The contemporary diagnosis of paraneoplastic neurologic syndromes (PNSs) requires an increasing understanding of their clinical, immunologic, and oncologic heterogeneity. The 2004 PNS criteria are partially outdated due to advances in PNS research in the last 16 years leading to the identification of new phenotypes and antibodies that have transformed the diagnostic approach to PNS. Here, we propose updated diagnostic criteria for PNS.A panel of experts developed by consensus a modified set of diagnostic PNS criteria for clinical decision making and research purposes. The panel reappraised the 2004 criteria alongside new knowledge on PNS obtained from published and unpublished data generated by the different laboratories involved in the project.The panel proposed to substitute “classical syndromes” with the term “high-risk phenotypes” for cancer and introduce the concept of “intermediate-risk phenotypes.” The term “onconeural antibody” was replaced by “high risk” (>70% associated with cancer) and “intermediate risk” (30%–70% associated with cancer) antibodies. The panel classified 3 levels of evidence for PNS: definite, probable, and possible. Each level can be reached by using the PNS-Care Score, which combines clinical phenotype, antibody type, the presence or absence of cancer, and time of follow-up. With the exception of opsoclonus-myoclonus, the diagnosis of definite PNS requires the presence of high- or intermediate-risk antibodies. Specific recommendations for similar syndromes triggered by immune checkpoint inhibitors are also provided.The proposed criteria and recommendations should be used to enhance the clinical care of patients with PNS and to encourage standardization of research initiatives addressing PNS. Yet, stated incidence varies from 1.6 to 8.9 per million person-years, suggesting that underdiagnosis and underreporting are still relevant issues.4 It is likely that the expanding use of immune checkpoint inhibitors (ICIs) in oncologic practice will lead to an increased frequency of similar syndromes.6Paraneoplastic neurologic syndromes (PNSs) are remote effects of cancer with an immune-mediated pathogenesis.7 Since then, several advances in the field of PNS suggest that this is an opportune moment to update the 2004 criteria: first, the characterization of new intraneuronal proteins as targets of autoantibodies in PNS; second, the discovery of pathogenic antibodies against neuronal surface antigens in neurologic syndromes that can occur with or without cancer, a group which emphasizes the need for a new definition of an onconeural antibody; and third, some of the 2004 fundamental criteria needed to be redefined or modified. For example, the definition of definite PNS solely based on the presence of onconeural antibodies is no longer adequate. Similarly, in the elderly population, where the prevalence of some tumors is high , the use of criteria that rely on generic tumor association may overestimate the real burden of PNS.In 2004, a set of recommended diagnostic criteria for PNS were defined by a panel of international experts and became the standard for clinical and research purposes.In September 2019, a group of international experts (PNS-Care panel) was convened and charged with revising the diagnostic criteria of PNS to benefit clinical decision making, epidemiologic, and research purposes and to address the ancillary issues outlined above. The following report, which includes a newly developed clinical scoring system (PNS-Care Score), represents the panel's consensus recommendations.The PNS-Care panel initially consisted of 14 investigators from 8 different countries; all members of the panel are neurologists with clinical and research expertise in PNS and related syndromes. The panel started with the premise that revised consensus diagnostic criteria for PNS were required to improve clinical care and support research. The group established 3 levels of certainty in the diagnosis of PNS  according to the coherence between clinical phenotype, antibody, and cancer. In assessing the diagnostic process, the panel reviewed the experience and caveats with detection and interpretation of neuronal antibodies. In addition, new recommendations were considered for neurologic syndromes developing in the context of ICI treatment. It was agreed that several neurologic disorders that can occur in association with cancer are not included in the current diagnostic criteria, such as inflammatory myopathies , myasthenia gravis, polyneuropathies associated with monoclonal gammopathies, and paraneoplastic retinopathy, optic neuritis, and cochlea-vestibulopathy. Well-designed diagnostic criteria already exist for most of these entities, which are historically not included within the spectrum of PNS.An initial draft of the guidelines was discussed during the inaugural meeting in Lyon (France) and subsequently underwent several iterations via electronic communication. The last version was then sent to all 14 members, in addition to 5 additional international experts, for final review and comment. All 19 PNS-Care panel members endorsed the final guidelines.Data sharing is not applicable to this article as no new data were created or analyzed in this study.PNSs are defined as neurologic disorders that (1) can affect any part of the nervous system, often presenting with stereotyped clinical manifestations; (2) occur in association with cancer; and (3) have an immune-mediated pathogenesis that is supported by the frequent presence of specific neuronal antibodies. The 3 parts of this definition represent the main axes of discussion by the panel and constitute the structure of the present guidelines.EncephalomyelitisLimbic encephalitisRapidly progressive cerebellar syndromeOpsoclonus-myoclonusSensory neuronopathyGastrointestinal pseudo-obstruction (enteric neuropathy)Lambert-Eaton myasthenic syndromeThere are no absolute pathognomonic neurologic presentations associated with PNS. However, the panel recognizes specific clinical presentations, here defined as “high-risk phenotypes” and previously known as “classical PNS,” frequently have a paraneoplastic etiology. In these phenotypes, cancer represents an important trigger, and therefore, their clinical recognition should lead to a search for an underlying cancer. The extent of cancer search may depend on the demographic characteristics of the patient  and the type of neuronal antibody (see below). Despite numerous advances in the field of PNS in the last 16 years, including the discovery of new antibodies and novel clinical manifestations, the panel agrees that there are no new descriptions of high-risk phenotypes, and therefore, the list is as below:7 These additional areas of involvement should be included in the description of the phenotype, for example, EM with dorsal root ganglionitis or sensory neuronopathy (SNN) or EM with peripheral neuropathy. EM almost always associates with small-cell lung cancer (SCLC) with Hu  or CV2/collapsin response-mediator protein 5 (CRMP5) antibodies.9The term encephalomyelitis (EM) should be used only in patients with clinical dysfunction at multiple sites of the nervous system, including also peripheral involvement such as dorsal root ganglia, peripheral nerve or nerve roots, as recommended in the 2004 PNS criteria.10 and in this phenotype, the most advances have been made in terms of antibody discovery since 2004. At that time, paraneoplastic and autoimmune LEs were clearly underdiagnosed, and the frequency of reported cases was substantially lower compared with rapidly progressive cerebellar syndromes and sensory neuronopathies.11 Importantly, some of the most frequent cell surface antibodies associate with typically nonparaneoplastic forms of LE, such as leucine-rich glioma-inactivated 1 (LGI1) or contactin-associated protein-like 2 (CASPR2) antibodies.10 Therefore, the historical concept of LE as a phenotype predominantly associated with cancer has changed dramatically in the last 10 years. 13 However, because of multiple variants of LE, such as less common forms almost always associated with cancer, this disorder has been retained as a high-risk phenotype. This is important for 2 reasons: first, because the neurologic presentation of paraneoplastic and nonparaneoplastic cases can be undistinguishable, and second, because some of the associated antibodies  can manifest as paraneoplastic LE in more than 50% of the cases. Although the presence of onconeural antibodies, such as anti-Hu and anti-Ma2, almost always occurs in adults and associate with an underlying cancer, the detection of Hu antibodies in children with LE is exceptionally rare and usually does not associate with cancer.18Limbic encephalitis (LE) usually presents with short-term memory loss, seizures, and psychiatric manifestations rapidly progressing in less than 3 months. The diagnostic criteria of LE were updated in 2016,20 but in general, the patients rapidly develop a severe and bilateral cerebellar syndrome limiting activities of daily living in less than 3 months. The panel decided to avoid the term paraneoplastic cerebellar degeneration when referring to the clinical syndrome because the presentation of the cases with or without cancer can be indistinguishable. Although gait ataxia may be the main or sole initial feature, truncal and limb involvement later in the course of the disease are needed to define it as rapidly progressive cerebellar syndrome. Extracerebellar dysfunction, predominantly involving brainstem, may accompany the cerebellar features. Isolated cerebellar symptoms are typical of Yo 21 and Tr/delta/notch-like epidermal growth factor-related receptor (DNER) antibodies.23 Unlike LE, newly identified antibodies for paraneoplastic (and nonparaneoplastic autoimmune) rapidly progressive cerebellar syndrome have been reported only in isolated case reports or small series of patients . Future research may help to clarify which aspects of cerebellar dysfunction correlate more with specific antibodies.This disorder, previously known as subacute cerebellar degeneration, is characterized by a rapidly progressive cerebellar syndrome, without substantial cerebellar atrophy at early stages of the disease. Cases with hyperacute onset, unilateral onset, or slowly progressive and insidious clinical course mimicking neurodegenerative diseases have also been reported,Opsoclonus-myoclonus syndrome (OMS) is characterized by involuntary, high-frequency, chaotic multidirectional saccadic movements without intersaccadic pauses, and nonrhythmic action myoclonus, often involving the trunk, limbs, and head. Additional features include cerebellar involvement (dysarthria and trunk ataxia) and encephalopathy (ranging from confusion to coma).24 Paraneoplastic OMS in adults frequently associates with SCLC or breast cancer. Patients with breast cancer and paraneoplastic OMS usually have Ri antibodies .26 Compared with adults with nonparaneoplastic OMS, those with paraneoplastic OMS are more likely to be older, develop encephalopathy, and have a poorer outcome.25 In young women, OMS may appear in association with ovarian teratomas without neuronal antibodies.27Two main etiologies for OMS include paraneoplastic and idiopathic mechanisms, although there is increasing evidence suggesting that the latter is usually an immune-mediated, postinfectious process. Paraneoplastic OMS in children accounts for 50% of cases and is closely associated with neuroblastoma.28 The potential causes of SNN are diverse, including Sjögren syndrome or platinum-based chemotherapy, but a paraneoplastic origin