Immunoglobulins are the potent effector proteins of the humoral immune response. In the course of evolution, immunoglobulins have formed extremely diverse types of molecular structures with antigen-recognizing, antigen-binding, and effector functions embedded in a single molecule. Polysaccharide moiety of immunoglobulins plays the essential role in immunoglobulin functioning. There is growing evidence that the carbohydrate composition of immunoglobulin-linked glycans, and especially their terminal sialic acid residues, provide a key effect on the effector functions of immunoglobulins. Possibly, sialylation of Fc glycan is a common mechanism of IgG anti-inflammatory action in vivo. Thus, the post-translational modification (glycosylation) of immunoglobulins opens up significant possibilities in the diagnosis of both immunological and inflammatory disorders and in their therapies. This review is focused on the analysis of glycosylation of immunoglobulins, which can be a promising addition to improve existing strategies for the diagnosis and treatment of various immuno-inflammatory diseases. Immunoglobulins, the potent effector proteins of the humoral immune response, possess both anti-inflammatory and proinflammatory activities triggered by antigen recognition based on an affinity for various fragment crystallizable receptors (FcRs) and complement factors ,2. StartFunctionally, an antibody consists of the following two parallel-evolving separate protein domains: A fragment responsible for binding an antibody  and a fragment interacting with the cell surface Fc receptor and certain complement system proteins, i.e., a crystallized antibody domain . The unique ability of antibodies to recognize antigens is provided by the mechanism of structural rearrangement of the antigen binding domain through somatic recombination and hypermutation of coding regions . Due to Light and heavy chains have a variable (V) N-terminal region (VL or VH) containing three hypervariable regions called the complementarity-determining regions (CDRs), as well as four framework regions. Three heavy chain CDRs and three light chain CDRs form an antigen binding domain ,21. The Glycosylation, the process of protein modification during and after translation, affects proteins’ properties and functions. Protein glycosylation is a very dynamic post-translational modification affecting cell surface-level molecular interactions, and secreted proteins activity. The aspects influenced by glycosylation are essential and involve secretion, solubility, stability, binding, conformation changes, and antigenic properties . ImmunogJoining of glycans with proteins occurs via one of two bonds, either at asparagine residues (N-glycans) or at serine/threonine residues (O-glycans). In contrast to other proteins that are able to interact with glycans, IgG glycans are represented by a rather conservative composition of N-linked sugar molecules .The structure of IgG glycosylation is shown in In general, changes in galactosylation have been observed in many physiological conditions of a human. In particular, the accumulation of agalactosylated antibodies has been observed in inflammatory diseases , whereas increased galactosylation has been associated, for example, with pregnancy .In addition, specific glycan changes are used in the field of monoclonal antibody therapy, thus, indicating the important role of glycans in the formation of the antibody effector function. For example, removal of fucose significantly increases the activity of the antibody-dependent cellular cytotoxicity system, whereas removal of GlcNAc alone has a more modest effect. Furthermore, sialylation reproducibly enhances the in vivo anti-inflammatory effect of immunoglobulin administered intravenously .The actual glycosylation of general or antigen-specific IgG is changed in many autoimmune diseases . StudyinMany studies have suggested that the development of certain autoimmune diseases was associated with a decrease in the overall level of IgG sialylation. In the cases of individual nosologies, a relationship has been found between total IgG or IgG autoantibodies decreased sialylation and the pathological activity of autoantibodies , and witSialylation is associated with an anti-inflammatory effect. An intravenous immunoglobulin used to treat inflammatory conditions has an anti-inflammatory effect due to a population of antibodies carrying 2,6-sialylated Ig glycans ,43.Contrary to autoimmune and alloimmune diseases, in which antibodies are usually involved in pathogenesis, they are called upon to exert protective activities. In most studies devoted to the study of IgG glycosylation levels in plasma/serum in infectious diseases, a decrease in galactosylation has been found, for example, there were distinct antigen-specific IgGs in hepatitis B and C that showed a specific glycosylation profile, which included a decrease in galactosylation, the degree of which was associated with the severity of the disease and liver damage . The antVariability of the Ig glycosylation profile was found in patients with cancer. Modifications of IgG glycans were first detected in patients with multiple myeloma, a malignant tumor of plasma cells related to paraproteinemic leukemia. In this case, decreased IgG galactosylation was observed as compared with healthy controls ,50. In aImportantly, some changes in the structure of glycans IgG can be associated with a higher risk of developing cardiovascular diseases, while other modifications can reduce CVD risk. The first group includes glycans with three GlcNAcs (GP6) or glycans with GlcNAc and sialic acid, whereas the second group includes sialylated glycans without a bisecting GlcNAc. In addition to aging, high levels of glycans with a core fucose and bisecting GlcNAc can be found in the serum of patients with type 2 diabetes, which is similar to indicators associated with the risk of cardiovascular disease. It has now been found that sialylation of N-glycans reduces CVD (cardiovascular disease) risk but also has a negative feedback, with elevated levels of very low-density lipoproteins and triglycerides in serum and the presence of atherosclerotic lesions in the carotid artery .Thus, we suggest changes in the relationship of the glycosylation patterns of immunoglobulins with various autoimmune, alloimmune, infectious, oncological, and other diseases and their association with a general inflammatory status. At the same time, apparently, B-cells have the ability to adjust the glycosylation profile of antibodies to increase antigen specificity, as well as to modulate effector functions.Apart from post-translational glycosylation, non-enzymatic glycosylation (glycation) of circulating proteins should be mentioned as a completely different type of protein and glycoprotein modification, which is driven by chemical interaction of glucose and free amino groups of lysine residues. Such a modification can sufficiently affect the chemical and functional properties of proteins, including immunoglobulins. An important aspect is that glycation or “glycoxidation” of proteins culminates in the generation of advanced glycation end products (AGEs) ,61. AGEsIn any case, non-enzymatic glycation is worth mentioning in the context of this review because it can exacerbate the damaging effects of deglycosylation of glycoproteins. For example, it has been shown that non-enzymatic glycation atop of desialylation of apoB-100, the major apoprotein of low-density lipoprotein, added much to its atherogenicity ,75,76; pRheumatoid arthritis and primary osteoarthritis were the first diseases for which a connection with a change in the composition of IgG glycol was uniquely established. Patients suffering from these pathologies showed a higher level of agalactosylated IgG glycans than in the control group . FurtherThe reduction of IgG galactosylation in RA correlated with interleukin (IL)-6 and C-reactive protein (CRP) . With reNon-galactosylated  IgG aAbs have been associated with proinflammatory activity in patients with RA ,82,84. IStudies of antigen-specific antibodies in RA have shown that their glycans differed in antibodies to the cyclic citrulline-containing peptide  of the IgG1 subclass from total IgG1 both in serum and in synovial fluid. The presence of ACPA in patients with differences in disease clinical course indicated important involvement of these autoantibodies in the pathogenesis of RA . GlycosyIt has also been demonstrated that ACPA glycoprofile was different in patients with positive and negative rheumatoid factor . More imThe reduced IgG galactosylation was associated with other autoimmune diseases, such as systemic lupus erythematosus, inflammatory bowel disease, and vasculitis-associated antineutrophil