Sarcoidosis is a rare, systemic inflammatory disease whose diagnosis and management can pose a challenge for clinicians and specialists. Scientific knowledge on the molecular pathways that drive its development is still lacking, with no standardized therapies available and insufficient strategies to predict patient outcome. In recent years, oxidative stress has been highlighted as an important factor in the pathogenesis of sarcoidosis, involving several enzymes and molecules in the mechanism of the disease. This review presents current data on the role of oxidative stress in sarcoidosis and its interaction with inflammation, as well as the application of antioxidative therapy in the disease. Sarcoidosis is a systemic inflammatory disorder of unknown cause whose pathogenesis is mainly attributed to T-lymphocytes, mononuclear phagocytes, and the accumulation of non-caseating granulomas. Exposure to an unidentified foreign antigen, aberrant innate immune response and possibly a genetic predisposition are all suspected factors leading the pathobiology. There are variable disease manifestations, but the lungs are affected in over 90 percent of cases with pulmonary involvement accounting for most of the morbidity and mortality. Disease course is characterized by episodic flare-ups and remissions that are impossible to predict. Prognosis is extremely variable, and up to one third of patients may develop unremitting disease resulting in progressive organ impairment .Despite the recent advances in understanding the mechanisms underlying this disease, sarcoidosis remains a challenging diagnosis, and there are still no validated biomarkers that can assist the clinician in diagnosis or prediction of disease outcome ,3,4. AppDefined as an imbalance between the production and the elimination of oxidant compounds, oxidative stress may underlie some of the molecular mechanisms that characterize sarcoidosis pathogenesis. The role of oxidative stress has been extensively documented in several lung diseases, including chronic obstructive pulmonary disease (COPD) and lung fibrosis ,6. Even This review evaluates oxidative stress in sarcoidosis and discusses the potential role of antioxidants and possible targets to reduce oxidative stress in sarcoidosis patients. In living systems, oxidative stress is linked to the presence of reactive chemical species that are naturally produced at a cellular level. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the main protagonists of oxidant production.2−), hydrogen peroxide (H2O2), hydroxyl radical (OH) and singlet oxygen (1O2), among others. They represent a type of oxygen-derived intermediates that are partially reduced, highly reactive and unstable [ROS is a term that collectively indicates chemical species such as superoxide radical (Ounstable . ROS areunstable . ROS areunstable . However−). Peroxynitrite can cause nitrosative damage to various biological targets, but it can also react with other molecules to form additional types of RNS, including nitrogen dioxide (NO2) and dinitrogen trioxide (N2O3). Increased RNS production has been shown in acute lung injury and adult respiratory distress syndrome (ARDS), suggesting a role in the pathophysiology of inflammatory lung diseases [RNS are a family of molecules that are produced starting with the rapid reaction of nitric oxide (NO) with superoxide to form peroxynitrite , inflammation, or when antioxidant defenses are impaired or limited. ,13. Unde2) is administered clinically [Of all human organs, the lung is particularly susceptible to damage caused by OS. For a start, the respiratory surface area is exposed to higher oxygen tension than other tissues. The direct injury to cells caused by air oxygen is exemplified by the notorious detrimental effects of hyperoxia that can be observed when an increased fractional inspired oxygen  to CD4+ T lymphocytes. In turn, T lymphocytes amplify the immune response through release of Th1-type cytokines such as interleukins (IL) 12, 18 and 27, interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) at sites of inflammation. These mediators promote macrophage accumulation, activation, and aggregation, while others such as IL-6 and IL-8, which are produced by macrophages, act as lymphocyte and neutrophil chemotactic factors, respectively . These eIn sarcoidosis, the activation of the macrophage capability to produce toxic oxygen molecules might play a crucial role in developing the tissue damage. In this regard, the first pieces of evidence regarding increased oxidative burden in sarcoidosis date back to over 30 years ago. Pioneering studies on lung alveolar cell populations demonstrated that resident macrophages from sarcoid patients with a high-intensity lymphocytic alveolitis produce higher amounts of superoxide anion, both spontaneously and in the presence of a triggering agent. This capability is dependent on IFN-γ levels, and macrophages can alternate states of high or low activity depending on local environment ,28,29. WIn the study of lung diseases, oxidative stress biomarkers can be evaluated in several biological matrices. In COPD, for example, evidence of OS has been gathered from markers in the peripheral blood and urine, as well as from bronchoalveolar lavage fluid (BALF), induced sputum and exhaled breath condensate (EBC) . In sarcIn vivo measurement of products of lipid peroxidation such as malondialdehyde (MDA), exhaled volatile alkanes, and prostaglandin-like compounds has received much attention over the last decades. Lipid peroxidation is, in fact, a major effect of oxidative damage, and there is abundant evidence showing the association between the levels of these biomarkers and the development of various diseases .Malondialdehyde (MDA) is a product of radical-induced peroxidation of fatty acids and is one of the most studied biomarkers of oxidative stress in several diseases, usually with thiobarbituric acid (TBA) assay methods (TBARS) ,37. In aIsoprostanes have emerged as reliable markers of oxidative damage in a wide variety of diseases and are one of the most used approaches to assess free radical injury in patients with lung disease. It has been postulated that isoprostanes may have a potential role as mediators of oxidant injury, a theory that has obvious therapeutic implications ,42. 8-epThe presence of 8-isoprostane in EBC has also been investigated with great interest, as this procedure has the advantage of being non-invasive and thus may represent a suitable candidate for routine disease follow-up. However, there are conflicting studies regarding the ability of 8-isoprostane in EBC to correlate with disease activity in sarcoidosis. In their study published in 2004, Psathakis et al. measured increased concentrations of this marker of oxidative stress in the EBC of 30 patients with sarcoidosis compared to 12 control subjects. Furthermore, 8-isoprostane levels correlated with disease activity as measured by serum angiotensin-converting enzyme (ACE) level, serum calcium level, and pulmonary function test results. 8-Isoprostane levels in patients with non-active disease did not differ from those in healthy subjects . A laterVolatile organic compounds (VOCs) such as ethane and pentanes are products of the interaction of ROS with biological components and can be measured in the exhaled breath . DespiteOxidative damage to the DNA has been studied in sarcoidosis, although to a lesser extent than other OS biomarkers and specifically in cardiac sarcoidosis. 8-hydroxydeoxyguanosine (8-OHdG), an oxidized nucleoside, is the most frequently studied DNA alteration, and is considered a marker that can be measured in urine and serum for assessing total oxidative burden . UrinaryIn the mitochondria, ROS are normally generated as by-products of aerobic metabolism of oxygen during electron transport chain activity. Inappropriate ROS production resulting from mitochondrial impairment is thought to underlie various chronic lung diseases, potentially contributing to lung cell dysfunction and disease progression ,63.The role of mitochondrial dysfunction in sarcoidosis has only just started to be unveiled. It was observed that mitochondria do show notable morphological changes in the capillary endothelial cells in patients with sarcoidosis, as demonstrated in the study carried out by Mochizuki et al., who in 2011 examined tissues of the bronchus and lung obtained from 16 patients and 11 controls and observed a close relationship between mitochondria and lipid droplets in capillary endothelial cells of the respiratory tract. The authors postulated that this relation may be involved in the pathogenesis of sarcoidosis . The genThe lung has an efficient built-in antioxidant defense system that operates through an enzymatic cascade that prevents the production of oxidant species. At the same time, scavengers convert oxidants to fewer toxic compounds, blocking the secondary production of intermediate species. The lung epithelial cells are rich with constitutively acting antioxidant enzymes such as glutathione (GSH) peroxidase, catalase (CAT), redoxins, and superoxide dismutase (SOD) that maintain the redox balance within the cells. In the airway secretions, albumin and mucin also show spontaneous antioxidant activity through their exposed sulfhydryl groups. Furthermore, the epithelial lining fluid contains several other non-enzymatic antioxidants such as ascorbic acid (vitamin C), alpha-tocopherol (vitamin E) and uric acid.Diminished antioxidants defenses have been demonstrated in sarcoidosis, both locally and systemically. The most studied antioxidants in sarcoidosis are listed in 2O2 scavenging capacity that have been described as key modulators of oxidative stress response pathways through their ability to rapidly regulate the activation of transcription factors involved in the antioxidant defense system [Redoxins are small redox enzymes with He system . The rese system . In the e system .The involvement of redoxins in pulmonary sarcoidosis has been investigated in the early 2000s with the help of immunohistochemistry. In the year 2000, Koura et al. first associated thioredoxins with sarcoidosis . Their a2O2 into water and oxygen), CAT can also catalyze the oxidation of various metabolites and toxins, including formaldehyde and alcohols [2− to H2O2 and oxygen. H2O2 is then reduced to water by CAT and GPx. SOD also inhibits the oxidative inactivation of nitric oxide (NO). In studies conducted by Lakari et al. on granulomatous and interstitial lung diseases, manganese-SOD (MnSOD) and CAT levels were compared to control subjects. In a work published in the year 1998, the group examined lung biopsies of granulomatous diseases: 22 patients with pulmonary sarcoidosis and 10 with extrinsic allergic alveolitis [CAT is the oldest known and first discovered antioxidant enzyme. It is expressed in every organ, particularly in tissues containing relatively high activity such as liver, kidney, and red blood cells . In addialcohols . In the veolitis . Intenseveolitis ,82,83. Hveolitis . A more veolitis . The stuGlutathione is a low-molecular-weight antioxidant that is essential to balance oxidative stress in most living systems and the most abundant thiol in animal cells. It exists in reduced (GSH) and oxidized (GSSG) states. In the reduced state, the thiol group is a source of one reducing equivalent and glutathione disulfide (GSSG) is thereby generated, with the GSSG/2GSH ratio serving as a good indicator of the cellular redox state . GSH proSerum paraoxonase-1 (PON1) is a glycoprotein which binds to circulating high-density lipoprotein (HDL) particles and preserves their function through inactivation of lipid peroxidation. It was also shown to inhibit low-density lipoprotein (LDL) oxidation, thus protecting endothelial cells from apoptosis . Thanks In sarcoidosis, downregulation of PON1 has been observed by Uzun et al. in 2008. Their work focused on blood analysis of patients with active (26) and inactive (37) sarcoidosis plus 48 healthy subjects. Serum PON1 activity, MDA levels and oxidized LDL were determined. Results showed that PON1 levels were significantly lower in the active disease state group than both the inactive form