should be especially considered if patients have inflammatory CSF or motor involvement.29 The terms sensorimotor/sensory neuropathy, polyradiculopathy, or polyradiculoneuropathy should be used when the clinical and electrophysiologic findings indicate additional involvement of the peripheral nerves or nerve roots. The most frequent antibody specificity for SNN is Hu, followed by CV2/CRMP5 and amphiphysin antibodies.31SNN refers to a phenotype caused by involvement of the sensory neurons of the dorsal root ganglia and manifesting with sensory deficits sometimes accompanied by motor symptoms due to additional involvement of motor nerve roots of peripheral nerves. SNN diagnosis (regardless of etiology) should follow previously reported criteria.32 An abnormal gastric emptying or small bowel manometry confirms the diagnosis. Gastrointestinal pseudo-obstruction is due to a myenteric plexus dysfunction and may occur along with other features of autonomic involvement, SNN, or EM. The identification of Hu antibodies suggests a paraneoplastic origin,32 whereas antibodies against ganglionic acetylcholine receptor are more frequently seen in nonparaneoplastic cases.33This term applies to a clinical picture characterized by recurrent episodes of abdominal pain, distension, constipation, and/or vomiting, without evidence of mechanical obstruction.34 Clinical suspicion must be confirmed with electrophysiologic studies.35Lambert-Eaton myasthenic syndrome (LEMS) is characterized by the progressive development of proximal muscle weakness that usually starts in the lower limbs and follows with involvement of the upper limbs, distal muscles, and finally the ocular and bulbar muscles. About 90% of patients have symptoms of autonomic dysfunction, which is a hallmark of LEMS, including dry mouth, erectile dysfunction, and constipation. In addition to muscle weakness and dysautonomia, patients have decreased or absent muscle reflexes, which improve after repeat exercise or maximal voluntary contraction.34 Conversely, antiglial nuclear antibodies (or SOX1 antibodies) are strongly associated with SCLC or paraneoplastic syndromes associated with SCLC; therefore, their detection in patients with LEMS strongly suggests the presence of an underlying SCLC.36 In addition, the Dutch-English LEMS tumor association prediction score is based on clinical criteria and is useful in the discrimination between paraneoplastic and nonparaneoplastic LEMS.37Antibodies against P/Q type voltage-gated calcium channels (VGCCs) are present in nearly 90% of the patients, although their detection is not needed for the diagnosis. These antibodies occur similarly in the paraneoplastic and nonparaneoplastic forms of the disease.Intermediate-risk phenotypes are neurologic disorders that can occur with or without cancer. The recognition of these phenotypes should prompt consideration of a PNS, particularly when no alternative explanation is found, and patients should be tested for neuronal specific antibodies.The Panel proposes to consider a possible intermediate-risk phenotype when the onset is rapidly progressive (<3 months) or there are inflammatory findings in the CSF or brain/spine MRI. The panel acknowledges that the list of possible intermediate-risk phenotypes is far from complete but listed below are some of the most suggestive ones:10 This applies especially for those cases with multifocal or diffuse involvement not restricted to the limbic system, such as anti-mGluR5 ,38 or anti-GABAAR encephalitis .39Encephalitis other than well-defined LE can be considered as intermediate risk phenotype if diagnostic criteria for possible autoimmune encephalitis are fulfilled and antibodies of high or intermediate risk are detected , sometimes accompanied by abnormal movements or cerebellar dysfunction. Brainstem encephalitis may co-occur with LE and is strongly associated with Ma2 antibodies, usually with underlying testicular tumors or non-SCLC (NSCLC).52 Morvan syndrome is almost always associated with CASPR2 antibodies, sometimes with concurrent LGI1 and netrin 1 receptor antibodies.52Morvan syndrome is defined by peripheral nerve hyperexcitability along with encephalopathy characterized by behavioral change, hallucinations, dysautonomia, and sleep disorders, especially agrypnia excitata. Importantly, the co-occurrence of LE and neuromyotonia should not be considered as a synonym of Morvan syndrome. Malignant thymoma is the tumor more commonly associated with Morvan syndrome, frequently accompanied by myasthenia gravis.53 However, patients may not have neuronal antibodies, and in these cases, the possibility of a paraneoplastic origin should be considered when MRI is suggestive, and there are no alternative diagnoses.Isolated myelopathy as a paraneoplastic manifestation of cancer may have a variable clinical evolution and usually presents with longitudinally extensive, symmetric, tract or gray-matter specific abnormalities in MRI studies. It is mainly associated with breast and lung carcinomas, and with CV2/CRMP5 and amphiphysin antibodies.54 Although some patients with anti–GAD65-associated SPS may have cancer, a paraneoplastic etiology should not be considered unless GAD65 is found expressed by the tumor cells. Besides focal variants of SPS (such as stiff-leg syndrome) that show the same antibody and tumor associations than classic SPS, another disorder lying within the SPS spectrum is progressive encephalomyelitis with rigidity and myoclonus, which usually presents with hyperekplexia, brainstem dysfunction, and dysautonomia, and is related mostly to glycine receptor antibodies in a nonparaneoplastic context.56Stiff-person syndrome (SPS) is characterized by painful muscular spasms that can be spontaneous or triggered by activity or external sensory stimuli and occurs in association with stiffness due to coactivation of agonist and antagonist muscles. Paraneoplastic SPS is mostly associated with amphiphysin antibodies and breast cancer. Compared with the nonparaneoplastic SPS, usually associated with glutamic acid decarboxylase 65 (GAD65) antibodies, patients with amphiphysin-related paraneoplastic SPS are older and frequently have neck and upper limb involvement.30 amphiphysin,31 and PCA-2/microtubule-associated protein 1B,57 usually in the context of SCLC, or breast cancer also in association with amphiphysin antibodies. In patients with cancer, the development of neuropathies that fulfill the criteria of Guillain-Barré syndrome or chronic inflammatory demyelinating polyneuropathy should not be considered paraneoplastic unless a high-risk antibody is identified  are variable. The most frequent antibodies are CV2/CRMP5,entified .58 in clinical practice, this link is suggested by:The panel agreed that the demonstration of a causal, not coincidental, association between the underlying tumor and the neurologic phenotype is crucial for the definite diagnosis of PNS. Although some progress has been made in the characterization of this pathogenic link, such as the identification of specific mutations or amplifications in the genes encoding for onconeural antigens in tumors of patients with PNS,Clinical series indicate that distinct types of PNS preferentially associate with certain types of cancers, regardless of the presence or absence of antibodies, and type of antibody. For example, rapidly progressive cerebellar syndrome in postmenopausal women is frequently paraneoplastic, and the tumors more frequently involved are breast and ovarian cancer . Another example is OMS in children with neuroblastoma .Antibodies are important to guide the search for an underlying tumor. In the context of PNS, 3 groups of antibodies can be considered according to the frequency of cancer association regardless of their eventual pathogenic effect. The first group includes antibodies that occur very frequently (>70%) in patients with an underlying cancer . In the BR antibodies have SCLC,15 more than 50% of patients with AMPAR antibodies have thymoma, lung, or breast cancer,17 and most (>85%) patients with Hu antibodies have SCLC.8In clinical practice, the indicated antibody associations with cancer have important clinical implications. For a specific phenotype, for example, LE, the presence of one antibody vs another suggests the likelihood of having a tumor or not and directs the tumor search. For example, patients with LE and LGI1 antibodies rarely have a tumor, whereas at least 50% of patients with GABA40 Very rarely, Hu18 or GABABRe3 antibodies can be identified in children with epileptic encephalopathy, and these patients almost never have cancer.These antibody-tumor associations can show age and sex dependence; for example, NMDAR antibodies rarely associate with tumors in young children or young adult male patients.e4 Similar studies are needed when there is limited experience with the PNS or associated antibody .e5In some clinical research settings , the demonstration that the tumor expresses the cognate antigen is critical to pathogenically associate it with the PNS .e6 Recommendations according to the type of suspected tumor are shown in table e-2 (links.lww.com/NXI/A491).Cancer screening should be promptly undertaken when a PNS is suspected and should be guided by the type of phenotype or antibody . Patient21-; however, this is a general recommendation that should be adapted to every individual case according to risk factors, clinical evolution, and medical resources. The same applies to patients with high-risk phenotypes along with intermediate risk antibodies , and the utility of tissue immunohistochemistry is unclear (pending to be better defined) for SOX1 antibody. Recommendations for antibody testing are shown in Although PNS can be diagnosed without neuronal antibody testing , the demonstration of neuronal antibodies is of extraordinary help in the diagnosis of PNS, and these antibodies have become very important biomarkers of PNS. Gold standard detection methods include rodent brain tissue immunohistochemistry/immunofluorescence (IHC/IF) accompanied by confirmatory studies using immunoblot with recombinant proteins (for most antibodies directed to intracellular antigens) or cell-based assays .Sensitivity and specificity for serum or CSF analysis vary among different antibodies; it is therefore recommended to perform antibody testing in both samples. Laboratory studies using CBA with serum only have similar problems of false-positive and -negative results. For all suspected autoimmune or paraneoplastic encephalitis associated with antibodies against neuronal surface antigens, screening of CSF should be obligatory to avoid mistakes. Patients with neuronal surface antibodies detected in serum only (CSF negative) should be re-examined in a research laboratories or with confirmatory tissue IHC/IF before considering a definite diagnosis. On the other hand, some antibodies  are best detected in serum, with CSF showing lower sensitivity. Despite the indicated gold standard techniques mentioned above (brain IHC/IF and CBA), very few laboratories use both techniques.e21-e25 Unexpected antibody results based on the type of neurologic phenotype, tumor, or patient's age and sex should raise concern for false-positive results and be reassessed with additional studies, preferably in research laboratories. Similarly, reference laboratories should perform the antibody testing in patients with high suspicion for PNS but negative routine screening of antibodies in clinical or commercial laboratories.e21,e22Commercial kits that test multiple antibodies may be helpful. However, often, the kits detect