cytoplasmic antibodies (antineutrophil cytoplasmic antibody-associated vasculitis), and also with the progression of these diseases, as well as the activity and severity of symptoms ,94,95,96Agalactosylated IgGs have been shown to mobilize the complement system more actively . In turnSystemic inflammation contributes significantly to the development of premature atherosclerosis in RA. It has been established that the main cause of high premature mortality in immuno-inflammatory RA was associated with cardiovascular complications (CVC) caused by accelerated progression of atherosclerosis and the development of chronic heart failure. Most often, CVC develope in patients with RA with low or moderate cardiovascular risk, but with high clinical and immunological activity of the disease. Attention is drawn to the similarity of mechanisms of immunopathogenesis of RA and chronic low-grade inflammation in atherosclerosis. According to modern views, chronic inflammation associated with the uncontrolled activation of both innate and acquired immunity play a fundamental role at all stages of RA and the atherosclerotic process. Protein–glycan interactions play a crucial role in the relationship between pathogens and the immune system and altered IgG glycosylation affects its inflammatory properties . CertainAGEs are recognized as nontraditional risk factors that play crucial roles in increased cardiovascular mortality and morbidity in RA . It is eAs stated above, immunoglobulins are the most important serum proteins that demonstrate changes in their glycosylation pattern; these changes can, in turn, affect their immunoeffector functions . IgG modIn healthy people, 15 to 25% of IgG Fab fragment is glycosylated . NeverthA change in the degree of glycosylation of IgG Fab fragment is also noted in human pathological conditions. For example, an increase in the carbohydrate content in the Fab fragment of autoantibodies with rheumatoid arthritis, up to 80%, was recorded with a simultaneous two-fold decrease in the ability of autoantibodies to bind antigen . In B-ceModels of inflammation in an in vitro experiment that stimulated lipopolysaccharide (LPS) or phytohemagglutinin (PHA) revealed that removing sialic acid residues from F(ab’)2 fragment reduced the anti-inflammatory activity of the antibodies . SialylaIn comparison to the Fab fragment, much more research has been devoted to glycosylation of the IgG Fc fragment, due to the possibility of modifying the functional activity of therapeutic antibodies by changing the glycosylation profile of the Fc domain. The structure of the Fc fragment of human IgG allows the antibody to perform its effector functions, i.e., act as an inducer of phagocytosis, participate in the implementation of the antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell phagocytosis (ADCP) programs, and thus modulate anti-inflammatory activity . Humans The IgG Fc sugar moieties, known as N-glycans, affect the affinity of the Fc domain in FcRs and complement factors, ultimately initiating to an array of inflammatory responses. Glycosylation of IgG Fc N-glycans is altered during pathological or changed physiological conditions, which influences structure and stability of aAbs ,2,111.A pregnancy-related phenomenon of IgG galactosylation has been observed in healthy women and women with chronic inflammatory disease such as rheumatoid arthritis (RA). In a detailed study of Fc glycosylation in IgG, during and after pregnancy, IgG galactosylation increased gradually during gestation, peaked in the third trimester, and decreased postpartum in healthy women and in RA patients. In these patients, increased Fc galactosylation in IgG correlated with overall improvement during pregnancy, whereas postpartum flare was associated with decreased galactosylation of Fc in IgG .IgG glycans rich in sialic acid can modulate the survival and activation of B cells. This process is mediated by signaling through a CD22 receptor on B cells surface. Researchers of this mechanism have found that apoptosis of B cells was enhanced in the presence of intravenous immunoglobulin (IVIg). This proved the modulation of the adaptive immune system depending on the level of sialylation of IVIg .The use of artificially sialylated Fc fragments has confirmed the assumption of anti-inflammatory activity of antibody sialylation . The mecA significant addition is the data on the change in age of the content of agalactosylated forms of Fc IgG fragment in human serum. As levels of G0 forms increase, and the proportion of sialylated forms decreases . InteresGlycosylation changes in IgG proteins isolated from the sera of patients with inflammatory arthropathies possess the potential to be crucial elements for the development of new therapeutic strategies . Highly-Currently, immunoglobulins and Fc fragments are used in the treatment of certain diseases, for example, thrombocytopenic purpura (ITP) , rheumatThe anti-inflammatory effects of sialylated IgG aAbs were first reported for the intravenous immunoglobulin (IVIg). IVIg is a complex heterogeneous mixture of human antibodies  and non-IgG proteins that is indicated for use as an immune replacement therapy in patients with primary and secondary immune deficiencies, and as an immunomodulatory treatment for several autoimmune and inflammatory diseases. Human IgG, which is composed of IgG1, IgG2, IgG3, and IgG4 subclasses, is one of the most dominant and significant glycoproteins in human serum . Since tFurther studies have described that the anti-inflammatory activity of IVIg was associated with a certain group of IgG molecules with α-2,6-sialic acid residues on polysaccharides linked with Fc . Such a The sialylated IVIgs modulate arthritis in mice via binding to the SIGN-R1 receptor (specific ICAM-3 grabbing non-integrin-related 1)  and in tWhen applied, sialylated IgG autoAbs in small doses reduced joint swelling in a CIA mouse model . EndogenSambucus nigra agglutinin (SNA)-agarose column, expressed much higher anti-inflammatory activity than the original IVIg. It was also found that injection of the Fc fragment, but not the Fab fragment of IVIg, induced the anti-inflammatory activity, and the SNA binding fraction of the Fc fragment was 10 times more active than the intact Fc fragment. Accordingly, IgG acquires anti-inflammatory properties upon Fc sialylation. Kaneko et al. also showed that the Fc sialylation of IgG was reduced by the induction of an immune response. Therefore, this mechanism can be characterized as a turning point from anti-inflammatory to proinflammatory activity.Kaneko et al. found that the anti-inflammatory and proinflammatory activities of IgG and Fc came from differential sialylation of the carbohydrate chains of the Fc region .They repGuo et al. showed that repeated injection of ovalbumin in mice caused production of specific IgGs and an increase of fucose content in the N-linked carbohydrate chains of the IgGs .Some of the most effective treatments for inflammatory arthropathies involving anti-TNF therapy affect the IgG glycosylation status. The effects of infliximab on IgG glycosylation status, in patients with CIA, RA, and spondyloarthropathy (SpA) have been studied recently ,129. TheThere is a strong correlation between serum glycosylation in patients with RA and SpA and inflammation, as changes in patients’ serum glycome is a measure of the anti-TNF effect on the immune system . ElevateThe active use of drugs based on immunoglobulins and their derivatives poses new challenges for specialists at all levels. Understanding the mechanism of the underlying therapeutic effects is impossible without studying the basic principles of the functional effects of these molecules on a particular physiological aspect and the pathophysiology of disease. Thus, the post-translational modification (glycosylation) of immunoglobulins expands the possibilities in the diagnosis of both immunological and inflammatory disorders and in their therapies. Known data indicate that this process is actively controlled at the molecular level and plays one of the determining roles in the regulation and functional activity of antibodies. However, the mechanisms underlying this control are not fully understood. Future studies should focus on the study of signal transmission during the production of immunoglobulins by B cells, which would help to unravel the mechanisms underlying the specific glycosylation of antibodies. This would establish the keyways to realize the effector activity of antibodies. Thus, obtained data would give new impetus to the development of therapeutic drugs and vaccines for the next generation, i.e., the next chapter in the treatment of a wide range of infectious, inflammatory, oncological, and autoimmune diseases.