and control groups, with a negative correlation with MDA levels. Oxidized LDL levels were significantly increased in both the active and inactive groups than the control group, a result suggesting that the increasing oxidative stress in sarcoidosis might be due to increased lipid peroxidation . In thisThe relevance of redox imbalance to the pathogenesis of sarcoidosis, coupled with the correlation of many biomarkers with disease activity, have understandably induced the scientific community into evaluating oxidative stress as a potential novel source of therapeutic targets. Current therapies for sarcoidosis focus primarily on pharmacologically limiting inflammation, and include corticosteroids, immunosuppressants and anti-TNF-α agents, which are all associated with variable response and important side effects. Oxidative stress provides a set of pathways in the pathogenesis of sarcoidosis that are yet to be fully explored and that are intertwined with inflammation. The high production of oxidant species that occurs in sarcoidosis consumes antioxidants. For this reason, it can be hypothesized that antioxidant supplementation might be beneficial in sarcoidosis treatment, but there have been very few studies on the subject.N-acetylcysteine (NAC), a thiol-containing mucolytic agent and the precursor of glutathione, has a well-established antioxidant activity . ConsequIn recent years, antioxidant research worldwide has particularly focused on quercetin. Quercetin is a natural compound of the flavonoid group of polyphenols. It is abundant in many fruits, vegetables, seeds, and grains, and is also used as a dietary supplement . QuercetAnother drug that has been shown to have antioxidant activity is Pirfenidone, a medication used for the treatment of IPF that works primarily through downregulation of the production of growth factors and procollagens I and II, although the exact mechanisms are elusive. Pirfenidone shows superoxide radical scavenging activity beyond its well-known antifibrotic effect and has shown a potent antioxidant role in experimental cirrhosis . CurrentSarcoidosis is an inflammatory condition of unknown cause that usually occurs in the lungs and lymph nodes, but it can affect almost any organ. Some individuals develop disabling disease despite corticosteroids and immunosuppressants, with an impelling need for effective tools to predict disease progression and drug efficacy and for better therapeutic approaches to preserve organ function. Over recent years, persuasive evidence has accumulated demonstrating that oxidative stress is a critical component of the pathology and progression of sarcoidosis and is intertwined with pathways of inflammation that are implicated in the disease see . Increas


Saccharomyces cerevisiae represents a valuable model organism for understanding human genetic variability. In the present review, we show how S. cerevisiae has been used to study variants of genes involved in different diseases and in different pathways, highlighting the versatility of this model organism.At present, the great challenge in human genetics is to provide significance to the growing amount of human disease-associated gene variants identified by next generation DNA sequencing technologies. Increasing evidences suggest that model organisms are of pivotal importance to addressing this issue. Due to its genetic tractability, the yeast  The advent of next generation DNA sequences technologies allows us to identify individual genetic variants, and this revolutionized the strategy for discovering the cause of several genetic and multifactorial diseases. The association between genetic variants and disease risk can potentially overturn our understanding of common disease. The discovery of pathways and processes that are causally involved in a disease, so far not linked, may open the way towards the development of targeted therapies and prevention strategies. However, the knowledge of individual genetic variants is not sufficient for determining the causality or predisposition to a particular disease without a clear picture of the impact of the genetic variants on the protein activity. The analysis of genetic variants resulted in the successful identification of impaired proteins underlying many Mendelian disorders. However, our knowledge is far from complete, especially for rare variants . While fSaccharomyces cerevisiae is a unicellular eukaryote representing an invaluable tool for uncovering much of the basic biology considering that it has helped to characterize pathways common to human cells. An example is the discovery and mechanistic understanding of autophagy, a fundamental process involved in cancer, neurological disorders, and other diseases, allowing Yoshinori Ohsumi to be awarded with the Nobel Prize in Physiology in 2016 ..SOD1 encAPRT), an enzyme involved in the salvage pathway of the purine metabolism. Purine metabolism plays an important role in nucleic acid synthesis, energy-dependent reactions, and cellular signaling. The interest towards this gene arises from the fact that mutations in APRT determined adenine phosphoribosyl transferase deficiency (APRTD), a rare monogenic disease [ade2∆ apt1∆ aah1∆ since it is defective for de novo purine synthesis  and not able to grow without additional purine   of trio kinase (TKFC), a bifunctional enzyme component of the fructose metabolism pathway associated with a fatal infantile multisystem disorder . The impne (DHA) . On the genicity .MARS1) identified in Charcot-Marie-Tooth disorder type 2U (CMT2U); in glycyl-tRNA synthetase 1 (GARS1) associated with CMT (CMT2D) and with Spinal muscular atrophy; and infantile, James type (SMAJI), and valyl-tRNA synthetase (VARS1), associated with developmental encephalopathy with microcephaly have been successfully studied in yeast [MARS1, GARS1, and VARS1, complement the function of strains deleted for the yeast orthologs, mes1∆, grs1∆, and vas1∆, respectively . The 5-FOA acid is toxic for URA3 positive cells because the enzyme coded by URA3 gene will convert it into a toxic compound causing cell death. Interestingly, Siekiersk et al. demonstrated that VARS1 variants are loss of function in yeast and precisely recapitulated some of the key neurological disease traits in Zebrafish [MARS1 variants and other aminoacyl transferase genes [In the previously described assays, the yeast ortholog was not an essential gene. Many reports demonstrate that is possible to use yeast to also study genes that are essential. This is the case of aminoacyl-tRNA transferases. Mutations in the gene methionyl-tRNA synthetase  in RNA biology is undisputed , POLR2A was not involved in human disease until 2016, when somatic mutations were discovered in a subset of meningioma [de novo variants in POLR2A were identified in 16 cases of neurodevelopmental syndromes characterized by profound infantile-onset hypotonia and associated with the disease [RPB1, is an essential gene; therefore, to study POLR2A variants in yeast, they used the strategy described above. Gene variants were then studied by constructing yeast strains carrying the yeast RPB1 mutagenized in order to create the amino acid substitution observed in the human gene. In addition to the growth assay in rpb1∆ strain, the POLR2A variants where further studied in yeast deleted in transcription factors DST1 (yeast ortholog of TFIIS) or SUB1 (yeast ortholog of PC4). This assay allowed determining how mutations in this subunit of RNA polymerase affect transcription fidelity. Altogether, on the basis of the results of both predictive and functional analyses, eleven variants were classified as probably disease-causing [POLR2A variant classification was leveraged to study other cases of individuals with neurodevelopmental syndromes, strengthening the association of POLR2A with this disease [Similar strategies have been adopted to study the variants of ningioma . Recentl disease . The imp-causing . Recentl disease .NADSYN1, coding for NAD synthetase 1 which is the final enzyme of the nicotinamide adenine dinucleotide (NAD) de novo synthesis pathway associated with congenital NAD deficiency disorder [QNS1 deletion may be overcome through exogenous supplementation of nicotinamide riboside (NR)  . In a caynthesis .PYROXD1. PYROXD1 encodes a protein with an N-terminal pyridine nucleotide-disulfide oxidoreductase domain and a C-terminal NADH-dependent nitrite reductase domain. Recently, biallelic PYROXD1 gene mutations were identified by exome sequencing in nine patients with congenital myopathy from five unrelated families. The mutations segregated with the disorder in the families, suggesting that they are responsible for the disease. Unfortunately, at the time of variants’ discovery, the function of the protein was not determined. In order to study the activity of the protein and the effect of variants on protein activity, O’Grady et al. developed a complementation assay in yeast. They observed that the human PYROXD1 cDNA rescues the oxidative stress defect observed in yeast glutathione oxidoreductase glr1∆ deletion mutant strain, indicating that PYROXD1 has an oxidoreductase activity in vivo in yeast cells. PYROXD1 variants expressed in glr1∆ grown in medium containing H2O2 had a lower growth rate in the presence of H2O2 compared to wild-type PYROXD1  at low copper concentrations and in the post-Golgi compartments, and the plasma membrane at higher concentrations. A hypothesis formulated on how the mutations could affect the transporter has been verified in the ccc2Δ yeast strain, deleted for the yeast ortholog of ATP7A . The assembled SDH complex is involved in two essential energy-producing metabolic processes of the cell, the Krebs cycle and the electron transport chain [sdh1Δ (ortholog of SDHA) that do not have a functional SDH complex, and thus non-functional mitochondria. The sdh1∆ mutant is able to grow in the presence of a fermentable carbon source (glucose) but not in the presence of a non-fermentable carbon source (glycerol). Since the expression of the wt Sdh1 restores the growth on glycerol, this assay was the readout for measuring the activity of SDHA variants. In order to further corroborate the results of variants functionality from growth assays, they tested the functionality of mitochondria. By means of yeast assays and computational model of protein structure of variants, they were able to draw conclusions on functional impact of variants [In addition to the mutations of genes involved in cell cycle and growth, cancer cells also carry mutations in genes involved in metabolism. This is the case of succinate dehydrogenase (SDH), a complex of four-subunit with mitochondrial localization encoded by nuclear genes (rt chain . Loss-ofrt chain . Tumor frt chain , data frvariants . The use of yeast has, of course, some limitations. We have to consider that some human proteins expressed in the model organism will not be as functional as in their native context of a human cell. This can be determined by the fact that some variants may alter interactions with proteins not present or too divergent in yeast; some may impact protein expression/activity by altering transcript splicing or binding sites for miRNA or mRNA binding proteins, which are conditions that cannot be recreated in yeast expressing the cDNA. Moreover, as a unicellular model organism, yeast fails to capture phenotypes resulting from inter-cellular interactions so it may not be suitable, for instance, for studying genes involved in development . HoweverWe showed here a significant selection of assays developed in the yeast S. cerevisiae for understanding human genetic variation. Moreover, we showed that yeast assays can be developed and further validated for studying human proteins with different functions and involved in several pathways and diseases. A precondition for setting functional assays is the identification of a measurable phenotype and, as shown here, yeast is a simple and tractable organism, sharing conserved pathways with higher eukaryotes; this allows facilitating the identification of the experimental conditions in order to study a specific protein. An added value is the availability of tools such as strain collections, databases, molecular toolkits and methods that have accumulated over the years ,89,90. TThe results from yeast functional assays cannot stand alone; hence, they are considered as indications complementary to additional clinical and experimental data. Yeast functional assays can be also considered as a method for rationally prioritizing experiments to be conducted in more complicated and expensive model organisms ,94. In a