antibodies of limited clinical value for diagnosis of PNS along with antibodies with well-known clinical and cancer associations that have been validated across different specialized centers. Although commercial line blot kits for antibody detection are useful for the initial screening of patients, the number of false-positive and -negative results is particularly high for line blots assessing Yo, Ma2, CV2/CRMP5, and SOX1 antibodies.links.lww.com/NXI/A491). Further studies involving larger series are needed to confirm the clinical and oncologic associations of these new antibodies and require input of research laboratories to forward the accuracy of patient diagnoses.Several antibodies, mostly related to rapidly progressive cerebellar syndrome, are not well characterized yet, because they have been described recently, in small series or isolated case reports, or there is limited experience across different research laboratories . After that, 3 levels of diagnostic certainty are proposed  based on a scoring system (PNS-Care Score) that considers the type of clinical phenotype, presence or absence of neuronal antibodies, and presence or absence of cancer  includes the presence of a high- or intermediate-risk phenotype (as previously described) along with a high- or intermediate-risk antibody, and the presence of cancer. The presence of cancer is mandatory to define definite PNS. If the cancer is unusual for the type of antibody found, the diagnosis of definite PNS requires the demonstration of antigen expression by the tumor.The panel proposed as exception the OMS associated with neuroblastoma or SCLC in which there is no specific antibody association. Therefore, although this syndrome provides a score of 7, it should be considered definite PNS when associated with these tumors. The panel also acknowledges that the present criteria do not identify as definite PNS neurologic syndromes associated with cancer and low-risk antibodies even if tumor cells express the neuronal antigen recognized by the antibody .Note that the diagnostic level of probable or possible PNS  may chane26 The adoption of ICI treatment in oncologic practice has led to increased survival and long-term remissions, even in patients with extensive metastatic cancer.e26 The major downside of ICIs is the possibility of developing immune-related adverse events (irAEs),e27 including severe (grade 3 or higher) neurologic syndromes (1%–3% of the cases). These include the worsening of preexisting and de novo development of autoimmune neurologic diseases. The panel recommends that the first step in approaching these disorders is to determine whether the syndrome fulfills the above-mentioned criteria for PNS, after having excluded other alternative etiologies .e29 Both peripheral and CNS complications have been described.e30,e31 There is already evidence that specific neurologic syndromes  can be triggered by cancer immunotherapy. Nevertheless, a substantial proportion of cases remain seronegative despite comprehensive screening, and the detection of antibodies is not required for the diagnosis of irAEs. Although classical PNSs are known to precede the discovery of cancer, neurologic syndromes triggered by ICIs by definition develop when the cancer is already diagnosed, in general shortly after the initiation of ICIs.ICIs enhance antitumor immunity by blocking immune checkpoint molecules expressed in T lymphocytes and tumor cells including programmed cell death protein 1, its ligand, and cytotoxic T-lymphocyte–associated antigen 4. similarly to what it was observed in 3 cases of ICI-triggered myasthenia gravis.e36-e38Of interest, for the few patients who developed antibody-associated irAEs and samples taken before ICI introduction were available, the retrospective analysis revealed the presence of Ma2 or Hu antibodies before the onset of PNS in 4 cases,e39; (3) future studies should address the potential value of assessing neuronal antibodies before starting ICIs, particularly in patients harboring cancers with tendency to associate with PNS ; (4) close neurologic follow-up of antibody-positive cases is recommended.The optimal management of ICI-induced neurologic autoimmunity has not been established and is beyond the scope of this diagnostic guidelines, but the panel recommended the following considerations: (1) neuronal antibody testing needs to be routinely performed in all patients developing neurologic irAEs resembling high or intermediate risk PNS; (2) patients with current or previous PNS are at a higher risk of developing neurologic worsening if treated with ICIs, and therefore, the risk/benefit ratio of ICI should be carefully weighted in this setting. For example, 50% of cases with preexistent PNS worsened during ICI treatment in a recent studyThe evaluation of suspected PNS and their management requires detailed (and evolving) knowledge, so as to permit timely and accurate diagnosis of these uncommon disorders. Unambiguous diagnostic criteria facilitate both timely diagnosis (which may affect the neurologic and oncologic outcome) and avoidance of overdiagnosis and unnecessary treatments. In addition, these criteria represent an important research tool for epidemiologic studies and to analyze the value of new antibodies for the diagnosis of PNS. For the reasons stated in the introduction, modification of the 2004 criteria was necessary to accommodate the new knowledge generated in the last 16 years. The update criteria presented here (1) include novel phenotypes and immune-mediated pathogenic mechanisms identified since 2004; (2) emphasize a causal  association with cancer; and (3) require the demonstration of neuronal antibodies using gold standard techniques. These 3 elements represent the core of the present criteria of PNS that we hope will be of help to clinicians and researchers.


Giardia duodenalis, intestinal permeability is increased in part due to disruption of the intestinal mucus barrier. Intestinal mucus is composed primarily of the heavily glycosylated MUC2 mucin. Glycosylation of mucins is important for maintaining the structure of the mucus gel, and glycans are key mediators of host-microbe interactions, as they provide binding sites to microbes and can be degraded as a nutrient source. Changes to mucin glycosylation patterns have been noted during intestinal inflammation and bacterial infection, and may contribute to altered intestinal permeability and dysbiosis. We hypothesized that intestinal mucosal glycosylation patterns may be disrupted during Giardia infection, and that this may contribute to Giardia-induced barrier dysfunction.Disruption of intestinal barrier function is important in the pathogenesis of numerous intestinal diseases and can lead to increased intestinal permeability. During infection with the protozoan parasite in vivo Giardia infection.Characterize changes to mucosal glycosylation patterns in the small and large intestines during Giardia duodenalis strain GS/M for 7 days. Tissue sections from the jejunum and colon were collected and stained with various fluorescein-coupled lectins  and fluorescence was quantified and normalized to tissue area. Quantitative PCR (qPCR) was performed for glycosyltransferase genes in the jejunum and colon.3–4 week old C57BL/6 mice were infected with In the jejunum, abundance of N-acetylglucosamine increased upon infection, while sialic acid and fucose abundance were significantly decreased in infected mice compared to controls. Conversely, in the distal colon, mannose and sialic acid abundance increased significantly upon infection.Giardia infected mice. In the jejunums of infected mice, expression of the sulfotransferase Chst4 decreased, while the fucosyltransferase Fut2 and the sialyltransferase St6GalNAc1 increased in comparison to controls. In both the jejunum and distal colon, expression of the core 2 synthase C2GnT1 increased, while expression of the core 1 synthase C1GalT1 was similar between control and infected mice.Expression of mucin-associated glycosyltransferase genes was also altered in the small and large intestines of Giardia-infected mice. Disruptions to mucin glycans appear to be regiospecific, suggesting that unique mechanisms are involved in the regulation of mucosal glycosylation during parasitic infection throughout different regions of the gut. These findings uncover a novel mechanism in the pathogenesis of Giardia infection, and may be important in understanding intestinal barrier dysfunction in a variety of different gastrointestinal diseases.Glycosylation patterns and the expression of glycosyltransferase genes are altered in both the small and large intestines of CCC


Several studies reported that metformin, the most widely used drug for type 2 diabetes, might affect cancer aggressiveness. The biguanide seems to directly impair cancer energy asset, with the consequent phosphorylation of AMP-activated protein kinase (AMPK) inhibiting cell proliferation and tumor growth. This action is most often attributed to a well-documented blockage of oxidative phosphorylation (OXPHOS) caused by a direct interference of metformin on Complex I function. Nevertheless, several other pleiotropic actions seem to contribute to the anticancer potential of this biguanide. In particular, in vitro and in vivo experimental studies recently documented that metformin selectively inhibits the uptake of 2-[18F]-Fluoro-2-Deoxy-D-Glucose (FDG), via an impaired catalytic function of the enzyme hexose-6P-dehydrogenase (H6PD). H6PD triggers a still largely uncharacterized pentose-phosphate pathway (PPP) within the endoplasmic reticulum (ER) that has been found to play a pivotal role in feeding the NADPH reductive power for both cellular proliferation and antioxidant responses. Regardless of its exploitability in the clinical setting, this metformin action might configure the ER metabolism as a potential target for innovative therapeutic strategies in patients with solid cancers and potentially modifies the current interpretative model of FDG uptake, attributing PET/CT capability to predict cancer aggressiveness to the activation of H6PD catalytic function. Since the 1950s, the biguanide metformin has been the most widely used antihyperglycemic drug to treat patients with type 2 diabetes. This drug exerts its effects by reducing hepatic gluconeogenesis . BesidesAs an obvious consequence, these expectations increased the interest in metformin up to contaminate the field of basic research activity, as documented by the PubMed database reported in Metformin anticancer potential has been attributed to the interference with key pathways, described by several excellent reviews ,6,7, andNevertheless, this wide literature did not solve yet the debate about the real potential of metformin in cancer therapy, since concerns exist about its effectiveness in the different cancer types preventing any possible definition about its clinical indications in the clinical practice.Despite this uncertainty, the evident metformin effect in several experimental models of solid tumors indicates that the approach to energy metabolism might represent a new, potentially effective approach to neoplastic patients ,9. This At the molecular level, the most recognized target of metformin action is the Complex I of the respiratory chain ,11. IndeThe inhibition of OXPHOS inevitably results in a reduced production rate of ATP and thus in an evident impairment of cell energy asset, the consequent decrease in the ATP/AMP ratio eventually activating energy sensor mechanisms shared by virtually all eukaryotic cells and able