Cardiovascular diseases are the first cause of death worldwide. Among different heart malfunctions, heart valve failure due to calcification is still a challenging problem. While drug-dependent treatment for the early stage calcification could slow down its progression, heart valve replacement is inevitable in the late stages. Currently, heart valve replacements involve mainly two types of substitutes: mechanical and biological heart valves. Despite their significant advantages in restoring the cardiac function, both types of valves suffered from serious drawbacks in the long term. On the one hand, the mechanical one showed non-physiological hemodynamics and the need for the chronic anticoagulation therapy. On the other hand, the biological one showed stenosis and/or regurgitation due to calcification. Nowadays, new promising heart valve substitutes have emerged, known as decellularized tissue-engineered heart valves (dTEHV). Decellularized tissues of different types have been widely tested in bioprosthetic and tissue-engineered valves because of their superior biomechanics, biocompatibility, and biomimetic material composition. Such advantages allow successful cell attachment, growth and function leading finally to a living regenerative valvular tissue in vivo. Yet, there are no comprehensive studies that are covering the performance of dTEHV scaffolds in terms of their efficiency for the calcification problem. In this review article, we sought to answer the question of whether decellularized heart valves calcify or not. Also, which factors make them calcify and which ones lower and/or prevent their calcification. In addition, the review discussed the possible mechanisms for dTEHV calcification in comparison to the calcification in the native and bioprosthetic heart valves. For this purpose, we did a retrospective study for all the published work of decellularized heart valves. Only animal and clinical studies were included in this review. Those animal and clinical studies were further subcategorized into 4 categories for each depending on the effect of decellularization on calcification. Due to the complex nature of calcification in heart valves, other in vitro and in silico studies were not included. Finally, we compared the different results and summed up all the solid findings of whether decellularized heart valves calcify or not. Based on our review, the selection of the proper heart valve tissue sources , decellularization technique  and implantation techniques  could provide a perfect anticalcification potential even without in vitro cell seeding or additional scaffold treatment. Cardiovascular diseases are the first cause of death worldwide. Among different heart malfunctions, like myocardial diseases, arterial atherosclerosis, aneurism and coronary artery infarction, native heart valve failure due to degenerative calcification is still a challenging problem. Calcification is divided into dystrophic and metastatic. While the first category is associated with localized damage or infection with no Ca/P abnormalities in the blood serum, the second one is more associated with an abnormal systematic level of Ca/P in serum due to certain diseases .Heart valve calcification usually involves two stages: heart valves sclerosis (disease initiation) and valves stenosis  . A long Nowadays there are two main types of heart valves substitutes: Mechanical and Bioprosthetic heart valves. Mechanical valves are mechanical devices from metals and/or polymers working as non-return flow valves. The main disadvantage of this type of valves is the formation of thrombotic clots in the stagnation points and/or hemolysis which requires lifelong anticoagulation therapy for the patients . BioprosTo overcome the long-term malfunctions associated with both types of heart valve replacements, new types of heart valve substitutes have been developed over the past 20 years, generally known as tissue-engineered heart valves (TEHV) , 8. In tDespite the significant progress in this kind of dTEHV, they still can suffer from immunological rejection and calcification . The latIn this review article we sought to find answers to the following questions: (1) Do decellularized heart valves calcify in vivo? (2) which factors can induce, reduce, or prevent the calcification process? (3) what are the possible mechanisms behind their calcification?For this purpose, we did a retrospective study for all the English published literature of decellularized heart valves. Only animal and clinical studies were included in this review. Both animal and clinical studies were further subcategorized into four categories depending on the effect of decellularization on calcification: (A) Decellularization totally prevented calcification, (B) Decellularization reduced calcification, (C) Decellularization showed both pro-calcification and anticalcification potential based on the conditions involved and finally (D) Studies those do not mention clearly the effect of decellularization on calcification. All of those categories were tabulated and compared to each other’s depending on the host animal , the type of heart valves involved, the technique of decellularization, recellularization and type of cells used, duration of the study follow-up, the size of the group involved and finally the techniques used to evaluate calcification occurrence. Due to the complex nature of calcification of heart valves, other in vitro and in silico studies were not included in this study. Finally, we compared the different results and summed up all the solid findings of whether decellularized heart valves calcify or not.In general, calcification of the native heart valves is a complex and poorly understood process. However, it appears to share a lot of similarities with different physiological and pathological conditions in the human body like ossification (cell differentiation and trans-differentiation) and atherosclerosis (immune-inflammatory responses)  as shownIn which the cellular activity results in calcification of the native heart valves. This can take place through either differentiation or trans-differentiation of surrounding cells. In the first one, mesenchymal progenitor cells differentiate into osteoblast-like cells and subsequently forms a bone-like matrix within the natural soft valvular tissue. While in the latter one, different kind of cells  and endothelial cells) transdifferentiate into osteoblast-like cells .In which the response of the immune system to different stimulations resulted in calcification. It takes place through two different cross-related mechanisms  as shown(a) Atherosclerosis-like disease, according to which heart valve calcification can be considered an immune-inflammatory disease; consequently, all the atherosclerotic events will be seen in the heart valve calcification.(b) Angiogenesis and neovascularization; although healthy human aortic valves are avascular structures, in calcified valves, there is evidence of angiogenesis near calcified nodules, under the leaflet border, and in areas infiltrated with inflammatory cells . CalcifiMatrix Vesicle formation and microcalcific nodulesIn this calcification process, heart valve calcification is associated with matrix vesicles (MV) formation that serve as a nucleus for calcium deposition. Under normal physiological conditions, MV takes place in the process of the natural formation of bone and cartilage. On the other hand, under certain circumstances -like hypercalcemia- smooth muscle cells have shown a release of MV which could lead to microcalcific nodules formation. Noteworthy, such a process takes place under inflammation conditions suggesting that inflammation may be a considerable factor in the calcification process .This microcalcific nodule formation has also been shown to be a passive process meaning that no active cellular mechanisms are involved. This is supported by the presence of amorphous and crystalline calcific deposits at the sites of cell death, either by apoptosis or necrosis. Therefore, the residuals of the dead cells’ debris may be involved in the formation and propagation of calcific deposits. However, the underlying mechanism is not yet well understood . ConclusECM remodeling-mediated calcificationA mentioned earlier, one of the mechanisms involved in heart valve calcification is the abnormal remodeling of valvular tissue ECM. This is often a consequence of increased expression of some metalloproteinases and cathepsins, which may result in the degradation of collagen and elastin and the formation of pro-inflammatory peptides, provoking the inflammatory response and allowing the expansion of calcific deposits. Additional evidence for this cellular mechanism is the observed activation of VIC in the calcified heart valves. It leads to abnormal secretion of collagen, hyaluronan, and other extracellular components, consequently affecting the stiffness of the valves and modulating the transition of VIC into osteoblast-like cells .Lipid insudationThose two mechanisms exhibit their calcific action through one or more of the following ways:Atherosclerotic-like mechanism of calcification involves lipid accumulation on the formed calcific deposits, because of hypercholesterolemia. A significant relation has been found between the high levels of proatherogenic oxidized low-density lipoprotein and fibrocalcific deposits formation on aortic heart valves .Besides, it has been shown that the lipid level reduction in the blood after pharmaceutical treatment with HMG-CoA reductase inhibitors (statins), which decreased the osteogenic pathways signaling, led to reduced aortic valve calcification in hypercholesterolemic animal models, compared with control, non-treated animals . HoweverThe pathogenesis of bioprosthetic heart valve calcification is even less understood than that of the native aortic valve. Still, the general mechanisms of both valves seem to be close .Like in native valves, bioprosthetic calcification involves passive and active mechanisms. Concerning the active mechanism, there is a close similarity between both native and bioprosthetic heart valves like the valvular infiltration of cellular elements  and the expression of noncollagenous matrix proteins . The ass2+ concentration inside the cells and raises the chance for further interaction with phosphate present at different sites of the cell (like cell membranes and DNA), thus forming the critical nuclei needed for the initiation of the process .Tab.Tab80].T® , which showed serious problems in its early models [® , a decellularized porcine heart valve used since 2002 in nearly 200 cases. Another product of the same company, Matrix P plus N™ was recently approved [The first commercially available product in the market was Synergrafty models . Howevery models . Matrix approved . Lately,approved .Ross operation.Different clinical and animal studies showed successful early, mid, and long-term performance of decellularized pulmonary heart valve allografts without calcification for aortic heart valvesallografts without calcification.Different clinical and animals’ studies showed successful early, mid, and long-term results studying the function of decellularized Despite the limited number of successful decellularized heart valves xenografts in animal models, only one short and mid-term clinical study with successful results have been reported.Although the immunological factor in heart valve calcification is controversial in the literature, in most of the clinical and animal studies it was deemed as an important factor for calcification.In the case of calcification of decellularized heart valves, there was no unique pathway different from the calcification mechanisms taking place in native and glutaraldehyde fixed ones.The careful selection of the proper heart valve tissue sources , decellularization technique  and implantation techniques  could provide a perfect anticalcification potential even with no need for in vitro cell seeding or additional scaffold treatment.The different species of decellularized valves, decellularization techniques, recellularization or not, storage conditions, characterization tools, duration of follow-up, experimental population and even the confusion in the used terminology, constitute a vast body of complex data, from which it is very difficult to draw solid conclusions. However, still few conclusions may be drawn:


Lipids are essential components of cell structure and play important roles in signal transduction between cells and body metabolism. With the continuous development and innovation of lipidomics technology, many studies have shown that the relationship between lipids and cancer is steadily increasing, involving cancer occurrence, proliferation, migration, and apoptosis. Breast cancer has seriously affected the safety and quality of life of human beings worldwide and has become a significant public health problem in modern society, with an especially high incidence among women. Therefore, the issue has inspired scientific researchers to study the link between lipids and breast cancer. This article reviews the research progress of lipidomics, the biological characteristics of lipid molecules, and the relationship between some lipids and cancer drug resistance. Furthermore, this work summarizes the lipid molecules related to breast cancer diagnosis and prognosis, and then it clarifies their impact on the occurrence and development of breast cancer The discussion revolves around the current research hotspot long-chain non-coding RNAs (lncRNAs), summarizes and explains their impact on tumor lipid metabolism, and provides more scientific basis for future cancer research studies. Breast cancer is a malignant disease that occurs in breast tissue. It occurs more frequently in women. It is also the second leading cause of female mortality. According to the breast cancer report updated by the American Cancer Society in 2019, the incidence of breast cancer in women increased by 0.3% annually from 2012 to 2016 , ultrasoThe evidence from many studies has confirmed that the metabolism of proliferating tumor cells is different from the cells in normal tissues . The occIn this paper, we review the literature on lipid and its metabolites in various experimental models in recent years, and then we summarize the valuable and potential lipid biomarkers to provide the basis for further study on the pathogenesis, diagnostic methods, and adverse clinical prognosis.Lipids are means as small hydrophobic or amphiphilic molecules that are perhaps derived wholly or partially from the condensation of carbocation groups of thioesters and/or the condensation of carbocation groups of isoprene units ,25. BecaThe chain extension of acetyl Coenzyme A (acetyl-CoA) primer and malonyl-CoA  group synthesizes FAs. The functional groups are replaced by other atoms to form various molecular groups . Some coSignificant changes in the lipid molecular structure have created a diversity of biological functions . The comIn 2003, Han and Gross and others initiated the concept of “lipidomics” . LipidomThe research content of lipid metabolomics mainly includes four parts: lipid extraction, separation, analysis and detection, and related bioinformatics analysis . BecauseHigh-resolution bio-mass spectrometry is the core tool of lipidomics analysis at present that can close the gap between lipid metabolism and clinical disease manifestations. There are several commonly used methods. The first is electrospray ionization mass spectrometry (ESI) . ESI is Lipidomics, as a new research field, represents a new approach for diagnosis and treatment innovation in the next generation. Mass spectrometry, as a powerful analytical technique, provides convenience for lipidomics to explain disease mechanisms. Drug action  is an important treatment for cancer patients. However, long term use results in drug resistance that reduces the treatment’s efficacy and accelerates the progress of cancer, ultimately leading to the death of patients. Though the proper combination of anti-cancer drugs has dramatically improved the treatment of malignant tumors, drug resistance to chemotherapy often occurs, and the resistance of cancer to chemotherapy drugs remains the main clinical problem in preventing a recurrence. The ATP-binding cassette (ABC) transporter superfamily is a group of transmembrane proteins. The ABC transporter uses ATP to hydrolyze energy to transport a series of ionic compounds  into the cell in the opposite direction. In addition, the ABC transporter has the ability to transport heterologous biological substances and drug-conjugated compounds. P-glycoprotein, multidrug resistance-related protein (MRP1), adenosine triphosphate binding transporter G superfamily member 2 antibody (ABCG2), mitoxantrone resistance protein (MXR), and breast cancer resistance proteins 1 (BCRP1) can help the body block the transport of drug molecules, expel anti-cancer drugs from cells, and reduce the effective concentration in cells, thus leading to acquired multidrug resistance. Drugs targeting tumor vessels have a limited effect on the survival rate of patients, which remains attributed to the patient’s resistance to anti-angiogenic drugs (AADs) ,57. WhatThe rapid growth and expansion of a tumor depends on large angiogenesis . Blood vIt is widely known that the reduction of glycoproteins and the increase of alpha-fetoproteins and carcinoembryonic antigens on the surface of cancer cells also promote the spread and metastasis of tumor cells. However, the role of lipid molecules in tumor drug resistance is often neglected. In fact, lipid metabolism plays an essential role in regulating tumor sensitivity to anticancer drugs. However, it should be noted that whether it is a CTP1 inhibitor or a macrosomia blocker, it may not be sufficient for cancer treatment alone because this release may not eliminate malignant cells. Clinically, the combination of a cell inhibitor, targeted therapy, and a lipid metabolism inhibitor is more effective in treating cancer . This neLipids widely exist in human blood, urine, hair, and other tissues with convenient accessibility. Therefore, there have been many valuable lipid molecular screening experiments using lipidomics technology. Experiments have shown that a lipid is a useful biomarker for cancer diagnosis ,64, espeIn some studies, changes of phospholipids in plasma, serum ,81, and Changes in the concentration of lipid metabolites reflect changes in the disease state. Monitoring the fluctuations of metabolites in organisms is an innovation in the early detection of breast cancer, but it is still in its infancy. Both the separation and analysis of metabolites require more mature technical and theoretical support. While researching lipid markers, we must also develop new and more valuable lipid molecules to detect early breast cancer.The metabolism of most tumor cells differs from that of healthy cells. In cancer cells, in addition to changes in glucose metabolism, lipid metabolism also undergoes adaptive changes ,95. DuriThe conserved nuclear protein 54 kD Nuclear RNA-and DNA-binding protein (p54nrb) plays a role in RNA splicing and editing, DNA repair, and gene transcription . These fIn addition, the development and metastasis of cancer are also closely related to the imbalance of the body’s redox balance . This imFor breast cancer patients, lymph node status is the most accurate and important survival predictor. In addition, tumor size, the degree of differentiation, and histological type can also be used as prognostic factors. With the in-depth study of lipid molecules, people have discovered changes in lipid levels in the prognosis of different types of cancer. This abnormal change is associated with the survival risk of a variety of cancers, including breast cancer ,117,118.Triglycerides (TGs) and cholesterol (CHO) play important functions in biofilm composition and energy storage; 27-hydroxycholesterol (27HC) is the main metabolite of high cholesterol and is also a ligand for the estrogen receptor and liver X receptor (LXR) . It is cTriple-negative breast cancer (TNBC) is one of the subtypes of breast cancer with a high risk of early recurrence and a low survival rate. A study found that the content of PC (32:1) in recurrent TNBC was significantly higher than that in non-recurrent TNBC. Compared with estrogen receptor-positive and human epidermal growthfactor receptor 2 (HER2) breast cancer, TNBC contained more PC (32:1) and PC (30:0) , suggestLong-chain non-coding RNAs (lncRNAs) have shown immeasurable potential value as a hotspot in molecular research, especially in the rapid development of tumor metabolic reprogramming. Some studies have shown that lncRNA can affect cancer migration, invasion, and other pathological processes . lncRNAs are defined as transcripts of more than 200 nucleotides in length without a coding ability . LncRNAsIn short, lncRNAs participate in lipid metabolism in different ways to affect tumor progression. We have explored the effect of lncRNAs on lipid metabolism and understand the mechanism of tumor carcinogenesis. These all provide new ideas and strategies for tumor treatment.Lipids play an important role in the occurrence and development of cancer. They transmit information through communication between cells, regulate cell metabolism, and maintain an internal stable state. Studies have shown that lipid molecules are potential biomarkers for biological diagnosis. Combined with mass spectrometry as an auxiliary detection tool for mammography, they are expected to realize non-invasive early screening of breast cancer. In addition, with the continuous development of lipidomics technology, combining lipidomics with other omics technologies can give full play to the role of lipidomics in understanding the molecular mechanisms of disease. Though there have been many studies on the relationship between lipids and various diseases at home and abroad, there have been few studies on how lipids and their metabolism are involved in the occurrence and development of breast cancer. This is caused by both the structural diversity of lipid types and the immaturity of separation technology, which are challenges that need to be overcome in lipidomics research. Therefore, it is necessary to combine the existing biological analysis technology and emerging interdisciplinary theory to make a breakthrough in the research of lipidomics.