Growing evidence suggests that the presence of C4 photosynthesis alone is insufficient to account for drought resilience in grasses, pointing to other adaptations as contributing to tolerance traits. The majority of grasses from the Chloridoideae subfamily are tolerant to drought, salt, and desiccation, making this subfamily a hub of resilience. Here, we discuss the evolutionary innovations that make C4 grasses so resilient, with a particular emphasis on grasses from the Chloridoideae (chloridoid) and Panicoideae (panicoid) subfamilies. We propose that a baseline level of resilience in chloridoid ancestors allowed them to colonize harsh habitats, and these environments drove selective pressure that enabled the repeated evolution of abiotic stress tolerance traits. Furthermore, we suggest that a lack of evolutionary access to stressful environments is partially responsible for the relatively poor stress resilience of major C4 crops compared to their wild relatives. We propose that chloridoid crops and the subfamily more broadly represent an untapped reservoir for improving resilience to drought and other abiotic stresses in cereals.Grasslands dominate the terrestrial landscape, and grasses have evolved complex and elegant strategies to overcome abiotic stresses. The C Chloridoid grasses have evolved unique adaptations to adverse environments and represent an untapped reservoir for improving resilience to drought and other abiotic stresses in cereals. Pooideae is the largest subfamily of grasses and includes the temperate cereals wheat (Triticum aestivum), barley (Hordeum vulgare), oat (Avena sativa), and rye , as well as most pasture grasses. All BOP clade grasses utilize the C3 pathway of photosynthesis, and most independent origins of frost tolerance in grasses are found within the Pooideae , maize (Zea mays), sorghum (Sorghum bicolor), and various millets are in Panicoideae and the under-resourced grain crops finger millet (Eleusine coracana) and teff (Eragrostis tef) are in the Chloridoideae. In this review, we focus on the PACMAD grasses and the evolution of the abiotic stress tolerance that empowered their dominance and diversification. We ask which factors fostered the evolution of stress tolerance in these grasses, and why all C4 PACMAD grasses are not drought-tolerant.The majority of species in the grass family falls into two evolutionarily and phenotypically distinct clades, BOP  and PACMAD , named for the subfamilies they contain . Most spPooideae . Conversolerance . The agr4 grasses in the United Nations Food and Agriculture Organization global crop production database, only two, finger millet and teff, are chloridoid grasses  leading all other plants in terms of global production. Water availability is thought to have been a major driving force of C4 grass evolution and diversification  and Oropetium capense, are able to equilibrate to low atmospheric moisture for months without dying, while other C4 species such as switchgrass (Panicum hemitomon) are semi-aquatic, requiring regular flooding to survive , increased leaf hydraulic conductance results in lower WUE  catalyzes the reaction fixing inorganic bicarbonate (HCO3), which is in equilibrium with CO2, into organic acids. These organic acids are then transported to bundle sheath cells where they are decarboxylated, raising the bundle sheath CO2 concentration and allowing Rubisco to operate more efficiently , NAD-dependent malic enzyme (NAD-me), and phosphoenolpyruvate carboxykinase (PCK). In NADP-me plants, malate is the primary C4 acid being transported between mesophyll and bundle sheath cells, while aspartate is the primary transported acid in NAD-me and PCK C4 grasses. WUE is correlated with the C4 subtype, with NAD-me grasses having higher WUE under drought stress than NADP-me grasses  and fonio millet (Digitaria exilis) use far less water  in a semi-arid environment revealed that maize is the highest yielding crop, followed by sorghum, with pearl millet having the lowest yield , African rice (Oryza glaberrima), pearl millet, and cowpea (Vigna unguiculata) were domesticated around the Niger River, likely in the early Holocene when the “green Sahara” slowly desertified  and iburu (D. iburua) were domesticated in the central delta and Jos plateau of Nigeria, respectively, and drought-tolerant iburu is intercropped with fonio as a fail-safe under low rainfall  and by predoctoral training award T32-GM110523 from the National Institute of General Medical Sciences of the NIH (to J.P.).Conflict of interest statement. None declared.


Discovered in 2009, the DEP-domain containing mTOR-interacting protein, DEPTOR, is a known regulator of the mechanistic target of rapamycin (mTOR), an evolutionarily conserved kinase that regulates diverse cellular processes in response to environmental stimuli. DEPTOR was originally identified as a negative regulator of mTOR complexes 1 (mTORC1) and 2 (mTORC2). However, recent discoveries have started to unravel the roles of DEPTOR in mTOR-independent responses. In the past few years, mTOR emerged as an important regulator of skeletal development, growth, and homeostasis; the dysregulation of its activity contributes to the development of several skeletal diseases, both chronic and genetic. Even more recently, several groups have reported on the relevance of DEPTOR in skeletal biology through its action on mTOR-dependent and mTOR-independent pathways. In this review, we summarize the current understanding of DEPTOR in skeletal development and disease. The DEP-domain containing mTOR-interacting protein (DEPTOR), also known as DEP-domain containing 6 (DEPDC6), has gained more attention from the scientific community since its discovery in 2009 due to the increasing amount of data revealing its crucial role in diverse biological processes and pathological conditions. Early studies discovered its function as a member of the mechanistic target of rapamycin (mTOR) complexes, mTORC1, and mTORC2 . HoweverDEPTOR is encoded by a gene located on chromosome 8 (cytogenetic location 8q24.12) and is only present in vertebrates, sharing a high degree of homology between different organisms . DEPTOR DEPTOR is also regulated at the transcriptional level, but contrary to its posttranslational regulation, the transcriptional control of DEPTOR is more tissue and environment specific. Several growth factors such as transforming growth factor β (TGFβ) and epidermal growth factor (EGF) have been associated with changes in DEPTOR, decreasing its expression , a transcriptional regulator that responds to DNA damage, hypoxia, and glucose deprivation, promotes the expression of DEPTOR in cancer cells under hypoxia  of S6K1 and AKT , outputsDeptor was associated with bone mineral density (BMD)  . Given tty (BMD) . A very via membranous ossification, whereas endochondral ossification forms the appendicular skeleton and some parts of the axial skeleton. During the process of endochondral bone formation, chondrocytes acquire diverse shapes and express different genes that characterize the different stages of chondrocyte development. Several signaling pathways act in concert to regulate skeletal development such as wingless-related integration site (WNT), TGFβ, bone morphogenetic protein (BMP), parathyroid hormone (PTH), and parathyroid hormone-related peptide (PTHrP)  .Raptor at the early limb bud stage using Prx-Cre impaired skeletal growth through decreased chondrocyte size and matrix production and delay in chondrocyte hypertrophy and bone formation (Tsc1) induced increased chondrocyte proliferation and decreased differentiation, resulting in a chondrodysplasia phenotype   and in the PTH and PTHrP receptor, parathyroid hormone 1 receptor (PTH1R) genes, respectively (JMC). We observed a decreased activity of mTORC1 and mTORC2 due to the accumulation of DEPTOR, which was also found in JMC. Our data demonstrated that SIK3 is an essential positive regulator of mTOR signaling that functions by triggering DEPTOR degradation by the proteasome in response to PTH/PTHrP signaling during skeletogenesis  . These togenesis .We showed that the accumulation of DEPTOR resulted in a disorganization of the growth plate in JMC patients, which was characterized by a severe reduction in the number of hypertrophic chondrocytes. Also noticeable was the ectopic invasion of bone into the cartilaginous growth plate. Analysis of the localization of DEPTOR protein in healthy human growth plates determined that DEPTOR was mostly expressed in resting and proliferative zones and decreased in hypertrophic chondrocytes. In contrast, a decreased expression of DEPTOR was not observed in the hypertrophic zone of JMC patients’ growth plates, supporting that increased DEPTOR levels compromise the commitment of chondrocytes to hypertrophy .PTHrP transcription and that this control is essential to coordinate chondrocyte proliferation and differentiation  and from 30 studies analyzing total body BMD (TB-BMD). Integration of this information identified 13 novel genomic loci, nine for eBMD and four for TB-BMD; DEPTOR was located in the eBMD group (A recent genome-wide association study (GWAS) performed to identify new genomic loci associated with BMD identified MD group .Deptor. Analysis of DEPTOR KO mice revealed low bone mass and vertebral stiffness. Knockdown of DEPTOR in human osteoclasts stimulated multinucleation and resorption, suggesting that the decrease in bone mass caused by Deptor depletion might be due to DEPTOR action on bone resorption and not on bone formation. Interestingly, knockdown or deletion of phosphoinositide 3-kinase (PI3K) (a positive regulator of mTORC1) resulted in the opposite phenotype, decreased multinucleation and resorption, and increased bone mass, consistent with the opposite effects of DEPTOR and PI3K on mTORC1 (n mTORC1 . Unfortun mTORC1 .Tsc1 in osteoclasts with the use of Lyz2-Cre prevented bone loss in a mouse model of osteoporosis (induced by the receptor activator of nuclear factor kappa-B ligand injections). On the contrary, no effect on bone resorption was found in TSC1 CKO under normal conditions, suggesting that mTORC1 plays a role in bone resorption under pathological conditions , and osteocalcin. Furthermore, the knockdown of DEPTOR resulted in increased ALP activity and mineralization, indicating that DEPTOR is a negative regulator of osteogenesis  was found in DEPTOR KD hBMSC cells  . Further in mice . The DEP in mice . This wo in mice , it woulogenesis  also supThese studies altogether provide a hint that DEPTOR functions to regulate cartilage and bone formation and homeostasis, probably under specific environmental and pathological situations: ER stress, Ca deprivation, aberrant PTH signaling, osteoporosis, osteoarthritis, and genetic diseases. On the contrary, it seems clear that, under normal conditions, DEPTOR is probably not critical for proper skeletal development and maintenance. However, much work is required to fully understand how DEPTOR acts to control and coordinate all these processes and to identify new pathways where DEPTOR might function. Furthermore, it is becoming more and more clear that DEPTOR is a lot more than only an mTOR inhibitor; this is probably only one of its multiple functions which happened to be the first discovered. In conclusion, it is very exciting to be a part of a growing community that is dedicated to unraveling the multifaceted role of DEPTOR in a variety of biological processes and, maybe in the future, use it as a target for the development of new therapeutics of several diseases that show the dysregulation of DEPTOR.JP-T wrote the introduction and made the figures. FC wrote the rest of the manuscript. Both authors approved the submitted version.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.