to enhance catabolic processes while switching off the anabolic ones in order to restore the cell. Among these pathways, a relevant role is played by the activation of the tumor suppressor gene liver kinase B1 (LKB1)  that eveAs a further consequence of OXPHOS inhibition, metformin has been found to induce an increase in the lactate/pyruvate ratio, suggesting an impaired cell capability to re-oxidize the cytoplasmic NADH, possibly caused by a direct inhibition of the mitochondrial glycerophosphate dehydrogenase .Besides these effects and the consequent deceleration in gluconeogenesis, the impaired energy asset implies a switch-off of G6P-phosphatase expression through mechanisms at least partially independent of AMPK activation and most likely related to the inhibitory effect by the direct AMP-dependent inhibition of adenylate cyclase . AccordiIn cancer cells, the OXPHOS inhibition activates the LKB1–AMPK pathway, thus inhibiting the Raptor–mTOR complex ) and the tuberous sclerosis complex 2 (TSC2) . The effAccording to these considerations, the energetic stress directly caused by metformin might provide a favorable therapeutic index in a sizable number of cancers, mostly in those characterized by an overexpression of mTOR. The faster progression and poorer prognosis of these lesions might indeed be most sensitive to the downregulation of mTOR transcription factors and the reduction of insulin-like growth factors caused by this treatment .Finally, the response of whole-body metabolic pattern to metformin intake indirectly modifies the systemic signaling to cancer lesion. Indeed, the antihyperglycemic drug action eventually results in decreased plasma concentrations of growth-supporting hormones insulin-like growth factor 1 (IGF1) and insulin .Under several aspects, the severe consequences of metformin-induced OXPHOS inhibition on cancer cell biology might appear somewhat paradoxical. Indeed, since the seminal studies by Otto Warburg, cancer metabolism is known to display a high glycolytic flux and lactate release even under exposure to high oxygen tension (aerobic glycolysis) ,31. LikeIn silico studies indicate that metformin interacts with HK2 pocket for glucose, selectively inhibiting the catalytic function of this enzyme by preventing the connection between the glucose itself and ATP already at concentrations around 100 µM. This inhibition is paralleled by a detachment of HK2 from the mitochondrion. In the same way as G6P, the inhibitory effect of metformin on HK2 is inversely related to the ATP concentration. Thus, ATP depletion caused by the respiratory blockade coupled with the enzyme detachment from the mitochondrial outer membrane eventually results in a marked inhibition of HK2 activity further decreasing the availability of G6P .Altogether, thus, these data seem to suggest that the slow-down in cancer growth cause by metformin might represent the consequence of respiratory inhibition combined with a decreased fueling of G6P for both glycolysis and PPP.The wealth of studies and observations reported above indicate a powerful anticancer potential for metformin largely independent of its antihyperglycemic action. A similar consideration also applies to experimental models of tumor-bearing mice, in which metformin has been found to slow down cancer progression and to improve the effectiveness of both radiotherapy  and chemA major problem in this setting relies on the dosage schedule adopted in basic research studies. In vitro, metformin metabolic effect has been reported for very high drug concentrations ranging from 1 to 10 mM ,39,40,4111C]-labeled metformin has been found to be highly concentrated in gut, liver, kidneys and bladder -2-desossiglucose, 2-NBDG) co-localized with the ER membrane, identified by the fluorescent probe glibenclamide. This distribution pattern was markedly and comparably altered by both high metformin doses and H6PD silencing ,40,41.Altogether, these observations would configure H6PD function as a primary determinant of the metabolic adaptation to cell phenotype and differentiation. This concept apparently disagrees with the very low expression of this enzyme, whose level was 300 times lower than its cytosolic counterpart G6PD, in our preliminary proteomic analysis. Nevertheless, Cossu et al. used nuclear magnetic resonance spectroscopy to verify the metabolic response to the inhibition of H6PD or G6PD expression with siRNA in human cancer cell lines . SilenciBased on these considerations, we hypothesized that FDG uptake might reflect the degree of activation of H6PD-triggered ER-PPP also in normal tissues. Actually, FDG uptake in normal mouse brain was decreased by the prolonged treatment with high doses of metformin . This efClassical models of cell biochemistry define PPP as the main pathway able to preserve the NADPH levels ,60. The In the clinical setting, the relevance of these observations mostly applies to the high number of patients with type 2 diabetes submitted to PET/CT imaging. Indeed, the competition between FDG and unlabeled glucose for cell entry possibly leads to an underestimation of lesion glucose consumption when scanning is performed in the presence of hyperglycemia. This concept has been accepted by current procedural guidelines that discourage FDG imaging in patients with fasting glucose levels up to 200 mg/dL . To avoiDespite the wide use of the biguanide in this setting, this potential pitfall and its interference with the diagnostic accuracy of PET/CT imaging has been scarcely addressed in the literature. Actually, we observed a slight reduction of the avidity for FDG in cancers grown in chronically and mildly hyperglycemic mice, previously treated with streptozotocin. Nevertheless, this response was paralleled by a loss of sensitivity to metformin, which involved both FDG accumulation rate and lesion growth . On one As a final consideration, however, a large uncertainty still exists about the potential interference of metformin treatment in PET diagnosis of colorectal cancer. Indeed, in this setting, the drug-induced inhibition of FDG uptake combines with a marked increase in tracer retention of normal colonic enterocytes, both in patients and in experimental models ,65. As aAlthough not conclusively duplicated in the clinical setting, the capability of metformin to inhibit proliferating activity and migratory potential of cancer cells has been documented by a wealth of studies both in vitro and in experimental animals. In agreement with the mechanism underlying the drug antihyperglycemic action, this pharmacological effect involves the biguanide capability to bind the respiratory Complex I and thus to inhibit the OXPHOS. The resulting impairment in cell energy asset activates AMPK phosphorylation, switching off the reductive syntheses needed for cancer growth. Nevertheless, besides this well-documented action, metformin anticancer potential extends to involve its capability to slow down the rate of a specific PPP located within the ER, whose activity has been found to be represented in many solid cancers.The reactions sequence, the controlling mechanisms, the exchange with cytosol metabolites and the ultimate role of this reticular pathway are still largely undefined. Yet, its role in feeding the NADPH reductive power for the synthesis of both nucleic acids and cell membranes has been found to be unexpectedly relevant and might thus represent a new, largely unexplored, target in anticancer therapy. Thus, the investigation on metformin effect so far provided a new therapeutic approach to oncological patients. The missing adherence of clinical data with experimental findings might probably reflect several explanations and mostly the large difference in the used dosages. Nevertheless, obtained data indicate the need for studying the therapeutic potential of other compounds targeting the ER metabolism.


Basal cell carcinoma (BCC) is a highly prevalent epidermal neoplasm that most commonly occurs in regions of sun-exposed skin, though rare cases arise in sun-protected areas. BCCs of the vulva account for a small fraction of cases and can be mistaken for other cutaneous genital pathologies on clinical examination. Here we report a case of vulvar BCC that presented as a firm, tender bilateral lesion of the mons pubis and was diagnosed by histopathology and immunostaining for classical BCC markers. Basal cell carcinoma (BCC), a malignancy of epidermal keratinocytes, is the most prevalent human cancer, with more than three million cases reported in the U.S. every year . It is eA 51-year-old (G3P2012) female patient presented to the clinic with vaginal itching, irritation, and a tearing sensation near the top of the vaginal area that had been present for one year. She reported that her physical discomfort had waxed and waned over that time but had worsened over the last several months. She denied excessive vaginal odor, general pelvic pain, fever, or chills and had no concerns for sexually transmitted infection. She stated that her discomfort was worsened by physical activity and moisture upon daily swimming. Physical examination revealed a 1.5 cm firm bilateral vulvar lesion with shallow red ulcerations at the lower mons pubis.An initial 3 mm Keyes punch  biopsy of the lesion was performed and sent for both virologic and pathologic testing. Herpes simplex virus 1 and 2 (HSV1/2) testing by polymerase chain reaction (PCR) was negative. Microscopically, hematoxylin and eosin (H&E) staining showed both papillary and reticular dermal nests composed of hyperchromatic keratinocytes with peripheral cell palisading  for epithelial cell adhesion molecule (EPCAM/BER-EP4) and diffuse cytoplasmic staining for B-cell lymphoma 2 (BCL2)  signal in these cells  mutant hair follicle stem cells can give rise to BCC in the absence of ultraviolet (UV) irradiation ,7. This In addition to their poorly defined etiology, vulvar BCCs are challenging to diagnose clinically, owing both to non-specific clinical symptoms such as pain, pruritus and inflammation as seen in our patient, and the variable physical appearance of the lesion itself . It is nEfforts to improve initial diagnosis have included database mining to generate a typical clinical presentation and the utilization of advanced imaging techniques during an assessment. A recent query of the surveillance, epidemiology, and end results (SEER) database found that among instances of vulvar BCC where the primary site was specified (n=347), the labia majora accounted for nearly 90% of the cases . This prUpon biopsy, vulvar BCC is histopathologically similar to BCC on sun-exposed skin. Superficial and nodular subtypes, which account for the majority of vulvar BCC cases  and wereThe therapeutic strategy for vulvar basal cell carcinoma most commonly includes excisional biopsy, as was the case for the patient described above, or wide local excision ,4. The uOur patient presented to the clinic complaining of vaginal pruritus and pain stemming from a 1.5 cm bilateral, firm, ulcerated lesion of the lower mons pubis. Prompt Keyes punch biopsy and immunohistopathology were performed to make a diagnosis of vulvar BCC. We describe the use of an IHC panel for confirmation of BCC diagnosis  that has been reported in the literature to improve correct differentiation between vulvar BCC and HPV-associated SCC of the vulva. We also draw attention to the benefit of dermoscopic and RCM imaging of vulvar BCC and ask that readers consider incorporating these techniques in workups of cutaneous vulvar lesions as they may prevent delays in biopsy and excision.