One key aspect of human aging is the dysregulation of the immune system. As we age, our body’s defense system weakens, leading to declined responses to infection by new pathogens and a decreased ability to become immunized  cell, macrophage, neutrophil, monocyte (MC), dendritic cell (DC), etc., which can recognize and destroy the invading pathogen and infected cells nonspecifically. The adaptive immune response involves the T cell (TC) and B cell (BC) and mounts a response specific to the invading pathogen. Naive TCs and BCs express specific receptors (TCR and BCR) on their cell membrane that can recognize specific antigens. Upon binding to a specific antigen, immature B cells activate, proliferate, and differentiate into plasma cells to produce a specific antibody that can neutralize and guide the destruction of the pathogen. Naive T cells recognize the antigen presented by antigen presenting cells (such as DCs), proliferate, and differentiate into effector T cells, memory T cells, and regulatory T cells. Memory is formed so that the system can launch a swifter response upon future encounter of the same pathogen. At a finer resolution, the complexity is even more daunting as each cell type has multiple subpopulations that express specific sets of genes and are specialized to different tasks.Given the complexity of the immune system, it is unsurprising that the aging phenotypes at the cellular/molecular level are also very complex. Instead of simple failure of particular components, aging brings about changes that cause dysregulation of the system , including BCs, TCs, NK cells, MCs, and DCs. Using a number of single cell technologies, they were able to quantify subpopulations in these different types of cells, characterize the changes in gene expression and chromosome accessibility in the subpopulations, and measure the clonal types and diversity of TCRs and BCRs. To characterize the interaction between aging and COVID-19, they also collected samples from young and old patients, either in the onset or recovered phase, and characterize the change of cell composition and gene expression.+ and CD8+ naive T cell, as well as an increase of CD4+ effector memory T cells (Tem), CD8+ cytotoxic T cells (CTL), and exhausted T cells (Tex), in relative abundance in the respective subpopulation. The fraction of the classic CD14+ MC in PMBC clearly increased. Overall, the analyses of cell types and subpopulations indicate that aging shifts the immune cells towards more polarized and inflammatory populations. This general conclusion is consistent with previous findings in animal models and from human cytometry studies  and mass cytometry by time of flight (CyTOF). scRNA-seq measures genome-wide mRNA expression and the data can be used to identify different cell subpopulations, analyze differential gene expression, and infer transcriptional regulators. CyTOF is a variation of flow cytometry where antibodies are labeled by heavy isotopes and the abundance is measured through mass spectrometry. This method relies on antibody labeling of candidate proteins that are known to be present in different cell populations. The authors showed that the two independent methods both indicate that there is a decreased TC and BC populations and increased NK cells and MC populations . On a finer resolution, the authors found a decrease in CD4+ T naive, Tcm, Tem, Treg, and Tex cells, and found genes common/specific to different subpopulations.While the changes in the proportion of different cell types and their subpopulations undoubtedly contribute to the age-related decline of immune function, detailed characterization of the change of internal state of different subpopulations may provide mechanistic insight into the failure of different components and the mis-regulation of their interactions. To characterize the internal state of each cell subpopulation, the authors combined scRNA-seq and single cell ATAC-seq (the assay for transposase-accessible chromatin with sequencing) to map differential expression and differential chromosome accessibility in different cell subpopulations between the young and old subjects. They found differentially expressed genes (DEGs) common to all five immune cell types , as well as genes specific to a subset of these cell types. Zooming in with higher resolution, the authors found DEGs in different subpopulations of each of the cell types, e.g., DEGs in CD4Besides the conserved gene expression signature in inflammatory pathways and oxidative stress response across different cell types, the authors found increased effector genes in TCs, and decreased genes involved in antigen presentation in DCs, suggestive of functional changes (such as the declined antigen presenting ability of DC) driven by intrinsic gene expression changes. Since the chromatin accessibility at gene promoters generally correlates with the expression level, their scATAC-seq data also allowed classification of different cell types and identification of differentially expressed transcription factors (DETs) based on the differential accessibility to the binding site , decreased number of unique clones, and increased clonal expansion (increased size of the maximum clone). The detailed sequence information also revealed that the use of the variable region genes is skewed. Going to finer resolution, the authors found decreased diversity in CD8+ Tem, possibly indicating decreased memory T cell survival due to aging. These age-associated changes of TCR and BCR may underlie the declined adaptive immune response in elderly people.Previously, it was found that diversity of naive T cells shrinks considerably in the aged population, suggestive of declined adaptive immunity  compared with the non-CD16 expressing classic monocytes and are expanded in various inflammatory diseases (Ziegler-Heitbrock, Having quantified the changes in PBMC due to aging, the authors went on to analyze the interaction between aging and COVID-19. Through a four-way comparison of young and old subjects with and without COVID-19, the authors found that COVID-19 infections lead to a depletion of TC and an increase of MC population, and in particular a depletion of CD4To summarize, Zheng et al. systematically characterized the change of the human circulating immune cell landscape due to aging and COVID-19 and analyzed the interaction between the two. Using carefully designed experiments and various cutting-edge single cell technologies, they were able to quantify the changes in composition and the internal state of various immune cell types and their subpopulations. While many of their findings regarding the aging of the immune system are consistent with previous studies based on model organisms and flow cytometry studies of human immune cells, the authors presented a high resolution map with many new observations that potentially connect the changes at the cellular and molecular level to the macroscopic change of immune function. Their study also provided a vast amount of data that can be further explored by the research community to generate new testable hypotheses.The big challenge going forward is to construct a causal and dynamic model of immune system aging, and to devise interventions based on mechanistic understanding. Charactering changes in various immune cell types and their subpopulations is an important first step towards that goal. Much more work is needed in the future to delineate changes that are drivers of the functional decline vs. those that are adaptive responses and even passive followers. Larger cohorts with a broader age distribution and longitudinal samples with finer temporal resolution should allow a better mapping of the dynamics of the change. In addition, characterization of PBMC should also couple with the characterization of lymphoid organs and other circulating factors in the blood, to better understand how the functional decline is shaped up by the altered interactions between these different components of the immune system. Recent studies of model organisms and other human samples also provide a great opportunity for comparative analysis across human tissues and across different mammalian species, to delineate conserved aging signatures and tissue/organ/species-specific features. Ultimately, mechanistic hypotheses connecting cellular and molecular changes to their functional consequences will need to be tested by targeted perturbation-response experiments.


Paroxysmal sympathetic hyperactivity (PSH) has predominantly been described after traumatic brain injury (TBI), which is associated with hyperthermia, hypertension, tachycardia, tachypnea, diaphoresis, dystonia (hypertonia or spasticity), and even motor features such as extensor/flexion posturing. Despite the pathophysiology of PSH not being completely understood, most researchers gradually agree that PSH is driven by the loss of the inhibition of excitation in the sympathetic nervous system without parasympathetic involvement. Recently, advances in the clinical and diagnostic features of PSH in TBI patients have reached a broad clinical consensus in many neurology departments. These advances should provide a more unanimous foundation for the systematic research on this clinical syndrome and its clear management. Clinically, a great deal of attention has been paid to the definition and diagnostic criteria, epidemiology and pathophysiology, symptomatic treatment, and prevention and control of secondary brain injury of PSH in TBI patients. Potential benefits of treatment for PSH may result from the three main goals: eliminating predisposing causes, mitigating excessive sympathetic outflow, and supportive therapy. However, individual pathophysiological differences, therapeutic responses and outcomes, and precision medicine approaches to PSH management are varied and inconsistent between studies. Further, many potential therapeutic drugs might suppress manifestations of PSH in the process of TBI treatment. The purpose of this review is to present current and comprehensive studies of the identification of PSH after TBI in the early stage and provide a framework for symptomatic management of TBI patients with PSH. There is a subgroup of patients with simultaneously paroxysmal transient increases in sympathetic activity involved in heart rate, blood pressure, respiratory rate, temperature, sweating, and posturing activity, which may also persist over time, and are associated with worse