Viruses that transcribe their DNA within the nucleus have to adapt to the existing cellular mechanisms that govern transcriptional regulation. Recent technological breakthroughs have highlighted the highly hierarchical organization of the cellular genome and its role in the regulation of gene expression. This review provides an updated overview on the current knowledge on how the hepatitis B virus interacts with the cellular 3D genome and its consequences on viral and cellular gene expression. We also briefly discuss the strategies developed by other DNA viruses to co-opt and sometimes subvert cellular genome spatial organization. The hepatitis B virus (HBV) is a widespread pathogen and HBV infection is one of the major risk factors for hepatocellular carcinoma (HCC). Despite the development of an effective HBV vaccine, the number of chronic HBV-carriers has reached 257 million individuals worldwide, of whom it is estimated that ~25% will die of liver cirrhosis or HCC .HBV is a small 3.2 kb) enveloped DNA virus that replicates its genome in the cytoplasm by reverse transcription of encapsidated pre-genomic RNA (pgRNA) into a partially double-stranded relaxed circular DNA (RC-DNA). Upon internalization and import into the nucleus, the RC-DNA is repaired and converted into covalently closed circular DNA (cccDNA) that serves as a template for all HBV transcripts, including pgRNA  kb envel,5. HBV cHBV episomal DNA establishment and maintenance, or HBV linear DNA integration and expression of HBV genes, do not occur randomly, nor in a “neutral” chromatin context, and may have to adapt to the existing cellular mechanisms that govern transcriptional regulation. While it is well documented that viruses that transcribe their DNA in the nucleus (including HBV) modify or repurpose the host transcriptional machinery to induce viral gene expression, new questions have emerged about the mechanisms of viral transcriptional regulation in light of novel insights into the spatial organization of the cellular genome and on the close relationship between 3D genome organization and genome function ,18,19,20Groundbreaking discoveries in 3D genome organization and gene expression regulation have been made possible by the improvement and development of imaging techniques such as fluorescence in situ hybridization (FISH) and super resolution microcopy and biochemical and genomic approaches, such as chromosome conformation capture (Hi-C) methods ,22,23,24Another level of organization revealed by Hi-C studies is the partition of the genome into two compartments: compartment A, which contains the gene-rich and active euchromatin regions, and compartment B, which contains the gene-poor, repressed heterochromatin regions . A and BThe cellular genome is thus subject to a highly hierarchical organization that regulates gene expression. Recent studies suggest that viruses are no exception and viral gene regulation depends on viral genome positioning that occurs in the context of the 3D structure of the host genome. In this review, we will summarize the current understanding of how HBV co-opts cellular genome compartmentalization to transcribe cccDNA. We will briefly discuss strategies developed by other DNA viruses that transcribe their genome in the nucleus.In a recent study, we addressed the question of whether non-integrated viral DNAs distribute randomly in the nucleus, or target specific regions in the higher-order architecture of mammalian genomes . Two DNATo enrich for HBV/cellular DNA contacts, a capture approach (CHi-C), using biotinylated oligonucleotides covering the viral genome, was applied, showing that the HBV genome makes contact with the entire host genome, with a significant preference for active chromatin regions of compartment A enriched in active histone marks . Further analysis has demonstrated that HBV contacts preferentially CpG islands (CGIs). These regions, present in eukaryotic promoters, are mainly unmethylated and control transcription initiation through the establishment of an active chromatin state . UnmethyRegarding Ad5, we observed that while viral DNA also contacts preferentially active regions, contacts are enriched at TSS and enhancers of highly expressed genes, but also genes up-regulated during the infection, suggesting again that contacts may interfere with cellular gene expression. Regions contacted by Ad5 are enriched for motifs such as FOXO A1 and FOXO A2 and CAAT enhancer binding protein (C/EBP). While it is not known whether FOXA factors are involved in Ad5 transcription, they do, however, interact with factors such as the mediator, which is involved in Ad5 expression. C/EBP has been shown to regulate Ad5 major late promoter transcription. Altogether, our data suggest that DNA viruses infiltrate the 3D genome and target specific active regions that may provide an environment favorable for their own transcription/replication. Further studies will be needed to determine the mechanism driving the localization of viral DNA at these discrete locations.Other groups, using different approaches, have explored the spatial organization of HBV and have reached a similar conclusion that HBV does not randomly localize in the nucleus, but contacts favorable active regions. Yang and collaborators used high-throughput wide translocation sequencing coupled with chromosome conformation capture (HTGTS-3C) to study both HBV cccDNA and integrated HBV DNA 3D organization . This apContacts between HBV episomal DNA and cellular enhancers has also been observed using a 3C approach . In thissmc6, involved in the transcriptional repression of HBV X-, leads to the relocation of HBV DNA to active regions on chromosome 19. Altogether, these studies show that HBV DNA is not randomly localized in the nucleus. Active viral DNA contacts preferentially active chromatin, while transcriptionally repressed DNA is associated with repressed chromatin and contacts preferentially the B compartment. Some of the differences found in the aforementioned studies may result from using infection systems versus transfection, different cell lines (transformed hepatoma cells or primary human hepatocytes) leading to a different number of cccDNA molecules per cell, which can have an impact on the detection of viral/host contacts. Moreover, chromosome 19, found to be preferentially contacted in some of the studies, has some unique features: it is located in a central position in the nucleus, it has the highest gene density of all the chromosomes, a high density of repetitive sequences (particularly Alu repeats) and the highest GC content of all chromosomes with 2/3 of the genes having at least one CpG island [Finally, in a recent work, Tang and collaborators analyzed the spatial position of the HBV genome, which is deficient for HBx expression and was thus transcriptionally silenced using 4C analysis in HepG2 NTCP and PHH-infected cells . They obG island ,72.smc6, which suggests that HBx per se is not directly promoting the targeting of HBV DNA to active regions, as suggested by Hensel et al. [Of note is the fact that HBV X- cccDNA relocates at active regions in cells silenced for l et al. , but ratl et al. . SMC5/6 l et al. ,76. Condl et al. ,78.The mechanism of cccDNA repression by the SMC5/6 complex remains largely unknown and one can envision several possibilities. The SMC5/6 complex binds to the HBV genome due to its episomal nature and is responsible for targeting it to repressed regions, then promoting its transcriptional silencing. Alternatively, the binding of SMC5/6 contributes to the deposition on HBV X- cccDNA of H3K9me3 and HP1, which in turn are responsible for the subsequent repositioning at repressed regions ,51. AnotIn conclusion, while the different studies agree that HBV DNA genome is not localized randomly in the nucleus but contacts active regions, the mechanism responsible for cccDNA nuclear localization remains unknown . YY1 hasRecent studies have starting to address the role of 3D chromatin organization in viral replication and transcription. The viral protein NS1 of the parvovirus minute virus of mice (MVM) has been shown to be involved in the localization of MVM genomes to cellular DNA damage sites that occur naturally in cells as they progress through the S-phase and that are required for the establishment of viral replication ,86. InteAltogether, these studies highlight the fact that the DNA virus co-opts the rules pertaining to the highly hierarchical organization of the cellular genome for their transcription/replication. They infiltrate the 3D genome using different strategies and contact specific regions that likely provide an environment favorable to their own transcription/replication. In doing so, viral DNA/ host DNA contacts in turn may impact on the gene regulation of cellular genes. Indeed, we observed that CGIs contacted by HBV are often associated with highly expressed genes and genes deregulated during infection, suggesting that viral DNA contacts could interfere with cellular gene expression. Similar results were reported by Yang and collaborators, who observed that host DNA regions contacted by HBV are enriched for genes deregulated by HBV infection . Gene exTERT promoter leads directly to its up-regulation [TERT. In such circumstances, cellular gene deregulation may thus require an alternative mechanism, such as long-range interactions [As mentioned in the introduction, HBV integration is a frequent event that occurs early in infection ,5. The igulation ,94. Someractions . Alternaractions ,97,98. Cractions ,42,99. Hractions . These oractions . Similarractions . Recent ractions . Chromatractions . A long-ractions . Interesractions .The HBV genome contains binding sites for CTCF, YY1 and other transcription factors that, upon integration, can potentially disturb the local chromatin organization; for instance, this includes the formation of de novo loops. As a result, HBV regulatory regions could be repositioned in the vicinity of distal genes. Although the HBV genome is rather small compared to other viruses, the presence of regulatory elements (4 promoters and 2 enhancers) and the aforementioned binding sites for TFs known to organize the genome may impact on the proper folding of the host genome and deregulate the genes important for cancer development.HBV, like other viral pathogens, exploits the 3D genome organization for its own transcription . HoweverFinally, as viral DNA integration occurs in a context where gene expression and genome 3D organization are tightly linked and influence each other, HBV-induced HCC should be explored in the context of the 3D genome organization. Investigation of 3D genome disruption in cancer is gaining momentum and studying how DNA viruses alter the 3D genome organization will contribute to a better picture of how 3D structure deregulation can contribute to tumorigenesis. Moreover, deciphering the molecular mechanisms involved in liver cell transformation following HBV integration may bring useful insights into the rational development of targeted therapeutic agents.


