NLRP7, NOD1, NOD2, SCAF11, and TIRAP was significantly higher in blood leukocytes of COVID-19 patients, indicating that the level of pyroptosis was significantly increased , we studied changes in the biological function of leukocytes between the two types of patients. Mismatch repair, homologous recombination, replication, cell cycle, and p53 signaling are more enriched in leukocytes of COVID-19 patients, indicating severe cell damage during viral infection and ongoing damage repair  undergo pyroptosis via the AIM2-Caspase1-gasdermin D and the CARD8-Caspase1-gasdermin D axes , we also compared the proportions of 28 immune cell types between the two groups and found that the numbers of various immune cells are much higher in COVID-19 patients than in non-COVID-19 patients, indicating a highly active immune response in COVID-19 patients  , ventilator-free days and a lower proportion of mechanical ventilation than PYRcluster1 , Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments  using the “limma” package, with cutoff criteria of |logFC| >1 and AIM2, CXCR2 and UBE2W, which were significantly highly expressed in PYRcluster1, were included in the 20 genes associated with distinctly methylated CpG sites between mild and severe COVID-19 patients   regression analysis. In a ratio of 2:1, 100 COVID-19 patients were divided into a training group and a test group, and the model was obtained in the training group. “Lambda-min” was chosen as the best value in the cross-validation procedure (The area under the ROC curve (AUC) values of the model in the training and test groups were 0.907 and 0.879, respectively, indicating that our predictive model performs well .The dataset GSE157103 used in this study is available from: National Center for Biotechnology Information (NCBI), Gene Expression Omnibus (GEO), YT, PZ, and LC designed and supervised the study, PZ collected the data, PZ performed the first part of the analysis, and YT completed the analysis and drafted the manuscript. YT, PZ, and QL prepared the original draft and wrote, reviewed, and edited the manuscript. JX and LC revised the manuscript and provided analytical technical support. All authors contributed to the article and approved the submitted version.This research was funded by the National Natural Science Foundation of China (32170750).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hsp90 is a ubiquitous molecular chaperone involved in many cell signaling pathways, and its interactions with specific chaperones and cochaperones determines which client proteins to fold. Hsp90 has been shown to be involved in the promotion and maintenance of proper protein complex assembly either alone or in association with other chaperones such as the R2TP chaperone complex. Hsp90-R2TP acts through several mechanisms, such as by controlling the transcription of protein complex subunits, stabilizing protein subcomplexes before their incorporation into the entire complex, and by recruiting adaptors that facilitate complex assembly. Despite its many roles in protein complex assembly, detailed mechanisms of how Hsp90-R2TP assembles protein complexes have yet to be determined, with most findings restricted to proteomic analyses and in vitro interactions. This review will discuss our current understanding of the function of Hsp90-R2TP in the assembly, stabilization, and activity of the following seven classes of protein complexes: L7Ae snoRNPs, spliceosome snRNPs, RNA polymerases, PIKKs, MRN, TSC, and axonemal dynein arms. The Hsp90 molecular chaperone is a central regulator of protein homeostasis in eukaryotes under normal and stressed conditions. Hsp90 is involved in the final stages of client protein folding and maturation. In mammals, there are two cytoplasmic Hsp90 isoforms, Hsp90α and Hsp90β, while in yeast, Hsp82 and Hsc82 are the inducible and constitutively expressed Hsp90 isoforms, respectively . Hsp90 iIn addition to stabilizing tertiary structure, Hsp90 and its cochaperones stabilize the quaternary structure of various macromolecular complexes. In 2005, our group identified Tah1 and Pih1 as Hsp90 interactors in yeast . Tah1 anin cellulo . L. L155]. 50 values of 22 and 3 nM, respectively . T. T162]. ectively . In addiectively . NeverthThe TSC complex may be stabilized through the PAQosome. Co-IP experiments in HeLa cells showed that FLAG-tagged URI1 and RPAP3 interacted with endogenous TSC1 and TSC2 . TAP-MS Motile cilia are small microtubule-based organelles required for fluid transport and cell motility in many organisms. In humans, motile cilia are essential for the generation of left-right asymmetry during embryonic development, sperm motility, and the movement of fluid in the respiratory tract, brain ventricular system, and oviducts . Motile DNAAFs were discovered through genetic analyses of families with primary ciliary dyskinesia and mutation studies in animals ,178,179.In vertebrates, the PIH1 domain is present in at least four proteins: DNAAF2, DNAAF6, PIH1D1, and PIH1D2 ,182,183.DNAAF4 