outcomes . The uniThere are about 8–10% of TBI survivors affected by this complication . PreviouAlthough the natural course between autonomic dysfunction and outcome of patients after TBI is not clearly understood, the objective quantification of such complications seems to be associated with global patient outcome . Given tIt is generally accepted that poor long-term outcome in PSH patients is associated with a low level of consciousness recovery, and early appearance and long duration of severe dysautonomic symptoms . ConventThe purpose of this review is to provide an overview of the pathophysiological mechanisms, clinical features, and identification methods, and discuss the substantial commonalities of therapeutic options for PSH. We also present an algorithm for the identification and management of PSH after TBI.Several theories exist regarding the pathophysiology of PSH syndrome, although early epileptogenic theories have been abandoned for lack of empirical evidence . ExistinAlthough the anatomical basis of the pathogenesis of PSH is still undefined, research has indicated that specific TBI damage characteristics will increase in occurrence. The presence of focal parenchymal lesions within the brain increases the likelihood of developing PSH . More deRecently, neuroendocrine regulation disturbances observed in the maladaptive response provide new insights for the pathophysiology of PSH . In the The primary clinical feature of PSH is simultaneous, paroxysmal transient increases in sympathetic, as well as motor activity . AlthougClinically, up to 72% of PSH patients with the above symptoms are caused by the unavoidable non-noxious stimuli . Some TBIn the past, long-term clinical observations have revealed that PSH is more prevalent in men than in women , 9, 50. Symptom-based findings are used for the early identification of PSH in TBI patients. In 2014, the PSH assessment measure (PSH-AM) tool , a cliniPrevious and recent cases provide evidence that PSH-AM can not only serve as reliable diagnostic criteria, but also stratify the severity of PSH , 57. TheNevertheless, it is important for clinicians to avoid rejecting a diagnosis, due to the absence of a certain feature or inconsistencies with the proposed diagnostic criteria , 62. CurFurthermore, some clues have emerged through the use of imaging techniques, in predicting the onset of PSH in TBI patients. First, a previous study suggests that the presence of focal lesions on computer tomography (CT) images during the first 48 h was associated with a greater probability of PSH crises as compared with the presence of diffuse lesions or a normal CT in TBI patients . SubsequTraditional views hold that obstacles to the development of PSH treatment are the following: (1) insufficient understanding of brain regions, 2) no definite relationship between neurotransmitters or hormones and clinical symptoms, 3) lack of standardized measures to assess the curative effect, and (4) insufficient evidence from clinical trials regarding the benefits of intervention for long-term outcomes  occurs in some severe cases, a switch to intrathecal injection is recommended, not only in treatment after initial treatment failure, but also makes other drugs dispensable , 66.In view of the above understanding, there are three treatment approaches: symptom elimination, symptom prevention, and refractory treatment . With thHowever, an entirely persuasive meta-analysis is not possible because of heterogeneity and poorly documented published data . There aRecent studies have recommended some non-pharmacological treatment methods for PSH . Before Supportive therapy helps to improve long-term outcomes of PSH patients . A previGiven that the occurrence of PSH may result from the presence of cerebral hypoxia, hyperbaric oxygen therapy (HBOT) in the management after TBI has been reported in a series of refractory PSH cases. Patients benefit from the increased oxygen availability due to improvements in cerebral aerobic metabolism in injured tissue , 92. TheWe integrated conventional references and then designed the protocol named “an algorithm for the management of PSH” (For details, see Our primary motive was to decrease missed diagnosis and misdiagnosis, and further avoid excessive medication, to improve the prognosis of our TBI patients in our institution. Meanwhile, this algorithm will provide one standardization screening procedure to address the limitations in individual identification and management of PSH. Although our protocol is simple and has not been certified by experimental findings, it will enormously raise our general awareness, enhance different levels of diagnosis and treatment ability, and possibly promote the development of guidelines for PSH management in the future.It is generally known that PSH is mostly prevalent in TBI patients, and the understanding of advanced pathophysiological, clinical features, and recognition methods of PSH in TBI patients is necessary.Although previous studies have not given a definite explanation, the disconnection theory and excitation:inhibitory ratio model were established foundations for the subsequently pathophysiological exploration. The findings of neuroendocrine disorders reveal a new sight of pathological analysis. Nowadays, more and more attention has been put in quantifying the frequency, severity, and duration of paroxysmal episodes. Given the complexity and diversity of PSH manifestations, early identification of the location and severity of TBI is essential for clarifying the neuropathology of PSH symptoms. Some features detected from the evidence-based clinical practice will provide predictors for early identification of PSH in TBI patients. Moreover, some risk factors such as age, early fever, GCS score, and the use of tracheostomy have been reported to be associated with the development of PSH. The development of laboratory examination and neuroimaging findings would provide more objective basis for the early diagnosis of PSH. The PSH-AM tool, which has been approved increasingly by clinicians, was the most optimum in early identification and prospectively detects the rate and stratifies the symptomatic severity of PSH by far. With the help of PSH-AM, a decline in the prevalence of PSH was revealed in TBI patients. In the future, rigorous investigations and prospective studies are needed to present more reliable data, for example, to establish whether individual management modulates the relationship between the severity of PSH and long neurological outcomes in TBI patients and to stratify complications caused by PSH as an entirety or PSH associated with the TBI.The advances in treatment of PSH are collected from the relevant literature reports. However, compiling universal protocol, using uniformly evaluation criteria, and accumulating the multi-center medical reports to minimize data bias remain challenging. It remains to be seen whether we can co-opt the therapeutic experiences of acute TBI to ease PSH symptoms. On the basis of the therapeutic regime reported in previous case series and small trials, more scientifically and rationally designed clinical trials to judge the putative benefits of PSH are needed.Z-QL and R-ZY contribute to literature extraction and prepared the manuscript. R-ZZ drafted this manuscript. G-HH and G-MZ supervised the study. All authors read and approved the final manuscript.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.


ObesStrober and Brady reviewed recent findings on the role of increased dietary fructose consumption in shifting the metabolic state of the whole body and in specific tissues, which had profound effects on many diseases, including obesity, diabetes, and cancer.Diet induces alterations in systemic and cellular metabolic profiles. Western diet high in fats and fructose is one of the main causes for the prevalence of many metabolic diseases , 9. In aYee et al. summarized the central role of the insulin signaling pathway in diabetes and cancer by regulating glycolysis, ROS levels, inflammation, cell migration, and angiogenesis. The review has also pointed out the importance of treating insulin resistance for oncologists because the insulin signaling is critical for promoting cancer initiation and progression.Hyperinsulinemia or insulin resistance is common in diabetic or obese patients . After bBerdeaux and Hutchins discussed the challenge of specifically targeting the CREB (cAMP response element binding protein) and CRTCs  in the skeletal muscle without disturbing CREB-CRTCs in other organs or cancer cells. One solution is to identify skeletal muscle specific regulatory mechanisms controlling CREB-CRTC transcriptional activity to safely improve health in patients with obesity, diabetes, and cancer.Decreased skeletal muscle mass with reduced glucose uptake and catabolism contributes to whole body metabolic dysfunction in obesity, diabetes, and cancer , 14. TheDai and Jiang updated the metabolic roles of these GTPases in obesity, diabetes, and cancer by balancing fusion and fission of mitochondria and orchestrating mitochondrial number and function.The number and function of mitochondria is largely controlled by GTPases such as Mfn1, Mfn2, Opa1, and Drp1 which regulate the fusion and fission of the organelle . Since mde novo nucleotide biosynthesis, the PPP is critical for regulating the redox homeostasis and macromolecular biosynthesis  is drawing more and more attention. Being the major source for NADPH and ynthesis , 19. Ge Zhao et al. studied the function of Piezo1, an adipocyte-rich cation channel stimulated by membrane tension and stretch, in adipose tissues. Adipose-specific Piezo1 knockout mice were prone to diet-induced metabolic diseases including obesity, insulin resistance and hepatic steatosis by triggering pro-inflammatory responses.In order to explore the role of obesity-induced mechanical pressure in lipid metabolism and insulin sensitivity, Our series of reviews and original research summarized some recent progress in elucidating metabolic, hormonal and immune connections between obesity, diabetes and cancer. Given the epidemic of the diseases, reducing cancer occurrence in obese or diabetic patients, and in addition, reducing the prevalence of metabolic syndrome that associates with increased risk for cancer, will be crucial for greatly improving global health.XT, MK, and DT wrote the editorial. 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.