Cells reside in a dynamic microenvironment that presents them with regulatory signals that vary in time, space, and amplitude. The cell, in turn, interprets these signals and accordingly initiates downstream processes including cell proliferation, differentiation, migration, and self-organization. Conventional approaches to perturb and investigate signaling pathways  are often binary perturbations that do not offer precise control over signaling levels, and/or provide limited spatial or temporal control. In contrast, optogenetics leverages light-sensitive proteins to control cellular signaling dynamics and target gene expression and, by virtue of precise hardware control over illumination, offers the capacity to interrogate how spatiotemporally varying signals modulate gene regulatory networks and cellular behaviors. Recent studies have employed various optogenetic systems in stem cell, embryonic, and somatic cell patterning studies, which have addressed fundamental questions of how cell-cell communication, subcellular protein localization, and signal integration affect cell fate. Other efforts have explored how alteration of signaling dynamics may contribute to neurological diseases and have in the process created physiologically relevant models that could inform new therapeutic strategies. In this review, we focus on emerging applications within the expanding field of optogenetics to study gene regulation, cell signaling, neurodevelopment, and neurological disorders, and we comment on current limitations and future directions for the growth of the field. Cellular signaling is mediated by highly dynamic and intertwined pathways that control crucial cell behaviors. During organismal development, these signaling processes are spatially and dynamically regulated to orchestrate cell expansion, cell differentiation, and tissue morphogenesis. Likewise, complex signaling pathways enable and protect adult organismal function. Investigating these complex mechanisms has required the development of tools to perturb their activity.Conventional chemical perturbations can offer some levels of control but have limited capability in their spatial and temporal resolution  has a tightly associated C-terminal Jα helix in a folded state, but upon exposure to blue light, the bound flavin mononucleotide (FMN) chromophore undergoes a conformational change to displace the Jα helix from the protein core, leading to uncaging of an effector kinase domain , isolated from plant photosensor phototropin 1 (phot1). Phot1 molecule has two LOV domains, with the second domain (LOV2) primarily involved in kinase activation  that interacts with a partner CIB1 in a blue light responsive manner, due to a flavin adenine dinucleotide (FAD) bound to Cry2 as a chromophore cofactor , derived from the plant Arabidopsis thaliana, contains two structural domains , derived from s Quail, . The N-ts Quail, . This Phs Quail, . More res Quail, . In addiMagnets, an improved system that incorporates positive and negative amino acids into the Vivid (VVD) N-terminal helix, also enables heterodimerization with electrostatic attraction upon uncaging of the protein  and constitutively photomorphogenic 1 (COP1) offer an alternative excitation wavelength (~280–315 nm). UV response locus 8 (UVR8) can translocate and bind to COP1 in the presence of UVB  can be constructed for large surface illumination in a uniform manner, though spatial resolution can be limited in this design  Genome Editing: CRISPR/Cas9  can be engineered for targeted genome modification in mammalian cells at specific DNA loci, which enables the causal dissection of gene functions  system for site-specific genome recombination, where low intensity or short pulsed illumination enabled efficient activation with high spatial and temporal precision (ii) Gene Activation: Optogenetic control of gene expression has been achieved with several different approaches, and a typical strategy combines a photosensory module with a split transcription factor that can be reassembled upon light illumination. The first reported optogenetic system for regulating gene expression, published in 2002  than the previous constructs in HEK293T cells.Engineering DNA-binding became far easier with the advent CRISPR/Cas9 genome editing, and this capability can be harnessed for other applications. For example, Zhang and colleagues  after 4 d of illumination. Another research has induced functional neuronal differentiation using a far-red light-mediated system, which offers low phototoxicity and deep tissue penetration  Gene Repression: In addition to activating target gene expression, multiple systems have been developed to repress transcription. For example, repressive histone modifiers have been fused to light-sensitive proteins to suppress Grm2 and Neurog2 gene expression in primary neurons and Neuro2a cells with light illumination  1-silencing transcription factor (REST) in primary neurons , revealed the role of this pathway in promoting endodermal differentiation at the cost of mesodermal fate during a critical time window  family of RTKs, as they are known to be important for neuronal survival, neurite outgrowth, synaptic function, and synaptic plasticity  system  channels to trigger Ca2+ influx into the cell  in Drosophila enhanced axon regeneration and proper axon guidance in the peripheral nervous system (PNS) as well as axon regrowth and functional recovery in the CNS  the gene cassette size that can be packaged in vectors for transfection; (2) safe and efficient delivery vehicles that can target the cell or tissue of interest ; (3) potential inflammation and immunogenicity caused by optogenetic tools; (4) limited red-light or NIR light sensitive proteins available with high tissue penetration and low phototoxicity, especially in wavelengths ranges not currently well-explored; (5) potential long-term effects of permanent introduction of non-human proteins with optical properties; and (6) a lack of appropriate illumination devices that present narrower-wavelengths for mitigating surrounding tissue phototoxicity (Wojtovich and Foster, To overcome some of these limitations and further improve the light-responsive protein properties, systematic protein engineering processes such as directed evolution (Packer and Liu, In summary, state-of-the-art optogenetic systems are enabling numerous discoveries and insights into fundamental functions of cellular signaling networks. With the substantial acceleration in the range of applications, the role of optogenetics will broadly enable the examination of how genes, proteins, cells, and cellular connections modulate local and global network activity to develop complex tissue structures and encode behaviors. Such fundamental knowledge gained from optogenetic research will also potentially enable clinical therapeutics.DZ and DS substantially contributed to the conception and design of the article and interpreting the relevant literature. DZ wrote the first draft of the manuscript. HJ and JC wrote sections of the manuscript. DS revised the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.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.


What are the overall incidence and comparative rates of infusion reactions among commonly used intravenous iron formulations?In this multicenter cohort study of 35 737 iron infusions administered to 12 237 patients, the incidence of infusion reactions was 3.9%, with only 2 total documented epinephrine administrations. There were significantly different comparative rates of infusion-related adverse events among individual iron formulations, and the use of premedication was associated with greater risk of adverse events.These results suggest that intravenous iron administration is generally well tolerated, although it may differ among available iron formulations, with an exceedingly low risk of severe infusion reaction. This cohort study evaluates rates of infusion reactions among 4 commonly used intravenous iron repletion strategies and how readministration was managed in patients with history of reaction. Although iron deficiency is common, it remains unclear which iron repletion strategy is associated with the lowest rate of infusion-related adverse events, and how patients with history of infusion reaction should be managed.To evaluate rates of infusion reactions among 4 commonly used intravenous iron repletion strategies and determine how readministration was managed in patients with history of reaction.This cohort study included all patients receiving intravenous iron infusion from January 1, 2015, to September 7, 2021, at 6 centers in Portland, Oregon. Participants included a total of 12 237 patients with iron deficiency, not restricted by etiology. Statistical analysis was performed from September to October 2021.Type of intravenous iron formulation and concurrent administration of diphenhydramine, epinephrine, famotidine, and/or hydrocortisone, used as surrogate maker of infusion reaction.Incidence of adverse events, including severe events requiring epinephrine, stratified by type of iron formulation, and in patients who received premedication or with history of infusion-related reaction receiving subsequent doses.P < .001). Severe adverse events were exceedingly rare with only 2 documented epinephrine administrations, both associated with iron dextran. Incidence of adverse events among those who received premedication was 23-fold higher compared with those who did not . Among 873 patients with history of infusion reaction who underwent readministration, the majority received the same formulation, which was associated with significantly higher reaction rate particularly if premedication was administered , compared with those who received an alternate formulation  (P < .001).Among 35 737 unique iron infusions (12 237 patients ), comprising 22 309 iron sucrose doses, 9067 iron dextran total doses , 3147 ferumoxytol doses, and 1214 ferric carboxymaltose doses, incidence of adverse events was 3.9% . Rate of infusion events differed among iron formulations: 4.3%  iron sucrose, 3.8%  iron dextran , 1.8%  ferumoxytol, and 1.4%  ferric carboxymaltose (These data, and the preponderance of published evidence, suggest that intravenous iron is generally well tolerated with exceedingly low risk of severe reaction, use of premedication and test doses are unnecessary, and that optimal prevention and management of infusion-related reactions warrant further study. Some have estimated the rate of anaphylaxis with IV iron to be less than 1 per 250 000 administrations.9 Proposed mechanisms of IV iron-associated adverse events (AEs) include type I hypersensitivity reactions to dextrans, which can be used as plasma expanders, complement activated-related pseudo-allergy (CARPA), or other non-IgE mediated mechanisms of complement activation.11Intravenous (IV) iron is more effective and faster acting than oral iron; however, concerns persist among some clinicians regarding the overall safety of certain IV iron formulations.12 Likewise, evidence is lacking for the clinical utility of test doses of LMWID or the use of premedication to prevent infusion reactions, and few data are available to guide the management of patients who experience a reaction and require ongoing iron repletion. This ambiguity, as well as general discomfort with the use of newer infusion formulations, may lead to less patient-centric and cost-effective iron repletion strategies, such as those requiring multiple visits for low-dose iron infusions as opposed to single-dose total infusions.14There are several commonly used formulations of IV iron, including iron sucrose (IS), low molecular weight iron dextran (LMWID), ferumoxytol, ferric gluconate, and ferric carboxymaltose, as well as the recently approved ferric derisomaltose, although the comparative rate of reactions among available formulations has not been well described and warrants further investigation.To address this ambiguity, we used infusion data from 6 centers in Portland, Oregon. First, we collected the concomitant administration of reaction medications  post-IV iron to gauge the rate of all iron-related AEs. We next evaluated the utility of premedication and test dosing with certain iron formulation. Lastly, we evaluated practice patterns and outcomes in patients with prior history of infusion reaction who required ongoing iron repletion.STROBE) reporting guideline.We performed a multicenter cohort study of all adult patients (age ≥18 years) who received at least 1 infusion of a preselected IV iron formulation , at 6 centers in Portland, Oregon  from January 1, 2015, through September 7, 2021. This study was approved by the OHSU institutional review board prior to initiation, and a waiver of informed consent was granted owing to the retrospective nature of the study. The clinical indication of IV iron administration was determined by the individual treating physician’s discretion and was not restricted in this analysis. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology , where each IV iron infusion was defined as either 1 total dose or test dose infusion. Test doses which were administered alone were considered unique infusions, however test dose combined with total dose infusion were considered as 1 combined infusion event. Each infusion was defined as a continuous event from start of IV iron infusion through completion, followed by any subsequent monitoring for AEs. To identify patients who experienced any type of infusion-related AE, we collected the peri-infusion medication administration of diphenhydramine, epinephrine, famotidine, and hydrocortisone as a surrogate marker of reaction response. Administration times of iron and peri-infusion medications were also captured, but only medications that were administered post–iron infusion were considered to represent a true adverse reaction. A comprehensive review of the relevant electronic health record (EHR) was additionally conducted for all patients who received epinephrine to verify accuracy, characterize infusion-related symptoms, and determine the clinical outcome after epinephrine administration. No patients were required to return for follow-up (thus none were lost to follow-up), and all patients who started IV iron infusion were captured in the analysis.After identifying the cohort of patients who received IV iron from pharmacy administration records, baseline demographics and documented history of allergies to medications, food, or contact allergy were obtained from the EHR. The type of IV iron formulation, dose, and the use of test dose LMWID were recorded, as well as type and dose of peri-infusion medication. Test dosing was not offered for other iron formulations at the included institutions. In patients who experienced AEs who went on to receive further doses of IV iron, iron formulation, and the use of premedication were additionally collected. The indication for IV iron, the setting of administration (inpatient vs outpatient), and the characterization of infusion-related symptoms triggering the administration of peri-infusion medication were unable to be obtained.The primary outcome of interest in this analysis was the overall incidence of AE and SAEs among all patients who received at least 1 dose of IV iron. An AE was defined as the administration of diphenhydramine, epinephrine, famotidine, and/or hydrocortisone within 24 hours post–iron infusion. SAE was further characterized as the use of epinephrine within 24 hours of IV iron administration. Secondary outcomes included the incidence of AEs in those who received premedication, defined as the use of diphenhydramine, famotidine, and/or hydrocortisone within 24 hours preceding infusion, and in those who received test doses, defined as 25-mg IV LMWID prior to total dose LMWID infusion. Additional subgroup analyses were performed to measure the incidence of AEs in patients with documented history of allergy, as well as in patients with history of infusion-related reaction who received subsequent doses of IV iron, stratified by iron formulation and use of premedication.2 tests were performed to determine the statistical significance of infusion reactions based on type of iron formulation. Statistical significance for analyses was set at P < .05, and all testing was 2-tailed. Additional Pearson χ2 tests were performed to compare the association of the following variables with rates of AEs: premedication, prior documented allergy, test-doses of LMWID, or readministration of IV iron after reaction. All analyses and data visualizations were performed using R for macOS version 4.1.2  from September to October 2021.Descriptive statistics were used to summarize baseline and outcome characteristics, with categorical variables presented as number and percentage and numerical variables presented as mean and SD. After accounting for the number of premedications and postmedications administered, Pearson χA total of 35 737 unique IV iron infusions were administered to 12 237 patients during the 6.5-year study period. Baseline patient characteristics and demographic information are reported in P < .001). Notably, our study was not specifically designed with intent to compare the rate of AEs between individual formulations.The cumulative incidence of infusion-related AEs was 3.9%  among 35 737 IV iron infusions included in the analysis. Notably, the rate of infusion reactions differed significantly by IV iron formulation. We detected reaction rates of 4.3%  in those who received IS, 3.8%  in LMWID , 1.8%  in ferumoxytol, and 1.4%  in ferric carboxymaltose   were associated with the administration of at least 1 premedication . The incidence of AEs among those who received premedication was approximately 23-fold higher compared with those who did not  . PostmedThe incidence of AEs associated with the use of test dose LMWID was also analyzed, and outcomes reported in P < .001).We performed additional subgroup analyses using stricter cohort selection criteria, summarized in P < .001).To gain a greater understanding of practice variations in patients with prior history of an infusion reaction receiving ongoing iron repletion, an additional subgroup analysis of those who underwent readministration of IV iron was performed. Among 873 patients who met inclusion criteria and underwent subsequent iron administration, 755 (86%) received the same iron formulation (of whom 605 [80%] received premedication), whereas 118 (14%) were switched to an alternate formulation (of whom 39 [33%] received premedication). Rates of recurrent AEs were significantly higher in the cohort of patients who underwent retrial of the same IV formulation and were premedicated vs not , compared with those who were switched to an alternative IV formulation and were premedicated vs not   of all infusion events. The rate of SAEs, defined by the use of epinephrine within 24 hours postinfusion, was exceedingly low with only 2 occurrences in total, both associated with IV LMWID. Although we cannot be completely certain given the retrospective nature of this review, it is possible that intervention with diphenhydramine and epinephrine converted a minor reaction into a more hemodynamically meaningful event ostensibly because of iron. There was a statistically significant difference in rate of AEs among the 4 commonly used IV iron formulations that were included in the analysis, with the highest rate of infusion reaction occurring among those who received IS (4.3%) and lowest among ferric carboxymaltose (1.4%). Among those who received total dose LMWID, the majority were administered as total dose alone with only 19.5% (n = 1771) preceded by test dose administration, and 56 infusions were given as test dose only. Notably, there was no significant difference in infusion-related AEs between total dose LMWID administration approaches (with or without test dose). The findings in our study suggest that IV iron administration is generally well tolerated with a low adverse reaction rate, and the routine use of test doses and premedications are likely of limited benefit.15 Although not entirely elucidated, this has largely been attributed to IgE-mediated, type 1 hypersensitivity response to the dextran group leading to basophil degranulation with subsequent histamine release, as well as specific IgE and IgG antibody formation against the dextran group.17 This agent was subsequently removed from formulary owing to safety concerns.Our study adds to the already robust body of evidence supporting the safety of IV iron infusion. Older formulations of IV iron, particularly the high molecular weight iron dextran (HMWID) formulation Dexferrum, which was first made available in 1996, was historically associated with unacceptably high rates of allergic and hypersensitivity reactions.11 Also of note is the concept of CARPA via non-IgE mediation, where C3a and C5a bind to mast cells, basophils, and macrophages, triggering the release of cytokines responsible for the clinical features of a hypersensitivity reaction.11 Although traditionally the proposed driver of hypersensitivity reactions associated with monoclonal antibody administration, CARPA has also been associated with nanoparticle-containing drug reactions of which all available IV iron formulations now consist.18 Hempel et al19 attempted to analyze IV iron complement activation by comparing 5 IV iron formulations using functional complement assays both in vitro and ex vivo, of which both LMWID and ferric carboxymaltose demonstrated complement-activating potential. It remains unclear whether free iron itself or its carbohydrate shell is responsible for complement activation, although the known association between nanoparticle activation of complement suggests that the carbohydrate shell may in fact be the primary culprit.11With the advent of newer formulations of IV iron, infusion reactions have not only substantially declined, but alternate mechanisms driving these reactions have also been postulated. One theory suggests that the transient presence of free iron in circulation, caused by iron extrusion from the carbohydrate complex, can occur in situations when IV iron is infused too rapidly to be safely bound to transferrin.18 Our study also found a significantly higher rate of infusion reaction in patients with documented history of allergies, although it should be noted that we captured a higher-than-expected rate of documented allergies, likely owing to the inclusion of allergies to medications, food, and contact allergy. Because of the proposed risk of hypersensitivity to the dextran-component and an association of LMWID with complement-activation in vitro,19 dextran-free formulations of IV iron had gained favor due to a theoretical lower risk of anaphylactoid reactions, although oftentimes at the expense of sacrificing single-dose total infusion for multiple visits of low-dose iron. This theory, however, has largely been debunked by retrospective and meta-analyses of patients undergoing IV iron infusion, in which LMWID has consistently demonstrated similar if not decreased rates of AEs compared with similar formulations.22 The context of this bias is perhaps related to the historical use of HMWID and its association with SAEs, although it is now well known that higher molecular weight compounds confer a greater degree of immunologic propensity.23 Furthermore, there remains a paucity of high-quality evidence supporting the use of test doses of IV LMWID, which may mark a somewhat antiquated approach to administration driven by previous biases, with more recent data demonstrating the safety of single-dose IV 1000 mg administration.24 In fact, our study found no significant difference in infusion reactions between those who did and did not