HPRT1) and the β-amyloid precursor protein (APP) genes affecting the regulation of alternative APP pre-mRNA splicing in LND has been demonstrated. Furthermore, there were also some reported cases of LND developing thrombosis while APP is an important regulator of vein thrombosis and controls coagulation. Otherwise, the surface expression of HGprt enzyme was also observed in several somatic tissue cancers while APP and the APP-like protein-2 (APLP2) are deregulated in cancer cells and linked to increased tumor cell proliferation, migration, and invasion. The present review provides a discussion about these findings and suggests a potential molecular link between APP and HGprt via epistasis between HPRT1 and APP genes affecting the regulation of alternative APP pre-mRNA splicing. As a perspective, expression vectors for HGprt enzyme and APP are constructed as described in Ref. # 24  Lesch-Nyhan disease: I. Construction of expression vectors for hypoxanthine-guanine phosphoribosyltransferase (HGprt) enzyme and amyloid precursor protein (APP). Nucleosides Nucleotides Nucleic Acids 39: 905–922), and they could be used as tools for clarification of these issues. In addition, these expression vectors, especially the one with the glycosyl-phosphatidylinositol (GPI) anchor can be used as a model for the construction of expression vectors for any protein targeting to the cell plasma membrane for studying intermolecular interactions and could be therefore useful in the vaccines as well as antiviral drugs development  Lesch-Nyhan disease (LND) is a rare X-linked inherited neurogenetic disorders of purine metabolic in which the cytoplasmic enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. Despite having been characterized over 60 years ago, however, up to now, there is no satisfactory explanation of how deficits in enzyme HGprt can lead to LND with the development of the persistent and severe self-injurious behavior. Recently, a role for epistasis between the mutated hypoxanthine phosphoribosyltransferase 1 ( This APP207 isoform would be responsible for the neurobehavioral syndrome observed in these patients HPRT1 and APP genes affecting the regulation of alternative APP pre-mRNA splicing. As a perspective, expression vectors for HGprt enzyme and APP are constructed Lesch-Nyhan disease (LND) is a rare X-linked inherited neurogenetic disorder of purine metabolism affecting 1 in 380,000 people, and caused by deficiency of the soluble cytoplasmic hypoxanthine-guanine phosphoribosyltransferase (HGprt) enzyme . This enzyme plays a central role in the generation of purine nucleotides from degraded DNA through the purine salvage pathways 2.2.1.Immamura et al. 2.2.Townsend et al. 3.A major unsolved question is how the loss of HGprt enzyme function affects the brain to cause the neurobehavioral syndrome in LND/LNVs. Histopathological studies of autopsy tissues from LND patients revealed no signs suggestive of a degenerative process in any brain region - adhesion of HGprt-deficient neuroblastomas as well fibroblasts from patients with LND/LNVs exhibited dramatically enhanced adhesion compared to control - Alzheimer's disease (AD) shares gene expression aberrations with purinergic dysregulation of HGprt deficiency - role for the APP is a key developmental gene related to cell-cell or cell-substrate adhesion, generation of neurons, their differentiation and migration, neurite outgrowth, regulation of synaptic function, and is important for brain morphology and highly coordinated brain function such as memory and learning has been suggested 207 isoform was found in most of the LND patients In an attempt to search for a link between LND/LNVs and APP, examination of the APP-mRNA profile, the genomic APP-DNA from the fibroblast of normal subjects and LND/LNVs patients was then performed. The results obtained showed, for the first time, that different isoforms of APP-mRNA can exist 3.1.HPRT1 and APP genes. Indeed, (1) the KPI domain, which is located in exon 7: amino acid residues 291–341 of the extracellular domain of APP770 and APP751 isoforms in vitro to be a potent inhibitor of the coagulation FXa, FIXa, FXIa, and FVIIa: tissue factor complex 751 and APP770 isoforms are expressed 2+/calmodulin-dependent mechanism in vitro to be potent inhibitors of trypsin, chymotrypsin, epidermal growth factor binding protein, the γ subunit of nerve growth factor, and several key prothrombotic proteinases including factor XIa, factor IXa, factor Xa, and tissue factor: factor VIIa complex In vivo studies on APP/KPIR13I mutant mice  in vitro and in vivo to have inhibitory activity against these hemostatic enzymes factors and regulates thrombosis The findings as described in R13I207 isoform as described in Taking into consideration these findings, a miss regulation of alternative APP pre-mRNA splicing could lead to the presence of the most abundant APP-mRNA isoform that would be a defective one encoding consequently a defective APP protein isoform (or its proteolytic fragments) with mutation and/or deletion in the KPI domain such as the APP/KPI3.2.HPRT1 is cytosolic within all normal cells and maintained stable and low levels in normal tissue. Here, some questions remain to be elucidated such as (a) How HGprt is able to localize to the surface?; (b) Does it provide any functional advantage to the cancer cell?; (c) Determine the reason some cells that express HGprt on the surface while others do not? The observed surface expression of HGprt on certain malignancies makes it promising as a biomarker in the early diagnosis of cancer such as lung and colorectal cancer APP gene is under epigenetic regulation and a role for epistasis between HPRT1 and APP genes affecting the regulation of alternative APP pre-mRNA splicing was also suggested Salvage enzyme, such as HGprt and is known as a housekeeping protein, in which its important role is responsible for the production of nucleotides such as GTP and ATP that are necessary to providing energy for several cellular process as well as for regulating cell proliferation 4.http://www.genenames.org/genefamilies/ENDOLIG) Epistasis is important, ubiquitous, and has become a hot topic in complex disease genetics such as AD, schizophrenia, autism, cancer, etc. in recent years. A gene does not function in isolation and by itself, but rather acts with other genes in a network, to influence complex traits of the complex disorders. However, the data supporting epistasis in complex human diseases are emerging slowly. This is due to different difficulties that we face in detecting and characterizing epistasis, such as challenges of modeling non-linear interactions, and in the interpretation of results HPRT1 and APP genes, and highlight the impact of alternative splicing (AS) process on human disease, and clearly show that how AS is dynamically regulated and generates isoform diversity with critical functions and a misregulation of AS plays a large role in numerous human diseases. An accurate quantification of various APP-mRNA isoforms from different tissues for identification the most abundant APP-mRNA isoform that may be decisive for the normal status or disease risk is needed and antisense drugs are the potential treatments In conclusion, the examples discussed here suggest strongly a potential molecular link between APP and HGprt via epistasis between HPRT1 gene in cancer, especially exploring the effects for the surface expression of HPRT1 gene; (c) exploring the mechanism linking HGprt deficiency, purinergic pathways, and neural dysfunction of LND; (d) exploring the structure and the physiologic function of APP; (e) studying intermolecular interactions between APP and HGprt enzyme.As a perspective, for clarification of these issues, it is necessary to study the HGprt enzyme and APP using expression vectors for exploring their impacts on LND as well as other human diseases, especially the ones related to APP such as AD and cancer It is also important to note herein that these expression vectors, especially the one with GPI anchor can be used as a model for the construction of expression vectors for any protein targeting to the cell plasma membrane for studying intermolecular interactions and could be therefore useful in the vaccines as well as antiviral drugs development


Viral RNAs contain the information needed to synthesize their own proteins, to replicate, and to spread to susceptible cells. However, due to their reduced coding capacity RNA viruses rely on host cells to complete their multiplication cycle. This is largely achieved by the concerted action of regulatory structural elements on viral RNAs and a subset of host proteins, whose dedicated function across all stages of the infection steps is critical to complete the viral cycle. Importantly, not only the RNA sequence but also the RNA architecture imposed by the presence of specific structural domains mediates the interaction with host RNA-binding proteins (RBPs), ultimately affecting virus multiplication and spreading. In marked difference with other biological systems, the genome of positive strand RNA viruses is also the mRNA. Here we focus on distinct types of positive strand RNA viruses that differ in the regulatory elements used to promote translation of the viral RNA, as well as in the mechanisms used to evade the series of events connected to antiviral response, including translation shutoff induced in infected cells, assembly of stress granules, and trafficking stress. RNA viruses are a major threat to human health . Their r7GTP) residue (designated cap), while the long 3′UTR folds into a complex secondary structure that includes several stem-loops critical for viral RNA replication. However, other members of the Flaviviridae family differ in the type of regulatory elements controlling their translation and replication, as exemplified by hepatitis C virus (HCV), classical swine fever virus (CSFV), and bovine viral diarrhea virus (BVDV). The genomes of these viruses harbor a functional internal ribosome entry site (IRES) element on their 5′UTR, responsible for the cap-independent translation initiation of the viral RNA . T. T123].  the RNA ,134. Althylation ,136. Thi to m6A) . Adenine(ALKHB5) . The YT5nd eIF3B . 6A modification sites of the viral genome resulted in decreased virus replication, suggesting that m6A modification might enhance EV71 life cycle [Methylation of adenosine on viral mRNAs has multiple effects on virus replication ,141. Forfe cycle . In this6A has a wide range of effects on mRNA translation, it is reasonable to assume that mechanisms influencing viral protein synthesis could be affected by m6A methylation. A recent study reported that HCV IRES-dependent translation is stimulated by m6A methylation via YTHDC2 [6A methylation favors cap-independent translation of certain cellular mRNAs [6A modification suppresses HCV viral particle production through the action of YTHDF proteins [6A in ZIKV and CHIKV RNA decreases virus replication [6A modification could regulate RNA functions in various manners. However, there are open questions for unclear results in RNA viruses that need to be elucidated in future studies. Given that ma YTHDC2 , consistar mRNAs ,145. Howproteins , whereaslication ,147. TheTo accomplish the replication cycle, viruses have developed diverse strategies taking advantage of RNA regulatory elements that recruit RBPs and other host factors in all stages of the infection cycle. Most significantly, the regulatory elements of viral RNAs contain structural features relevant for their function, such that conserved structural elements associate RBPs connected to its relevance for the viral replication cycle. Highlighting the plasticity of RNA-protein interactions to achieve a large variety of specific functions, there are many ways to complete the replication cycle. Notably, the translation field has accumulated insights into the regulatory mechanisms occurring in both the host cell and the RNA virus translation during the entire viral replication cycle, and consequently, the antiviral state. Host factors are essential at all stages of the virus life cycle. Thus, besides performing critical roles in viral RNA translation and replication, host proteins participate in early infection steps, such as internalization and uncoating ,148 and 6A methylation in viral replication. The different combinations of RNA-protein complexes executing diverse mechanisms of action suggests that there is much to learn about virus–host interaction interface, highlighting the need to explore the multifaceted role of RNA-protein and protein-protein interactome in infected cells. Ultimately, dissecting the relationship between viral RNAs and host RBPs is expected to expand our understanding of virology and RNA biology in general, and also, to guide the development of novel antiviral approaches. In this review we have revisited representative examples of positive strand viral RNA translation initiation, the strategies developed to overcome the inhibition of protein synthesis in infected cells, the role of RBPs in the assembly and evading stress granules, the involvement of ER trafficking host factors targeting regulatory elements of viral RNAs to ribosome-rich compartments, and the influence of viral mRNAs m


FOXP3 mutations that impair its expression or function and result in an early-onset and life-threatening autoimmune disease: immune dysregulation, polyendocrinopathy, and enteropathy, X-linked (IPEX) syndrome. Since its first recognition in the early 2000's, IPEX syndrome has been a prototype autoimmune monogenic disease, primarily affecting regulatory T-cell mediated immune regulation. In turn, IPEX has revealed key differences between human and mouse FOXP3, thus posing unique challenges for diagnosis and treatment. Importantly, for most patients, IPEX remains a life-threatening and incurable disease.Forkhead box protein 3 (FOXP3) is an essential transcription factor that regulates the functional maintenance of self-tolerance and prevention of autoimmune diseases conferred by CD4+ T regulatory cells (Tregs). The importance of FOXP3 protein in the homeostasis of the immune system is exemplified by FOXP3 mutations in patients who present with immune dysregulations as underlying chronic or recurrent gut disease, subtle kidney pathology, skin diseases, recurrent cytopenia or Type 1 diabetes. Therefore, while severe “typical” early-onset IPEX remains the paradigmatic presentation of FOXP3 deficiency, the atypical forms of the disease, comprising late-onset, mild-IPEX or unusual, sometime monosymptomatic disease, are increasingly being observed. This new clinical heterogeneity of IPEX is nicely reviewed in Consonni et al., who summarize reported late-onset and mild forms of IPEX, as well as cases without enteropathies and with unexpected presentations. The authors provide an in-depth update of the genotype phenotype correlation, although the mechanistic impact of FOXP3 mutations remains largely unexplored. The functional impact of certain mutations located in non-coding and coding regions of the FOXP3 gene can be predictive of similar phenotypic outcomes. In contrast, several other FOXP3 mutations, including those in the leucine-zipper or forkhead (FKH) DNA-binding domain, cause variable unpredictable phenotypes in different patients. Moreover, unusual manifestations such as nephropathy or hypogammaglobulinemia are repetitively related to specific FOXP3 mutations in different patients. Curiously, a number of patients have FOXP3 mutations, but no clinical manifestations thus requiring no treatment; this cohort confirms the need for more informative genotype-phenotype diagnostic tools that can accurately predict disease severity. Barzaghi and Passerini present a comprehensive summary of the immunological alterations that could provide a more informed IPEX diagnosis when FOXP3 mutations are detected. These analyses comprise several important tests, such as quantification of the status of the FOXP3-specific epigenetic marker (TSDR) or assessing Treg-cell functional suppression, that are currently performed on a research basis in specialized centers. Our current knowledge of the pathogenesis of IPEX exemplifies the broader role of FOXP3 in causing immune dysregulation, and the direct effect of dysfunctional Treg cells. FOXP3 mutation in CD4+ T cells can also impact expression of the various FOXP3 isoforms revealing both a fundamental difference between the human and mouse protein and an additional diagnostic challenge. Indeed, as reviewed by Mailer, human, but not murine, FOXP3 is expressed as three major alternatively spliced isoforms. Notably, alterations in the relative expression of each isoform have been observed in immunological abnormalities leading to autoimmunity or inflammation, thus adding a potential level of complexity to the pathogenic impact of FOXP3 mutations in IPEX. Although further studies are required to assess the consequences of different FOXP3 mutations in altering FOXP3 isoform expression, and indeed to fully determine the functions of the isoforms themselves, the importance of maintaining expression of each is clear, and should be taken into consideration in developing new gene therapy approaches for IPEX. Lastly, a fascinating examination of the epigenetic and posttranscriptional regulation of FOXP3 is discussed by Grover et al., with findings supporting the development of a new potential therapeutic intervention to treat IPEX.In this Research Topic experts in the field have shared their contributions, providing updates on our current understanding of IPEX pathogenesis, diagnosis and clinical manifestations of the disease. Although still rare, the increasing awareness of the disease has expanded IPEX diagnosis to older children through the identification of in utero, as uniquely demonstrated by Carneiro-Sampaio et al. These observations begin to clarify the primary role of immune regulation at the molecular and system level in the developing embryo, and stimulate new questions about the origin of autoimmunity and its interrelationship with fetal cell and tissue development.In summary, studies of FOXP3 in IPEX have shown that altered genetic composition, biochemical features, epigenetic, and posttranscriptional regulation can cause a wide spectrum of disease manifestations ranging from no obvious impairments to the life-threatening early-onset form of IPEX that is the hallmark of diseases resulting from ineffective immune regulation. Intriguingly, in its worst form, IPEX can manifest Despite much progress and some success treating with hematopoietic stem cell transplantation, IPEX remains an incurable disease. The updated concepts summarized in this Research Topic will hopefully stem new targeted precision medicine treatments and inspire new gene therapy approaches that could in “one shot” reverse this disease.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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.