contains a C-terminal TPR domain, and a yeast two-hybrid screen showed that it interacts with Hsp70 and Hsp90 C-termini via the EEVD motif that binds TPR domains . These iDNAAF1 and DNAAF7, which lack Hsp90 binding domains, have been linked to Hsp90. Streptavidin-II/FLAG tandem affinity purification coupled with mass spectrometry (SF-TAP/MS) experiments using HEK293 lysates showed that DNAAF1 associates with several Hsps, including Hsp70 and Hsp90 . AlthougDNAAF11 (formerly named LRRC6) is another essential protein for dynein arm assembly ,190. HEKThe R2TP complex may be involved in late-stage dynein arm assembly. Similar to DNAAFs, the catalytic components of R2TP, RUVBL1 and RUVBL2, were demonstrated to be involved in dynein arm assembly through mutational analyses in animal models. Inducible deletion of RUVBL1 in mouse oviducts resulted in the absence of outer dynein arms and the appearance of undefined protein clusters . StreptaIn a conditional mouse model, loss of RUVBL1 resulted in immotile spermatozoa due to reduced ODA components, DNAI1 and DNAI2 . In mousChlamydomonas, experiments using insertion and truncation mutants showed that WDR92 is needed to stabilize ODA and IDA heavy chains during preassembly [Drosophila WDR92 was shown to interact with CG18472, the closest Drosophila orthologue of human SPAG1 [In addition to R2TP and R2TP-like complexes, proper dynein arm assembly requires WDR92 (recently renamed DNAAF10), which is also highly expressed in human testes . In Chlaassembly ,201. Co-assembly ,202, sugan SPAG1 ,203. A pan SPAG1 . These fDrosophila sperm. While investigating Drosophila spermiogenesis, Fingerhut and colleagues identified a novel RNP granule located at the axoneme distal end, the site of ciliogenesis [RUVBL1 and RUVBL2 were recently demonstrated to be involved in the synthesis of cytoplasmic cilia, in which the axoneme is exposed to the cytoplasm ,205. CytThese studies demonstrate that Hsp90 and R2TP have many diverse and essential roles in macromolecular complex assembly that often complement each other. Before complex assembly, Hsp90 initiates subunit expression  and regulates RNA levels . During complex assembly, Hsp90 stabilizes clients that compose multisubunit complexes , while R2TP mediates important interactions between Hsp90 and clients , adaptors and clients , and complex subunits . After complex assembly, Hsp90 is critical for the function of some complexes , and R2TP together with its associated prefoldin-like module are involved in the localization of assembled protein complexes . Thus, in addition to the canonical role of Hsp90 in client stabilization, these findings highlight the additional roles of Hsp90, together with R2TP, in quaternary complex assembly. Determining how Hsp90 integrates its clients into multiprotein complexes may facilitate the discovery of novel therapeutic drug targets. For example, inhibiting Hsp90-mediated TELO2-mTOR interactions may be an effective adjuvant against mTORC1-driven tumors. Thus, the role of Hsp90 during complex assembly and how it functions with its chaperones and cochaperones, especially TTT and R2TP, should be further investigated.


Expanding knowledge of the molecular mechanisms at the basis of tumor development, especially the cross-talk between oncogenic pathways, will possibly lead to better tailoring of anticancer therapies. Nuclear factor erythroid 2-related factor 2 (NRF2) plays a central role in cancer progression, not only because of its antioxidant activity but also because it establishes cross-talk with several oncogenic pathways, including Heat Shock Factor1 (HSF1), mammalian target of rapamycin (mTOR), and mutant (mut) p53. Moreover, the involvement of NRF2 in gammaherpesvirus-driven carcinogenesis is particularly interesting. These viruses indeed hijack the NRF2 pathway to sustain the survival of tumor cells in which they establish a latent infection and to avoid a too-high increase of reactive oxygen species (ROS) when these cancer cells undergo treatments that induce viral replication. Interestingly, NRF2 activation may prevent gammaherpesvirus-driven oncogenic transformation, highlighting how manipulating the NRF2 pathway in the different phases of gammaherpesvirus-mediated carcinogenesis may lead to different outcomes. This review will highlight the mechanistic interplay between NRF2 and some oncogenic pathways and its involvement in gammaherpesviruses biology to recapitulate published evidence useful for potential application in cancer therapy. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that protects cells from oxidative stress by regulating various phase II detoxifying/antioxidant enzymes, such as heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), catalase, superoxide dismutase (SOD), and glutathione (GSH) ,4. Of noNRF2 activation is tightly regulated by Kelch-like ECH-associated protein 1 (Keap1), an ubiquitin ligase that, in unstressed conditions, interacts with NRF2 triggering its proteasomal degradation in the cytoplasm  Figure a. Oxidatsequestosome 1 (SQSTM1), which induces a “non-canonical stabilization/activation” of NRF2 by triggering Keap1 proteasomal degradation [Another important molecule involved in NRF2 regulation is p62/radation ,10,11  (target of oncosuppressor p53) can bind to Keap1 and interrupt the Keap1/NRF2 interaction, activating NRF2 . MoreoveAnother fundamental function of NRF2 is to counteract inflammation that, along with ROS detoxification, plays a key role in preventing carcinogenesis, as evidenced by several studies performed in vitro and in vivo in animal models . The rolThe other common target of NRF2 and HSF1 is p62/SQSTM1, known to play a pro-tumorigenic role not only because it promotes NRF2 stabilization  but alsoNrf2 gene increase the susceptibility of cancer cells to the cytotoxic effects of mTOR inhibitors [Brucea javanica able to interfere with NRF2-mediated defense mechanisms [Another important pathway that may directly interact with NRF2 is the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR, which is involved in regulating a variety of vital cellular processes . This pahibitors . Regardihibitors , a molechibitors . The acthibitors ,34. The hibitors . In addihibitors . Furtherchanisms ,37, can chanisms ,39. Thischanisms .As anticipated above, a key role in NRF2 regulation is played by its phosphorylation mediated by several kinases, whose activation may be influenced, in turn, by NRF2, in feedback loops. Among those kinases, there are the above-mentioned PI3K/AKT and ERK1/2, but also protein kinase C (PKC) , c-jun NNRF2 may cross-talk with wild-type (wt) and mutant (mut)p53, inhibiting the wtp53 oncosuppressor functions and strengthening the mutp53 oncogenic functions. Both effects contribute to tumor progression and cancer cell resistance to the cytotoxic effects of anticancer therapies. The oncosuppressor p53 is the sensor of DNA damage that activates target genes involved in cell cycle arrest, senescence, or apoptosis, according to the extent of genotoxic damage ,50. In pWe have shown that NRF2 activation, by, for instance, high glucose or natural compounds , may reduce p53 apoptotic function ,38,57,58Besides being deregulated at the protein level, p53 is inactivated by gene mutations in almost 50% of all types of tumors; those missense mutations often diminish the p53 ability to bind specific DNA recognition sequences of wild-type target genes, losing their oncosupressor function . Mutp53 Nrf2 depletion decreases pancreatic carcinogenesis and cancer invasion [Among the several oncogenic transcription factors that interact with mutp53 to promote cancer progression  is NRF2,invasion . In anotinvasion . This coinvasion . In addiinvasion ,24,39. THypoxia is a hallmark of solid tumors, and it is responsible for activating hypoxia-inducible factor-1 (HIF-1) oncogenic signaling to promote tumor progression, invasion, and chemoresistance . There iBesides mutp53, other oncogenes have been reported to affect NRF2 activity, e.g., Myc has been shown to activate NRF2 and induce tumorigenesis in cells undergoing carcinogenic treatment . InteresWe have recently shown in vitro that the silencing of p62/SQSTM1 and NRF2 can counteract the Epstein-Barr virus (EBV)-driven B lymphocyte immortalization . This efRegarding gammaherpesvirus-driven carcinogenesis, it has been shown that, besides latent antigens, proteins expressed during the activation of the lytic cycle may contribute to cancer onset . This isThis update on the key role of NRF2 in cancer survival, progression, and chemoresistance highlights how several of its induced effects rely on the collaboration of NRF2 with oncogenic pathways, such as HSF1, mutp53, and mTOR, and with pro-tumorigenic molecules, such as p62/SQSTM1, sometimes in a feedback loop. Of note, we also highlight how NRF2 activation is exploited by gammaherpesvirus-driven carcinogenesis and immune suppression .Interestingly, the NRF2 detoxifying activity, hijacked by cancer cells as a protective mechanism, particularly in the course of anti-cancer treatments, is also important in cancer prevention. Therefore, NRF2 manipulation could be a valuable strategy both to prevent cancer and to inhibit its progression, e.g., through the restoration of wtp53 apoptotic activity. In addition, given the key role of NRF2 in gammaherpesvirus-driven carcinogenesis, targeting NRF2 could represent a promising strategy to counteract cancers associated with them. Indeed, NRF2 manipulation may help to reduce the capability of these viruses to infect target cells, to prevent the transformation of cells from which their associated cancers arise, to counteract viral lytic antigen expression and the inflammation that they promote, and, last but not least, to interfere with their-induced immunosuppression. Of note, in cancers associated with gammaherpesviruses, both NRF2 inhibition and NRF2 activation may represent a promising therapeutic approach, as ROS, unless they are too high, can sustain pro-survival, oncogenic pathways, such as STAT3 ,100,101.