receive a test dose, although 29 of the patients who had AEs to a test dose requiring postinfusion medications did not proceed to the full dose. Overall, the low and relatively similar rate of AEs between these strategies calls into question the overall clinical utility of the test dose approach.Risk factors that appear to both increase the incidence and severity of hypersensitivity reactions to IV iron include history of allergy or atopy, a fast infusion rate, and prior history of reaction to IV iron.22 Iron sucrose differs from other formulations in that the smaller carbohydrate core binds iron less tightly, resulting in significantly increased labile-free iron after administration and higher risk of hypersensitivity reaction.13 With the advent of multiple low doses of IS, most studies have demonstrated similar safety profiles when compared with LMWID.26 Additional studies have evaluated newer formulations of IV iron, including the FIRM study, a recent randomized, double-blinded, controlled trial of nearly 2000 enrolled patients comparing the safety of ferumoxytol and ferric carboxymaltose, which detected no significant difference in the primary end point of moderate-to-severe AEs or hypotension  and no anaphylaxis reported in either group.27 An often cited report issued by the European Medicines Agency in 2013 suggests that although available data shows a clear association with IV iron and hypersensitivity reactions, the data cannot be used to detect differences in rates of reactions between iron formulations.28 Furthermore, given the very low rate of SAEs, they conclude that the benefits of IV iron administration outweigh its risks, provided that measures are taken to minimize severe events.Randomized clinical trials (RCTs) comparing rates of infusion reactions among all available formulations are generally lacking, and results of various retrospective analyses often demonstrate conflicting results. Our study found significant differences in the rate of infusion reactions among IV iron formulations, with the highest rates of reaction occurring among those who received IS (4.3%) and lowest among those who received ferric carboxymaltose (1.4%). A retrospective study of 619 patients who received 5 different IV iron formulations reached similar conclusions, with a significantly higher odds ratio of AE occurring in those who received IS .30 These agents routinely cause somnolence, flushing, hypotension, and tachycardia that mimic infusion-related reactions leading to a higher frequency of reported AEs.5 Even second-generation antihistamines, though less sedating, have also been associated with flushing, palpitations, and dizziness. These conclusions are consistent with our study, in which premedications were administered in 6.0% (n = 2157) of all infusion events and were associated with a 23-fold higher rate of infusion reaction compared with no premedication . Interestingly, similar findings were seen when we evaluated the subset of patients with history of infusion reaction who received ongoing iron repletion. AEs were significantly greater in patients who received the same IV iron formulation compared with those who switched to an alternate formulation, although notably higher rates of AEs in both groups were detected in those who were premedicated, supporting the theory that many of the documented reactions may instead be because of premedication itself, and not iron. Adding to a growing body of data to support the safety of IV iron rechallenge following an AE is a recently published large, retrospective cohort analysis conducted in Australia by Stojanovic et al.31 Sixty-nine patients who experienced an AE to IV iron polymaltose who did not complete their initial infusion were subsequently rechallenged with ferric carboxymaltose, of which 68 patients tolerated completion without incident, including 3 patients who experienced SAEs during their first IV iron infusion. General conclusions from these and other studies suggest that routine use of premedications be limited only to those with substantial risk factors , rechallenge to an alternate IV iron formulation is safe, and ultimately, future RCTs are needed to adequately study the ideal approach to both prevention and management of infusion-related reactions among all currently available IV iron formulations.Similarly, the use of premedications, such as corticosteroids and antihistamines, to prevent or minimize infusion-related AEs remains highly controversial. Not only has the efficacy of premedications been questioned, but there is data to suggest that the first-generation of histamine receptor antagonists, such as diphenhydramine, account for a large majority of perceived mild hypersensitivity reactions to IV iron.Our study has several strengths. First, to our knowledge, this is the largest retrospective analysis comparing IV iron infusion-related reactions among 4 commonly used formulations. Second, we believe in the methods of our data accrual approach in detecting clinically relevant infusion-related reactions through the identification of perimedication administration. Furthermore, rates of adverse events in our study are consistent with prior estimates, instilling confidence that our findings are reproducible when compared with previously published data.This study had some limitations. There are limitations inherent to a retrospective analysis and data extraction, which include the inability to randomize patients by IV iron formulation and capture patient- and infusion-specific details, including type of clinical reaction to identified allergens and infusion rates of each IV iron administration. Second, we acknowledge that the methods used in this analysis cannot fully account for confounding by indication, which can lead to biased outcome estimates in retrospective studies and is of particular concern when comparing infusion reaction rates with the use of premedications, which has independently been associated with higher rates of AEs in those receiving IV iron. However, this concern is mitigated by a subgroup analysis of patients with prior reaction to IV iron who underwent readministration, in which we reached similar conclusions. Additionally, we are limited by the use of postmedication administration as a surrogate marker of AEs and were unable to verify the accuracy of these findings with individual chart review given the large sample size. However, we were able to undertake comprehensive chart review to verify the accuracy of SAEs in patients who received epinephrine, which may in fact have not been clinically indicated and potentially converted a minor reaction into a more hemodynamically significant event. Finally, although the higher rates of AEs detected among those who received premedication may be attributed to symptoms that mimic infusion-related reaction, it is also possible that we inadvertently captured some medications that were administered during hospitalization. This concern is tempered by the fact that the vast majority of IV iron is given during ambulatory encounters at our relevant institutions, and as such the included peri-infusion medications evaluated in our study are highly likely to be IV iron–related administrations. Based on our practice, we anticipate a very low rate of inpatient IV iron administration (likely less than a few percent), although unfortunately we were not able to measure this rate given limitations of retrospective data collection through the EHR.This cohort study found that patients receiving IV iron had very low rates of infusion-related reactions, with a near-zero rate of epinephrine administration. The routine use of premedications, particularly first-generation histamine receptor antagonists, may in fact cause more harm than benefit by inducing symptoms that could be perceived as a mild hypersensitivity reaction. Although further investigation is warranted to determine the ideal approach of readministration of iron in those who experience an infusion reaction, our data suggests that rechallenging patients with IV iron, perhaps with an alternate formulation and not preceded by sedating antihistamines, would be both safe and effective. Overall, IV iron continues to hold an undeniably essential role in the management of iron deficiency.


Cobalt is essential to the metabolism of all animals due to its key role in cobalamin, also known as vitamin B12, the primary biological reservoir of cobalt as an ultra-trace element. Current cancer treatment strategies, including chemotherapy and radiotherapy, have been seriously restricted by their side effects and low efficiency for a long time, which urges us to develop new technologies for more effective and much safer anticancer therapies. Novel nanotechnologies, based on different kinds of functional nanomaterials, have been proved to act as effective and promising strategies for anticancer treatment. Based on the important biological roles of cobalt, cobalt oxide nanoparticles (NPs) have been widely developed for their attractive biomedical applications, especially their potential for anticancer treatments due to their selective inhibition of cancer cells. Thus, more and more attention has been attracted to the preparation, characterization and anticancer investigation of cobalt oxide nanoparticles in recent years, which is expected to introduce novel anticancer treatment strategies. In this review, we summarize the synthesis methods of cobalt oxide nanoparticles to discuss the advantages and restrictions for their preparation. Moreover, we emphatically discuss the anticancer functions of cobalt oxide nanoparticles as well as their underlying mechanisms to promote the development of cobalt oxide nanoparticles for anticancer treatments, which might finally benefit the current anticancer therapeutics based on functional cobalt oxide nanoparticles. In the past few decades, cancer, a disease that is induced by the evasion of one’s own protective system against malignant cells, is causing continuing high morbidity and mortality worldwide. As is commonly known, radiotherapy, chemotherapy and immunotherapy are commonly used to fight cancer. However, like a double-edged sword, these methods can also produce lots of side effects while relieving cancer progress. For instance, the systemic cytotoxicity and severe side effects of radiotherapy are the biggest obstacle to their maximum therapeutic efficacy for cancer ,2. In ad2O3 NPs and Co3O4 NPs) have been applied in different fields because of their special physical and chemical properties . Mo. Mo3O4 Napeutics .3O4 NPs as drug carriers, it is necessary to verify whether they affect the structure of proteins when they are coupled with proteins, as the drug delivery system would directly interact with the proteins after administration. Human serum albumin (HSA) is the most abundant protein in plasma and is often used to carry a variety of drugs to meet various treatment requirements. After Arsalan et al. used different concentrations of Co3O4 NPs to interact with HSA molecules, the fluorescence results show that high concentrations of Co3O4 NPs could change the quaternary structure of HSA but did not affect the natural structure of HAS [One of the most accepted applications of nanomaterials is their potential to act as a vehicle for drug delivery . In ordee of HAS .Chattopadhyay S et al. linked N-Phosphonomethyliminodiacetic acid (PMIDA) coated cobalt oxide nanoparticles (PMIDA-CoO) with folic acid (FA) for cancer-cell-targeted delivery of anticancer drugs, such as doxorubicin and methotrexate. Flow cytometry analysis confirmed that anticancer drugs loaded onto the complex showed high cytotoxicity and could induce apoptosis of cancer cells .In recent years, researchers have been working on the development of a highly specific drug delivery system, nanoscale drug delivery system (NDDS) ,79, whicQ. Ren et al. proposed to use the