Citrussinensis L. (Osbeck)] is due to the presence of anthocyaninpigments that largely contribute to determine the high organolepticqualities and the nutritional properties of the fruits. The contentof pigments in sweet orange depends primarily on genetic factors andon environmental conditions. In particular, it has been extensivelyshown that cold temperature induces an increase of anthocyanin contentthat is achieved by the induction of the related gene expression.The purpose of our work is to understand the mechanism underlyingthe color variegation occurring inside the blood oranges during thecold induction of anthocyanin biosynthesis, despite the fact thatthe entire fruit is genotypically programmed to produce pigments.Therefore, the amount of anthocyanin and the expression of both structuraland regulatory genes have been monitored in either high-pigmented(HP) or not/low pigmented (NP) segments of the same fruit during thestorage at 4 °C for a total experimental period of 25 days. Ourresults clearly indicate that the anthocyanin content is directlycorrelated with the levels of gene transcription, with higher pigmentedareas showing higher enhancement of gene expression. Furthermore,we analyzed the reshaping of the DNA methylation status at the promoterregions of genes related to anthocyanin biosynthetic pathway, suchas DFR and Ruby. Our results unequivocallydemonstrate that in the promoter regions of both DFR and Ruby, the amount of cytosine methylation stronglydecreases along the cold storage in the HP areas, whereas it increasesin the NP areas of the same fruit, probably causing a partial blockof the gene transcription. Finally, by measuring the changes in theexpression levels of the Citrus DNA demethylases,we found that DML1 might play a crucial role in determining the observeddemethylation of DFR and Ruby promoters,with its expression induced by cold in the HP areas of the fruits.This is the first report in which different levels of gene expressionimplicated in anthocyanin production in blood orange fruit is correlatedwith an epigenetic control mechanism such as promoter methylation.Theblood red color of pigmented orange fruit varieties [ Citrus sinensis) L. Osbeck], which include Tarocco, Moro, and Sanguinello are ableto synthesize anthocyanins which confer to the fruit the characteristicbloody color and make them easily distinguishable from the nonpigmented,blond orange varieties.1 Anthocyanins aresynthesized via the flavonoid pathway, which is a ubiquitous and well-describedplant secondary metabolism pathway.2 Mostgenes encoding the enzymes that biosynthesize the pigment moleculeshave been cloned and characterized in various species,3 the Citrus species included.7 Phenylalanine is a direct precursor for the synthesis of anthocyanidinsand its conversion to the pigment core structure requires a seriesof reactions. The first of them is represented by the transformationof phenylalanine to trans-cinnamic acid catalyzedby the phenylalanine ammonia lyase (PAL). In the following steps,4-hydroxylation of cinnamic acid by cinnamate 4-hydroxylase (C4H)generates p-coumaric acid that is converted by the4-coumarate: CoA ligase (4CL) to the respective CoA ester. The firstspecific enzyme of the anthocyanin biosynthetic pathway is chalconesynthase (CHS) which condenses three malonyl-CoA and one p-coumaroyl-CoA molecules to produce the naringenin chalcone. Thenaringenin chalcone is isomerized by chalcone isomerase (CHI) to theflavanone naringenin, which is subsequently converted to dihydrokaempferolby flavanone 3′-hydroxylase (F3H). The dihydroflavonols, dihydroquercetin,and dihydromyricetin, are synthesized from dihydrokaempferol by sequentialhydroxylations catalyzed by flavonoid 3′-hydroxylase (F3′H)and flavonoid 3′,5′-hydroxylase, respectively. Dihydroflavonol4-reductase (DFR) can reduce the dihydroflavonols to their correspondingleucoanthocyanidins. Anthocyanidin synthase (ANS) converts the colorlessleucoanthocyanidins into the colored anthocyanidins, that, once formed,are immediately modified by the UDP-glucose-flavonoid glucosyl transferase(UFGT) that catalyzes the addition of a glucose molecule in the 3-OHpositions of anthocyanidins increasing their hydrophilicity and stability.1 A similar mechanism to the detoxification processwas proposed for anthocyanin transfer into the vacuole, this localizationbeing necessary to prevent oxidation.9 According tothat, anthocyanins need to be conjugated to glutathione by a specificglutathione transferase (GST)6 that isoften indicated as the real last enzyme involved in this metabolicpathway. Anthocyanin biosynthesis is regulated mainly at the transcriptionallevel by the MBW complex, composed by proteins of the Myb, basic helix–loop–helix(bHLH) and WD-repeat families of transcription factors.11 Specifically, pigmentation ofblood oranges originates from a retrotransposon insertion that allowsthe expression of Ruby encoding the MYB-type transcriptionfactor of the MBW complex that activates anthocyanin production in C. sinensis.11 Accordingly, themajority of differences in anthocyanin levels in Citrus are related with the alteration of Ruby expressioncaused by point mutations, or by indels resultingfrom transposable elements.12 Recently,a group of mutants have been identified in which anthocyanin pigmentationdoes not occur despite the presence of wild-type Ruby alleles. This subset of anthocyaninless variants is characterizedby the absence of proanthocyanidins in the seeds, and by fruit juicealmost completely lacking in acidity,13 together defining the “acidless” phenotype. A bHLHgene named Noemi has been found to have a crucialrole in the control of flavonoid biosynthesis as it interacts with Ruby to determine both anthocyanin production in citrusleaves and bud and the regulation of fruit acidity.13 However, the role of Noemi in pigmentedsweet orange fruit has not yet been investigated either in normalor in stressful conditions. In the pigmented varieties, the anthocyanincontent may also vary in response of different environmental conditionsas such as light exposure, nutritional status, xenobiotic or hormonetreatments, and temperature.19 The cold induction of anthocyanin biosynthesis has received greaterinterest than other inducing environmental challenges because theproductivity of commercially important Citrus varietiesis seriously laid down by low temperature that induces metabolic changessuch as elevated electrolyte leakage, reduced photosynthetic capacity,and respiration rate.20 Most interestingly,it has been shown that the pigment content of freshly harvested fruitssignificantly increases throughout cold storage due to ongoing biosynthesisof anthocyanins.17 This enhancement is accomplished by both the up regulation of thegenes involved in the anthocyanin biosynthesis17 and the increase of encoded proteins,19 as well as by the induction of the regulatory Ruby gene transcription.11 Furthertranscriptome study showed that cold stress induces transcriptomeadjustments absolutely orientated toward the enhancement of the flavonoidbiosynthesis pathway in blood oranges, including those genes belongingto upstream metabolic pathways, such as the shikimate pathway thatyields phenylalanine.16 Interestingly,the red color that is achieved in fruit flesh during both ripeningand in response to cold is not uniformly distributed in the fruitflesh. On the contrary, pigment accumulation is concentrated in particularareas of the entire fruit in which it is then possible to observehighly pigmented and low/not pigmented segments, despite the factthat they are programmed to synthesize anthocyanins by their genotype.DNA methylation, in combination with histone modifications and non-histoneproteins, defines chromatin structure and accessibility. DNA methylationcontributes to regulate gene expression, transposon silencing, chromosomeinteractions, and trait inheritance. Many early studies of abioticstress showed stress-induced DNA methylation and/or demethylationpatterns either genome wide or at specific loci. In some cases, thesechanges in DNA methylation may be associated with transcriptionalregulation of genes involved in plant stress responses24 suggesting that DNA methylation is important in mediating plantinteraction with the environmental signals. In this work, we evaluatedthe effect of cold temperature storage upon anthocyanin accumulationand the related gene expression both in high-pigmented (HP) and low/nopigmented (NP) areas of the same fruit, separately. Similarly, MethylationSensitive Amplification Polymorphism (MSAP) analysis was used to determinethe methylation status of the DFR and Ruby promotersduring cold storage in both NP and HP areas to correlate the DNA methylationlevels with gene expression. In this way, we tried an easy way toset to zero the genetic differences (same fruit) in order to highlightthe variation in epigenetic marks that can be responsible of differentgene expression levels. Moreover, the expression of DNA demethylasesinvolved in the dynamics of methylation rearrangements was also measured.Finally, the role of the BHLH Noemi gene in the variegationof pigmentation during cold stress was considered and discussed.Anthocyanins are water-solublepigments belonging to the flavonoidcompound family concerned in numerous aspects of plant developmentand defense. Several varieties of sweet orange [(C. sinensis (L.) Osbeck]were harvested in January 2019 from approximately 15 year old treesgrown at the experimental agricultural field of University of Catanialocated in Primosole, in the territory of Catania . Freshlyharvested oranges were washed with distilled water, gently dried withpaper towels, and then left to dry at room temperature overnight.Subsequently, orange fruits were placed in a box (60 fruits) storedin a ventilated cold room at 4 °C and 90–95% relativehumidity (RH). Samplings of the cold stored fruits were carried outevery 5 days for a total storage period of 25 days. During each sampling,9 fruits were randomly collected and divided into three subgroupsof three fruits each. After fruit peeling, orange wedges have beencut. The highly pigmented areas of the wegdes were separated fromthe low/non- pigmented areas and those belonging to different fruitsof the same subgroup were pooled to constitute three independent meansamples, and indicated as high-pigmented (HP) and low/no pigmented(NP). The orange flesh was then immediately frozen with liquid nitrogenand stored at −80 °C until used (both RNA and DNA extractionand anthocyanin determination).Pigmentedoranges (Tarocco Tapi)  and the methylation percentagewas calculated as methylation% = 100 – (100 × 2CT–Δ).DNA extraction wasperformed upon samples belonging to the HP and NP areas of the samefruit average sample using the Invitrogen Plant DNAzol reagent kit.The quality and integrity of extracted DNA was evaluated by spectrophotometricanalysis at NanoDrop and trough