Atherosclerosis is responsible for 20% of ischemic strokes, and the plaques from the internal carotid artery the most frequently involved. Lipoproteins play a key role in carotid atherosclerosis since lipid accumulation contributes to plaque progression and chronic inflammation, both factors leading to plaque vulnerability. Carotid revascularization to prevent future vascular events is reasonable in some patients with high-grade carotid stenosis. However, the degree of stenosis alone is not sufficient to decide upon the best clinical management in some situations. In this context, it is essential to further characterize plaque vulnerability, according to specific characteristics . Although these features can be partly detected by imaging techniques, identifying carotid plaque vulnerability is still challenging. Therefore, the study of circulating biomarkers could provide adjunctive criteria to predict the risk of atherothrombotic stroke. In this regard, several molecules have been found altered, but reliable biomarkers have not been clearly established yet. The current review discusses the concept of vulnerable carotid plaque, and collects existing information about putative circulating biomarkers, being particularly focused on lipid-related and inflammatory molecules. Stroke is a leading cause of death, disability, and dementia worldwide. It represents the third cause of mortality in western countries. In addition, more than 20% of patients suffer a stroke recurrence within 5 years of follow-up, increasing the risk of severe disability . Eighty Despite the recent advances in medical and surgical treatments, the cerebrovascular burden of atherosclerosis remains considerably high . The manBefore a symptomatic ischemic stroke occurs, an atherosclerotic lesion frequently suffers from asymptomatic microruptures that lead to microemboli . As a coThis review discusses the concept of vulnerable carotid plaque, and it presents the existing information on the putative circulating biomarkers for carotid atherosclerosis, with a focus on lipid-related and inflammatory molecules. This review also discusses the relationship between these biomarkers and the occurrence/recurrence of symptoms, as well as the putative use of these biomarkers in the clinical practice.Atherosclerosis is an inflammatory process triggered by lipoprotein retention in the artery wall. The overlying deposition of lipids leads to narrowing (stenosis) of arteries . AtherosGiven that atherosclerotic lesions exhibit a long-term development, they can be found in several stages, from an initial, asymptomatic, and non-stenotic stage to an advanced phase during which thrombi are formed, leading to embolism . In thisFatty streak: The earliest sign of atherosclerosis is endothelial dysfunction which favors low-density lipoprotein (LDL) entry into the subendothelial space. In this microenvironment, the generation of modified LDL triggers the recruitment of monocytes, their differentiation into macrophages, the formation of lipid-loaded foam cells, and the induction of an inflammatory response.Fibroproliferative lesion: Further inflammation allows the infiltration of more leukocytes, leading to plaque development. The fibrous cap is formed by the activation of smooth muscle cells that are recruited to the intima and that which secrete connective tissue.Advanced lesion: Inflammation is enhanced triggering cell apoptosis and death, and the formation of a lipid-rich necrotic core. Additionally, degradation of the extracellular matrix occurs.More advanced lesion: This condition is characterized by increased cell necrosis, severe inflammation, calcification, cholesterol crystals, neo-vascularization, and thinning of fibrous cap, leading to plaque rupture in the shoulder regions. In the latest stage of the process, a thrombus generation may be triggered by the exposure of the lipid core or by molecules released by cells.Therefore, in the earliest stage of atherosclerosis, LDL is retained within the subendothelial space, where it is modified by oxidative stress and enzymatic activities, and it acquires inflammatory properties. In endothelial cells, modified LDL induces the expression of adhesion molecules and chemoattractant cytokines (chemokines), both involved in the recruitment of circulating leukocytes to the lesion area. Infiltrated monocytes are then differentiated into macrophages, which, in response to modified LDL, elicit an enhanced inflammatory response by releasing more chemokines, other cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), and growth factors. This response promotes the recruitment and activation of SMCs, which secrete connective tissue and thus contribute to the generation of the fibrous cap. SMCs and mainly macrophages are able to uptake modified LDL becoming lipid-loaded foam cells. At this stage, when the counteracting response against inflammation is defective or insufficient, atherosclerotic plaques that had remained stable for a long time may progress and become unstable, as reviewed by Tabas et al. .A stable plaque shows a thick fibrous cap versus lipid core and exhibits a generally calcified state . By contThe most frequent mechanism involved in the pathogenesis of atherothrombotic stroke is the rupture of a carotid plaque with the subsequent thrombus formation and distal embolism . The treSymptomatic stroke patients with carotid arteriosclerosis usually have unstable rupture-prone lesions presenting specific morphological and histological features . These fIn spite of the ultimate advances in carotid imaging, predicting plaque vulnerability remains challenging. In this regard, plasma biomarkers could provide adjunctive criteria, allowing for a better diagnosis, risk stratification, and clinical management.One of the priorities in the field of ischemic stroke prevention is finding new biomarkers that may help in assessing the vulnerability of atherosclerotic plaques. The identification of biomarkers will help in answering the two key questions that arise whenever we find an atherosclerotic plaque in the carotid artery or in the intracranial circulation in the setting of an ischemic stroke: (1) Does a correlation exist between the plaque and the stroke pathogenesis? (2) What is the risk of stroke recurrence? The second question is extremely important in assessing whether a medical treatment alone will be sufficient to prevent new events or whether a revascularization therapy is necessary.Ideally, putative biomarkers must satisfy several criteria , the mosIn the specific case of atherothrombotic stroke, the nature of the biomarkers is expected to be inflammatory or lipid-related because of their involvement in atherosclerosis. An increased concentration of a biomarker or a set of biomarkers should indicate the occurrence or recurrence of ischemic stroke in asymptomatic and symptomatic patients, respectively. In the absence of atherothrombotic stroke, biomarkers are expected to show the lowest level compared with the levels in other conditions; however, biomarker levels may be higher in asymptomatic/symptomatic patients with stable plaques. When a plaque becomes vulnerable, biomarker concentration is expected to considerably increase, especially in symptomatic patients Evaluation of these biomarkers, together with the assessment of the plaques by neuroimaging, , will allow for a better stratification of the risk of atherothrombotic stroke. Therefore, these tools will allow the selection of high-risk patients who may become a candidate for CEA.Currently, individual biomarkers to be targeted routinely remain to be established. In this context, it would be more useful to find alterations in clusters of several biomarkers than to select rigid cutoffs for a single biomarker . On thisIn summary, in the field of atherothrombotic stroke, the identification in the plasma of reliable and specific biomarkers that are indicative of the carotid plaque vulnerability is essential. This review summarizes the up-to-date data on plasma biomarkers for ischemic stroke, with a focus on carotid atherosclerosis and lipid-related and inflammatory molecules. All the information specifically reported on the atherothrombotic subtype is summarized in Several lines of evidence have shown the pivotal role of cholesterol in carotid artery atherosclerosis. Atherosclerotic plaques are lipid-enriched, and their lipid content strongly determines the plaque vulnerability and the rate of stenosis progression. Extracellular cholesterol crystals accumulated in a carotid plaque promote inflammation and erosion of the fibrous cap, eventually leading to plaque vulnerability . SpecifiMost epidemiological studies have shown a direct correlation between lipid levels and risk of ischemic stroke ,115, andA role of lipoprotein(a) (lp(a)) as an ischemic stroke biomarker and as a predictor of recurrence has also been suggested . StudiesBesides size, the biological properties of HDL and LDL are highly influenced by their lipid and apolipoprotein (apo) composition and their content in hydrolytic enzymes. The plasma levels of these molecules have been suggested as putative biomarkers of ischemic stroke. Although their specific role in stroke subtypes is poorly defined, lipid-related biomarkers are suggested to be mainly involved in atherothrombotic stroke. Among apolipoproteins, the main candidates are: apoB/apoA-I ratio, apoE, and apoJ, also known as clusterin. A significant association between the increase in the carotid arterial intima-media thickness and increased apoB/apoA-I levels was found in patients with ischemic stroke ,128. IncSome enzymes associated with LDL and HDL have been proposed as putative ischemic stroke biomarkers, mainly, including platelet-activating factor acetylhydrolases (PAF-AH), also known as lipoprotein-associated phospholipase A2, and paraoxonase-1 (PON1). The circulating PAF-AH is bound to HDL and LDL, particularly in sdLDL . PAF-AH,PON1 is a calcium-dependent HDL associated esterase that protects LDL against oxidation. Low levels of PON1 activity increase ischemic stroke risk , and theAs discussed, sdLDL levels are associated with ischemic stroke. This phenomenon could be a consequence of the higher susceptibility of sdLDL to suffer from modifications compared with normal-sized LDL. The inflammatory properties of modified LDLs are a main topic in atherosclerosis research. Although LDL can be minimally modified in plasma circulation, it becomes more easily modified when trapped in the arterial wall where it is exposed to enzymatic activities and free radicals released by cells. As a result of these modifications, LDL aggregates and thus its return into the circulation is hindered. Extracellular aggregated apoB-containing lipoproteins with atherogenic properties have been found in human carotid arteries . The preApart from oxLDL, the presence of a circulating form of modified LDL with inflammatory properties, called electronegative LDL (LDL(−)) has been described. LDL(−) is an LDL subfraction with a high negative charge, and it constitutes about 3−5% of the total LDL in healthy subjects. An increased proportion of LDL(−) was found in pathologies associated with cardiovascular risk, in patients with acute myocardial infarction , and eveInflammatory factors have been studied