simple redox reaction between cobalt oxide and monodisperse hollow manganese to form adjustable hollow MCO NPs and encapsulate tumor therapeutic compounds (such as doxorubicin). Because the TME has a high concentration of glutathione (GSH), it can promote the release of DOX and achieve good tumor inhibition . The exp2O4 superstructure (ATS-MNS-HB) to achieve high sensitivity and high accuracy in the detection of tumor markers [3O4 NPs to form a new nano-probe. Finally, the obtained nano-probes were applied to the immunohistochemical (IHC) detection of EGFR expression in (NSCLC) tissues of non-small cell lung cancer with good stability and high sensitivity and specificity [In addition, in terms of tumor markers, Dai H et al. designed an enzyme-free integrated biological probe composed of a heme-modified magnetic NiCo markers . It showcificity . This wo3O4@CeO2-Au@Pt complex to detect SCCA [3O4@CeO2 nanoparticles have high catalytic performance and electron transport ability, while sea-urchin gold–platinum nanoparticles (Au@Pt nanoparticles) have high stability and strong biological binding force, which can be combined to construct the nanocomposite Co3O4@CeO2-Au@Pt. By the cooperation of Co3O4, CeO2-Au, Pt and D-Au nanoparticles, the detection limit of SCCA was determined as 33 fg/mL with very good reproducibility [Squamous cell carcinoma antigen (SCCA) is favored by many researchers as a tumor marker in different kinds of cancers , but it ect SCCA . Firstlycibility . The abo3O4 NPs to interact with two typical enzyme models: luciferase (Luc) and galactosidase  [3O4 NPs significantly activated β-Gal, which indicated that biochemical activity was unique between enzymes and cobalt oxide nanoparticles. In addition, it is proved that the complex formed by nanoparticles with Luc, β-Gal or other labeled enzymes can be used to label cancer cells with photoluminescence  . The resence (PL ). Thus, Up to now, cobalt oxide nanoparticles have shown great potential for anti-tumor application with increasing attention. Based on the above information, tumor cell membranes contain more phospholipids than normal cells. The presence of large amounts of phospholipids may affect the attachment of CS-CoO and PMIDA-CoO nanoparticles to the membrane of cancer cells, and the presence of acids in the phospholipids may help the nanocomplex release free cobalt ions into cells, thereby allowing cell apoptosis by activating different biochemical processes . The posCobalt oxide nanoparticles can be directly used as anticancer drugs to induce cancer cell damage and can also be used as chemical sensitizers and protective agents during anticancer therapy, which may be very helpful to enhance the anticancer effects and reduce the side effects of chemotherapeutics. Additionally, cobalt oxide nanoparticles also have a good performance in improving the effects of immunotherapy as anticancer vaccines. Interestingly, with very good photothermal conversion abilities, cobalt oxide nanoparticles could also act as photothermal agents for photothermal therapy of cancer. Moreover, similar to most nanoparticles, cobalt oxide nanoparticles could also act as drug carriers for cancer-cell-directed drug delivery, which may significantly improve the cancer-targeting effects of anticancer drugs. Moreover, the ability of cobalt oxide nanoparticles to construct nano-probes or biosensors can be applied for cancer-associated molecule detection, while the regulation effects of cobalt oxide nanoparticles on enzyme activity also indicate their potential uses as enzyme regulators during cancer treatment.3O4 NPs and found that Co3O4 NPs did not cause obvious necrosis of normal cells [3O4 NPs could interact with mitochondria, affect the expression of mitochondria-related genes, result in impaired mitochondrial function and decreased ATP production, which seriously inhibit the growth of normal cells. The mechanism of Co3O4 NPs to kill cancer cells was proved to be generally associated with plasma membrane damage or ROS accumulation, but this study did not find significant changes in ROS level and plasma membrane in normal cells, which needs to be further explored to explain these mechanisms [At present, most anticancer studies focusing on cobalt oxide nanoparticles use a low concentration of nanoparticles to treat cancer cells. The lowest concentration that has killing effects on cancer cells but no killing effects on normal cells is always used as the effective concentration for anticancer application. However, some scholars have found that although normal cells at these concentrations do not die, there is a certain extent of growth inhibition. Thus, there is an urgent need to further study the toxicity mechanisms of cobalt oxide nanoparticles both in cancer cells and normal cells at certain doses. Wang et al. treated normal cells with a sublethal concentration of Coal cells . Howeverchanisms . At pres3O4 NPs, in fact, more cobalt ions were released by chitosan-coated Co3O4 in acidic conditions than that in neutral pH conditions, and this effect was positively correlated with the cytotoxicity of the nanoparticles [The understanding of cobalt ion toxicity is very important to the use of cobalt-related materials for biomedical purposes. More mechanism studies are needed to explore the cytotoxicity of cobalt ions in different cancer cells and normal cells before the consideration of using cobalt oxide nanoparticles for clinic trials. With regard to the anticancer study of chitosan-coated Coarticles . TherefoThe concept of using nano-carrier systems for cancer-targeted drug delivery is still restricted by the development of functional targeting ligands and drug-encapsulating materials. For the drug carrier roles of cobalt oxide nanoparticles, how to construct functional cobalt oxide nanoparticle delivery systems with outstanding cancer-cell-targeting effects would be the most important issue. However, how to encapsulate drugs into cobalt oxide nanoparticles with high loading rates also remains a critical issue for their further applications.3O4-NP-B (bulk) has a stronger inhibitory effect on liver glutathione (GSH) activity than Co3O4-NP-S  [The surface properties of nanoparticles have been well known to play critical roles in their biomedical applications. In addition, it is worth noting that how the shape and size of cobalt oxide nanoparticles affect their anticancer activities should also be an important subject for the study of cobalt oxide nanoparticles. Raman et al. found that Coherical) . This woUp to now, although some reports about cytotoxicity of cobalt oxide nanoparticles against normal cells have been demonstrated, there are still no reports describing the biocompatibility of cobalt oxide nanoparticles, which is critical for their further clinical uses. Therefore, more attention should be paid to the biocompatibility of cobalt oxide nanoparticles, such as their hemolytic properties and sensitization responses.As one of the trace elements, cobalt is essential for normal physiological activities of the body, especially for its role as a metal constituent of vitamin B12. However, we cannot deny that excessive exposure to cobalt would induce various adverse health effects, causing unavoidable damage to the body and affecting hearing, vision, nervous, cardiovascular and endocrine systems ,89. TherIn our opinion, the best future development of cobalt oxide nanoparticles for anticancer therapy should be focused on the following: (i) the anticancer mechanism exploration of cobalt oxide nanoparticles; (ii) the drug delivery roles of cobalt oxide nanoparticles against cancer; (iii) the roles of cobalt oxide nanoparticles in rebuilding tumor microenvironment; (iiii) the ability and mechanisms of cobalt oxide nanoparticles to sensibilize current chemotherapy; (iiiii) the development of cobalt oxide nanoparticles for photothermal therapy; (iiiiii) the evaluation of potential adverse effects induced by cobalt oxide nanoparticles; and (iiiiiii) the systemic metabolism of cobalt oxide nanoparticles in vivo. With the increased attention paid to these fields, we believe that we can build a strong network to estimate the potentials of cobalt oxide nanoparticle based anticancer strategies.Overall, although lots of work is still needed to further extend cobalt oxide nanoparticles for anticancer treatment, the promising abilities of cobalt oxide nanoparticles indeed present strong potential to fight cancer by acting in different roles. With the increasing attention paid to cobalt oxide nanoparticles, we believe that there would be more and more strategies coming out for anticancer treatments based on cobalt oxide nanoparticles. The continued development of functional cobalt oxide nanoparticles would finally benefit the current therapies for cancer, as well as other biomedical fields.


Dear Editor,With great interest we read and appreciated the recently published article by Goossens and colleagues . They ofWe would like to raise some points of interest that merit discussion. First, the authors measured serum cholesterol levels in patients on ICU admission and day 3, whereas muscle strength was assessed at day 8. What was the rationale for the choice of these timepoints? Ideally, comparison of muscle strength should be made against contemporaneous cholesterol levels to reduce risks of confounding and to support causality arguments.Second, did the authors consider measuring endogenous production of 3-HB and plasma levels in their septic animals? Endogenous hydroxybutyrate levels are altered in sepsis due to a shift towards fat metabolism and increased ketone body production . UnderstThird, the authors present the fate of supplemented 3-HB as cholesterogenic or tricarboxylic acid cycle (TCA cycle) substrate . A complementary pathway worth considering is fatty acid synthesis which also relies upon acetyl-CoA as a basic substrate. Again, temporal measurement across the catabolic and recovery phases of sepsis would be informative.Fourth, to our knowledge, the liver produces ketone bodies but cannot directly metabolize them due to a lack of ketoacyl-CoA transferase. The observed elevation in mevalonate within the liver after exogenous 3-HB administration may relate to indirect metabolic pathways, e.g. via fatty acids transported back to the liver. et al. for the interest in our work on the role of cholesterol in ICU acquired weakness and the impact thereof on exogenous 3-hydroxybutyrate (3HB) and for raising several interesting points [We thank Hofmaenner D. g points .The first point addressed the difference in timing between measurement of serum cholesterol and of muscle strength in the human study (EPaNIC RCT). In this study, the impact of omitting parenteral nutrition for one week in ICU on ICU acquired weakness was assessed in a matched subgroup of 600 patients on post-randomization day 8 . Muscle et al., endogenous production of 3HB is increased in the acute phase of critical illness, but this is abolished as soon as parenteral nutrition is initiated, as we have previously shown both in the septic mouse model [Second, the authors asked whether data on endogenous production of 3HB and plasma concentrations were available for the septic mice. Indeed, as mentioned by Hofmaenner D. se model , and in se model , 8. We aThe third and fourth points addressed the metabolic fate of the exogenous 3HB and whether use as substrate for fatty acid synthesis could be involved. Indeed, 3HB can in theory be converted into fatty acids. In the mouse model, 3HB supplementation has previously shown not to alter plasma fatty acid concentrations or triglyceride content in muscle or liver , whereaset al. that more research is needed to better understand the role of cholesterol in ICU acquired weakness and to assess the clinical potential of 3HB supplementation in human ICU patients.We totally agree with Hofmaenner D.