gel electrophoresis, respectively. DNA digestion was carriedout using the methylation-sensitive endonuclease McrBC  which cleaves DNA containing methylcytosineand 5-hydroxymethylcytosine on one or both strands. Sites on the DNArecognized by McrBC consist of two half-sites of the form (G/A)mC.These half-sites can be separated by up to 3 kb, but the optimal separationis 55–103 base pairs.p < 0.05) followed by Tukey’s testfor multiple comparison procedures using the statistical softwarepackage Statistica v. 13.0 .Data were analyzed by one-wayANOVA  and low/no pigmented (NP) areas. The effect of cold storage uponthe anthocyanin content of both HP and NP segments of orange fruitsis reported in pal, chs, dfr, ans, and ufgt was investigated in the above-mentioned HP and NP segmentsof orange fruits exposed to cold using the real time RT-PCR. The resultsare illustrated in CT–ΔΔ  reachinga peak after 15 days of cold storage in the HP areas at which theexpression level was more than 4 times higher with respect to theNP areas. Similarly, the transcripts of chs, dfr, ans, and ufgt enhancedup at the second sampling reaching at the third sampling (after 15days of storage) expression levels ranging between 3 and 4 times higherthan the NP samples .32 Promoter DNA methylation directly repressesgene transcription by affecting the binding of transcription activatorsto DNA or promoting the binding of transcription repressors or, indirectly,by inducing repressive histone modifications.34 In Ruby and DFR promoters during cold stress is shown in HP and NPfruit segments by MSAP, as described in the Ruby, we choseDFR as it represents an important regulatory point in the anthocyaninbiosynthetic pathway, also controlling the flux into biosyntheticpathway branches leading to distinct anthocyanin profiles.5 The analysis of methylation status of Ruby promoter highlighted that the percentage of DNA methylationincreased in the NP segments from 15% to 35% during cold stress, whereasa dramatic reduction of the Ruby promoter methylationis observed in the HP areas during the experimental period  in the NP areas, whereas a decreaseof methylation status is registered in the HP segments reaching the1% value at the end of the experimental period , DEMETER (DME), DEMETER-LIKE 2 (DML2),and DML3.36 By searching for DNA demethylasegenes in the orange genome, four AtROS1 orthologshave been identified indicating that distinct demethylases are involvedin removing the DNA methylation mark in Citrus genus.29 They include orange1.1t01511 (CsDME), Cs6g15500 (CsDML1), Cs5g04950 (CsDML4), and Cs3g07800 (CsDML3). The expression ofthese four genes was monitored in both HP and NP areas of cold stressedsamples. As shown in 30 In conclusion, we assessed thatcold stress actives the anthocyanin production in blood oranges, whichare genotypically programmed to do so, not evenly, but determiningdifferently pigmented segments inside the same fruit (Ruby) and structural  gene expression.The analysis of the pigmentationof the blood orange varietieshas been subject of many researches and has clarified the pathwayleading to anthocyanin biosynthesis and its regulation. samples 2B–E. The samples 1 and it  samples 2. Figurel period 4A. Althol period 4B. Thereme fruit 1B. The cRuby and DFR promotersindicated that the level of cytosinemethylation sharply decreases in HP segments thus suggesting thatDNA demethylation might represent a reversible epigenetic mark thatis removed during cold stress in the HP areas. Finally, by the analysisof gene expression, it is likely that DML1 is the demethylase involvedin the cold induced DNA demethylation occurred in the HP areas. Finally,the role of Noemi in fruit flesh under cold stresshas been investigated indicating that it is not involved in cold inducedfruit pigmentation. As far as we know, this is the first report regardingthe role of the epigenetic control of fruit anthocyanin synthesisinduced by cold that might be correlated with its dappled pigmentation.Moreover, the analysis of methylationstatus of


Individuals with type 2 diabetes mellitus are at greater risk of developing cancer and of dying from it. Both diseases are age-related, contributing to the impact of population aging on the long-term sustainability of health care systems in European Union countries. The purpose of this narrative review was to describe, from epidemiological, pathophysiological and preventive perspectives, the links between type 2 diabetes mellitus and the most prevalent cancers in these patients. Multiple metabolic abnormalities that may occur in type 2 diabetes mellitus, particularly obesity, could explain the increased cancer risk. In addition, the effectiveness of drugs commonly used to treat type 2 diabetes mellitus  has been broadly evaluated in cancer prevention. Thus, a better understanding of the links between type 2 diabetes mellitus and cancer will help to identify the contributing factors and the pathophysiological pathways and to design personalized preventive strategies. The final goal is to facilitate healthy aging and the prevention of cancer and other diseases related with type 2 diabetes mellitus, which are among the main sources of disability and death in the European Union and worldwide. How long we live and what proportion of that life is spent in good health have important implications for individuals and societies. Currently, one-fifth of the 512.4 million inhabitants of the 28 European Union countries (EU-28) are at least 65 years old—an increase of 0.3 percentage points in one year and 2.6 percentage points over the past decade. Moreover, recent projections suggest an increase in this population to 29.1% by 2080, reflecting an additional 53.3 million older inhabitants in the EU-28 [The aging process is the main risk factor for the incidence and progression of type 2 diabetes mellitus and cancer, two of the most common causes of death and disability in the EU-28 and worldwide ,4. WhileThere are several biologically plausible explanations for the association between type 2 diabetes mellitus and cancer development. In cancers, malignant transformation typically involves a process of initiation followed by promotion and progression, stimulating cell growth and development. Patients with both diabetes and cancer share multiple common risk factors associated with initiation, including age, male or female gender depending on cancer type and location, obesity, limited physical activity and poor dietary and lifestyle habits . In addiThe objective of this narrative review was to consider the links between type 2 diabetes mellitus and cancer from three distinct perspectives: (1) Epidemiological, to describe the burden of cancer attributable to type 2 diabetes mellitus; (2) pathophysiological, to describe subjacent pathological mechanisms that increase the risk of cancer in individuals with type 2 diabetes mellitus; and (3) preventive, to describe potential preventive strategies aimed to prevent both type 2 diabetes mellitus and cancer.Diabetes constitutes a worldwide public health problem that affected 463 million people in 2019, considering all different subtypes of the disease; this number is projected to reach 578 million by 2030 and 700 million by 2045 . The lifSeveral cohort studies have assessed the magnitude of the association and the risk of cancer in different locations. In 2011, the Emerging Risk Factors Collaboration published data on 123,205 deaths reported in 97 prospective studies   and in SMetabolic abnormalities observed during the onset and progression of diabetes  may haveHyperglycemia is responsible for induction of oxidative stress and DNA damage, which may trigger the first phases of tumorigenesis ,26. In vEndogenous hyperinsulinemia, mainly observed in type 2 diabetes mellitus patients in response to reduced insulin sensitivity of peripheral tissues, may also contribute to the relationship between diabetes and cancer by activating insulin receptors, IGF-1 receptors and hybrid insulin/IGF-1 receptors, all of which can stimulate cancer cell proliferation and survival and promote metastasis, thus favoring cancer progression . InsulinChronic inflammation may establish a tumor-supporting micro-environment favoring the neoplastic process , charactOxidative stress is another player tightly linked with inflammation and cancer risk in diabetes. Patients with diabetes usually have increased levels of all biomarkers of oxidative stress, which can mediate damage to cell structures, including lipids, membranes, proteins and especially DNA . DNA mutThe scientific evidence points out excess weight as a common risk factor for both type 2 diabetes mellitus and cancer . ObesityMultiple factors potentially contribute to the progression of cancer in obesity and type 2 diabetes mellitus, including hyperinsulinemia and insulin-like growth factor I, hyperglycemia, dyslipidemia, adipokines and cytokines and the gut microbiome. These metabolic changes may contribute directly or indirectly to cancer progression . Thus, oInsulin resistance is also associated with lipid abnormalities, characterized by elevated very low-density lipoproteins (VLDL) and triglycerides, decreased high-density lipoprotein (HDL) cholesterol and a decline in the hepatic production of sex hormone binding globulin (SHBG), which may lead to an increase in the levels of free hormones (including estrogen and testosterone). Furthermore, excess adiposity may lead to increased local aromatization of androgens to estrogens, which may affect tumor growth ,36. ThisSeveral epidemiological studies have explored the relationship between the clinical stages of hyperinsulinemia and the risk of cancer before diabetes onset ,54,55. OFinally, a review studied the relationship between the generation of lactic acid and the subsequent acidification of the surrounding environment. The findings could explain the negative prognosis of cancer in cases of extracellular lactic acidity. In diabetes, acidity affects insulin capacity to join the receptor, increasing the peripheric resistance and exacerbating symptoms. As a result, cancer progression acquires high invasive capacity and metastasis together with an inhibition of immune surveillance. Thus, acidity could be a relevant therapeutic objective .With the introduction of metformin in the mid-1990s and the subsequent advent of thiazolidinediones, the treatment paradigm of type 2 diabetes mellitus changed. These drugs provided an opportunity to address and directly reverse, at least in part, the defects in insulin action seen in individuals with type 2 diabetes mellitus . BiguaniMetformin is the first-line type 2 diabetes mellitus treatment that works by reducing insulin resistance and fasting plasma insulin levels, leading to a reduction in blood glucose concentrations without causing overt hypoglycemia . SeveralThe insulin-dependent mechanism is based on metformin’s effect on lowering serum levels