extensively within the context of stroke. However, their use as biomarkers is hindered by the fact that these immunological factors are pleiotropic and that their levels may be elevated non-specifically in other conditions that mimic stroke, or in other diseases associated with an inflammatory response. In addition, some inflammatory biomarkers are associated with stroke per se and are directly related to infarct volume. However, stroke subtype may partly determine the inflammatory profile . These pSeveral acute-phase proteins and inflammatory cytokines are increased in blood from patients with ischemic stroke, particularly the atherothrombotic subtype. High-sensitivity C-reactive protein (hs-CRP) levels are particularly high in symptomatic atherosclerosis , and theElevated levels of other cytokines, such as monocyte chemoattractant protein 1 (MCP-1), IL-2, IL-6, IL-9, IL-12, IL-18, and chemokine (C-X-C motif) ligand 1, are associated with ischemic stroke ,149,150.MMP are endopeptidases that degrade the extracellular matrix and thus they play an important function in inflammation, remodeling, and angiogenesis. Plasma MMP-2 levels have been found to be associated with stroke outcome ; howeverCell adhesion molecules (CAMs) are located on cell surfaces and are involved in binding with other cells or with the extracellular matrix. In the inflammatory state, due to tissue damage, CAMs can be released and can activate the secretion of cytokines. The soluble (s) forms of selectins, including E-selectin and P-selectin, are elevated in ischemic stroke . In atheSome cell receptors related to cell inflammation are increased in stroke patients. Lectin-like oxidized LDL receptor-1 (LOX-1) is a scavenger receptor found in atherosclerotic carotid lesions, the shedding of which is promoted by inflammatory molecules and by oxLDL . The solBesides sLOX-1, the soluble forms of other scavenger receptors have been proposed as biomarkers for ischemic stroke. The elevated soluble cluster of differentiation (CD) 163 (sCD163) in plasma has been reported to be the result of macrophage activity within atherosclerotic plaques . In ischThere is evidence demonstrating the role of inflammatory adipokines in ischemic stroke, although it remains controversial . AdiponeThis review focuses on biomarkers that are related to inflammation and lipids. It is important to mention that not only their concentration but also genetic mutations and epigenetic mechanisms can strongly influence the predictive value of these biomarkers. Therefore, genetic analysis may add complementary relevant information to analyze protein levels or may be informative by itself. Single nucleotide polymorphisms have been found in several genes related to inflammation and ischemic stroke ; these pApart from the previously mentioned biomarkers, the involvement of other biomarkers, such as those related to angiogenesis, thrombosis, and calcification has already been extensively reviewed elsewhere, in the context of carotid atherosclerosis . Other pOther biomarkers that are gaining interest in their potential role in the diagnosis and monitoring of several diseases are microRNAs (miRNAs). However, at the moment, lack of research in large clinical trials may delay its implementation in the clinical setting. miRNAs are short, single-stranded, non-protein-coding RNA sequences that function as key post-transcriptional regulators. miRNAs are stable and detectable in peripheral blood, and they play a role in atherosclerotic plaque formation and stability  and in sThe miRNAs described herein have been found to be upregulated. miR-21 is one of the most validated miRNAs in carotid atherosclerosis. It is up-regulated in the serum from patients with ischemic stroke and asymptomatic atherosclerosis  and in hBesides their diagnosis and prognosis use, blood biomarkers help in gaining a better understanding of the molecular mechanisms underlying ischemic stroke; also, they allow for the determination of putative targets for novel therapeutic interventions. In atherothrombotic stroke, targeting the initial specific factors that trigger the atherosclerotic process and an excessive inflammatory response is a promising therapeutic option. As an example, the toll-like receptor (TLR) pathway is essential in the activation of the innate immunity system, and it was found to be the most important activated pathway in the peripheral blood of stroke patients . An incrIn the context of novel therapies, immunotherapy-based approaches that aim to improve stroke outcome are being developed. They are designed based on the inhibition of the acute innate immune response (to limit excessive tissue damage) and post-acutely stimulation of the adaptive immune system to limit post-stroke complications. A first line of therapy would be the use of anti-inflammatory drugs. In this regard, there are promising studies regarding the beneficial role of specific anti-inflammatory molecules, such as IL-10, in atherosclerosis, but they need to be further studied and are still not clinically used in the treatment of patients. Regarding the inhibition of acute innate immune response, in some preliminary studies, the action of inflammatory cytokines is blocked by using different approaches: the use of soluble receptors, the inhibition of their synthesis, the use of anti-inflammatory molecules, or the use of monoclonal antibodies against specific inflammatory mediators. In most cases, however, those studies are preliminary, and the treatments yielded modest beneficial effects due to the complexity of the immune response; conducting a risk-benefit analysis is challenging, and the clinical testing of these treatments has only just started. In this context, clinical trials involving the use of antibodies to block the interaction of leukocytes with endothelial cells have demonstrated limited clinical translation . The CANIt has been hypothesized that some chronic bacterial infections may be associated with the development of atherosclerosis and the risk of complications, such as myocardial infarction or stroke. These bacterial infections seem to play a role in the initiation, progression, and the destabilization of atherosclerotic plaques. In this regard, sub-antimicrobial dosages of antibiotics could be beneficial in inhibiting inflammation. This is suggested by some studies demonstrating the benefits of antibiotics in selected patients. A study from Sander et al. showed a protective effect of roxithromycin treatment in the progression of arteriosclerosis . Once agRegarding the pivotal role of lipids in atherothrombotic stroke, LDL and modified LDL are potentially important targets. A recent study has reported that PCSK9 administration in combination with statins has efficiently lowered LDLc levels and has reduced the operative complications of carotid stenting probably by stabilizing carotid artery plaque . Other f(1)Testing the association between circulating biomarkers and clinical outcomes in big cohorts. Although little observational studies are useful to drive hypothesis, they have to be tested ultimately in big cohorts, in order to increase the sample size. In this regard, we would encourage basic and translational researchers to partnerships with clinicians in big clinical cohorts or trials. Biomarkers substudies within big cohort studies or trials are warranted to increase the evidence of the predictive value of circulating biomarkers.(2)Studying the association between circulating and imaging biomarkers. There are many validated imaging biomarkers that are currently used to predict carotid plaque vulnerability. Studying the association between these validated imaging biomarkers and circulating molecules can be a useful intermediate step for selecting good candidates before testing their predictive value in big cohorts.(3)Including circulating biomarkers in predictive scores. In the field of stroke, there are several examples that demonstrate that merging information from clinical, imaging, and laboratory variables increases the predictive power of some scales created to predict stroke recurrences. We believe that this approach should be taken into account in future research as it may increase the chances of a hypothetical implementation of circulating biomarkers in the clinical practice.Currently, there is no evidence for using a specific plasma biomarker as a predictor of carotid plaque vulnerability or as a predictor of stroke recurrence in patients with carotid atherosclerosis. However, the CANTOS trial represented a proof-of-concept that a plasmatic inflammatory biomarker (in that case CRP) can be used for selecting patients with atherosclerosis who may be candidates for immunomodulation therapies. Despite the little specificity of the CRP, elevated levels of this inflammatory biomarker identified patients that benefited from treatment with canakinumab. These results encourage the research on plasmatic biomarkers. We think that future translational research comprising plasmatic biomarkers and carotid atherosclerosis should be focused on three main frameworks:In summary, circulating biomarkers profiling is a promising tool that may help in the future in identifying plaque vulnerability in patients who are at high risk of recurrence or even first-ever atherothrombotic stroke and would benefit from carotid revascularization. Unfortunately, in spite of the extensive up-to-now search for circulating biomarkers in stroke, no single reliable individual biomarker has demonstrated enough sensitivity and specificity to be established in the clinical practice yet. The most feasible possibility would be to find a combination of several molecules in order to develop a panel of biomarkers. In atherothrombotic stroke, these biomarkers are expected to be lipid-related and inflammatory molecules, which are distinctive features of this etiology, and they may be released from the carotid plaque into the circulation as a consequence of plaque instability or rupture. In this scenario, there is growing evidence of the role of inflammatory biomarkers, as they arise as a consequence of acute stroke, but without being predictive of the progression of the disease. Moreover, their concentration may be non-specifically elevated in other concomitant pathologies. In the context of atherothrombotic stroke, biomarkers related to lipoproteins may be more specific and predictive than inflammatory molecules. In this regard, it would be important not only to measure the concentration of lipids and lipoproteins but also to study the role of alterations in the qualitative properties of lipoproteins, particularly those leading to the presence of modified forms of LDL. However, a main drawback is that the assessment of some of these properties is still technically difficult to implement in routine laboratories. Prospective large clinical studies applying a combination of circulating and imaging biomarkers together with clinical data are essential. Taken together, there is still a long way in the search for circulating biomarkers easily measurable in the clinical practice that may be useful to select patients at high risk of atherothrombotic stroke. In a broader perspective, some of these biomarkers might also be useful in other cardiovascular diseases associated with atherosclerosis.