of insulin and IGF-1, thus reducing the stimulus for growth. Given the potential importance of the Warburg effect in cancer metabolism, a glucose-rich environment could provide favorable conditions for aerobic glycolysis . TreatmePlausible insulin-independent mechanisms also exist. First, activation of LKB1/AMPK signaling by metformin could inhibit aerobic glycolysis in cells containing functional LKB1/AMPK pathways. In cells lacking functional LKB1/AMPK pathways, metformin could also induce tumor cell death by reducing ATP levels, which makes susceptible cells unable to respond to energy stress . Thus, tA review of the most recent meta-analyses showed divergent results based on cancer locations. On the one hand, metformin treatment in patients with diabetes significantly reduced the risk of colorectal ,73,74,75New studies have shown the potential role of metformin in pathologies having common physiopathological pathways with cancer. For instance, AMPK pharmacological activation by metformin plays a key role in inhibiting inflammation and improving endothelial dysfunction . AdditioAdditionally, known as glitazones, thiazolidinediones are the most important intracellular agonists of peroxisome proliferator-activated receptors γ (PPARγ) and are used to reduce hyperglycemia in patients with type 2 diabetes . PPARγ iShafiei-Irannejad et al. reviewed several studies showing that thiazolidinediones can be used alone as adjuvant therapy or in combination with other chemotherapeutics to sensitize tumor cells to the cytotoxic effects of anticancer agents . NeverthThere are no conclusive results about the risk of cancer associated with sulfonylureas, a group of drugs that act by increasing insulin release from the beta cells in the pancreas . Althougp = 0.007 and Hazard ratio for cancer mortality = 0.60; 95% confidence interval 0.36–0.98, p = 0.04) [The antitumoral capacity of statins has been highlighted; however, the results are not conclusive . A Japan = 0.04) .The social and economic consequences of type 2 diabetes mellitus are felt by all countries in terms of decreasing productivity and increasing poverty . PreventThe increasing adoption of personalized eHealth solutions  encourages individual responsibility for health-related decisions and for appropriate self-care and self-management of health conditions . Some inIndividuals with type 2 diabetes mellitus are at greater risk of developing cancer and of dying from it. Multiple metabolic abnormalities that can occur in type 2 diabetes mellitus may explain the increased risk. Understanding more about the links between these two diseases, particularly the role of obesity, could help to identify the contributing factors. In addition, this knowledge will help to design personalized preventive strategies. The final goal is to facilitate healthy aging and the prevention of cancer and other diseases related with type 2 diabetes mellitus, which are among the main sources of disability and death in the EU and worldwide.


Non-shivering thermogenesis is an energy demanding process that primarily occurs in brown and beige adipose tissue. Beyond regulating body temperature, these thermogenic adipocytes regulate systemic glucose and lipid homeostasis. Historically, research on thermogenic adipocytes has focused on glycolytic metabolism due to the discovery of active brown adipose tissue in adult humans through glucose uptake imaging. The importance of lipids in non-shivering thermogenesis has more recently been appreciated. Uptake of circulating lipids into thermogenic adipocytes is necessary for body temperature regulation and whole-body lipid homeostasis. A wide array of circulating lipids contribute to thermogenic potential including free fatty acids, triglycerides, and acylcarnitines. This review will summarize the mechanisms and regulation of lipid uptake into brown adipose tissue including protein-mediated uptake, lipoprotein lipase activity, endocytosis, vesicle packaging, and lipid chaperones. We will also address existing gaps in knowledge for cold induced lipid uptake into thermogenic adipose tissue. Endotherms maintain their body temperature by producing heat through both shivering and non-shivering thermogenesis. The cells primarily involved in non-shivering thermogenesis include brown and beige adipocytes, both of which stimulate heat production through disruption of the mitochondrial proton gradient by uncoupling protein 1 (UCP1)    were cold intolerant. The reliance of BAT thermogenesis on circulating lipids was further demonstrated by genetic ablation of the first enzyme in intracellular TG lipolysis, adipose triglyceride lipase (ATGL) encoded by Pnpla2. Whole body or adipose tissue specific ATGL KO led to accumulation of TGs in BAT and an inability to maintain body temperature  1 and 2 , cold exposure led to reduced levels of TGs and cholesterol in the plasma accompanied by an influx into the BAT , which has been proposed to facilitate the desorption step. However, FABPpm has also been proposed to function as a buffering system, maintaining the concentration of unbound fatty acids across the plasma membrane (FA transport is divided into 3 steps: (1) adsorption, (2) translocation, and (3) desorption . First described in 1985, the putative fatty acid transporter FAPBPM was shown to have a high affinity for fatty acids  to expedite movement through membranes. There are currently 9 FABPs named for the tissue of discovery, but they are not exclusively expressed in the tissue for which they are named. The cytoplasmic FABP family differs in sequence, structure, and function from the plasma membrane associated FABP  is the best characterized in its family. FABP4 was first isolated from mouse embryonic fibroblast (3T3)-derived adipocytes, although it is highly expressed in macrophages as well  has been shown to be the dominant isoform in BAT upon cold activation. Expression of FABP3 was significantly increased in BAT after 4 h of cold despite a basal level of expression 6-fold lower than FABP4. Further, FABP3 KO mice were cold intolerant but displayed similar changes in thermogenic transcriptional programs to WT mice, such as induction of the coactivator PGC1α, which regulates mitochondrial biogenesis. Expected physiological changes were observed in FABP3 KO mice as well, such as depletion of TGs in BAT and increased FFAs in plasma , is an integral membrane protein belonging to the scavenger receptor superfamily. SRs bind a host of ligands including LDL, oxidized LDL, phospholipids, cholesterol esters, collagen, thrombospondin, carbohydrates, and microbial pathogens. There are 11 classes of scavenger receptors categorized based on primary sequence (A-L). CD36 belongs to the scavenger receptor B class (SR-B), the members of which contain two transmembrane domains and a single extracellular loop  receptor and transporter in rat adipocytes  recognition sites to aid in endocytosis. Lipoproteins fall into four main classes depending on their relative composition of proteins and lipids: high density (HDL), low-density (LDL), very low-density (VLDL), and chylomicrons. In addition to the characteristic structure of each class, different lipoproteins also serve different functions. For example, LDL carries circulatory cholesterol and easily enters arterial walls, while chylomicrons are essential for dietary lipid transport to the liver and peripheral tissues. Nonetheless, each class has a vital role in lipid transport, endocytosis, and hydrolysis . Following recognition, a clathrin-coated pit forms a vesicle around the endocytosed lipoprotein. The vesicle is directed to endosomes where a drop in pH causes release of LDL from LDLR. Acidic lipases in the newly formed lysosome hydrolyze the contents of LDL to release FAs from TGs and unesterified cholesterol from cholesteryl esters . CholestLdlr−/− mice displayed a reduction in plasma TG upon activation of BAT by cold or with a β3 adrenergic receptor agonist but did not show a reduction in plasma cholesterol normally observed after BAT activation  and LDL. Like chylomicrons, VLDL is utilized by tissues after lipolysis by LPL.TGs are the neutral storage form of FAs and travel between tissues through the bloodstream via chylomicrons and VLDL. Chylomicrons are the largest form of lipoprotein and are assembled by the small intestine following emulsification of dietary fats. TGs packaged into chylomicrons mainly enter adipose tissue and skeletal muscle due to high lipoprotein lipase (LPL) activity. The remaining TG is taken up by the liver and can be repackaged into VLDL for use throughout the body. Alternatively, FAs are synthesized by the liver through 20 LPL is ferried and anchored to the endothelium via glycophosphatidylinositol-anchored HDL-binding protein (GPIHBP1). Mice lacking GPIHBP1 have increased plasma TGs due to reduced tissue uptake  or treatment with heparin to release LPL from the vascular wall in mice prior to cold exposure almost completely abolished labeled TRL uptake into BAT.3H from labeled triolein in BAT, and increased expression of CD36 in BAT  and suppressed when substantial nutrients are available or needed, such as postprandially or during cold exposure , and fatty acid esters of hydroxy fatty acids (FAHFAs). Little is known about how theses lipids are transported across plasma membranes, what functional roles they serve in non-shivering thermogenesis, and in what complex structure they are mobilized in the circulation. While this remains an emerging field, it is rapidly progressing with new mass spectrometry-based technology such as ion mobility, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry, heavy isotope labeling, and several new chemical probes  is embedded on the outer-surface of the mitochondrial membrane and esterifies the fatty acid from an acyl-CoA to carnitine. This acylcarnitine can then diffuse into the porous outer mitochondrial member. Acylcarnitine is then brought into the inner mitochondria by carnitine acylcarnitine transferase (CACT) and de-carnitylated by CPT2. Besides their cellular role for fatty acid transport, acylcarnitines are also found in the blood plasma. Plasma acylcarnitines increase with chronic diseases such as type 2 diabetes, cardiovascular disease, and inborn errors of metabolism as well as in acute metabolic stresses such as fasting, exercise, and cold exposure  and cardiolipins  and the National Cancer Institute (NCI) under Award Number K12HD101368. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The work was also supported in part by startup funds from the University of Wisconsin-Madison School Department of Biochemistry to JS and NIH RO1 DK118093 to JN. Other funds that supported this publication include funds from the Diabetes Research Center at Washington University in St. Louis of the National Institutes of Health under award number P30DK020579.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.