Tumours are characterized by a state of chronic inflammation and are regarded as wounds that never heal. Mesenchymal stromal/stem cells (MSCs) are being considered as a possible treatment option. While MSCs can regulate the immune system, migrate to sites of inflammation, and are naturally immune-privileged, there have been contradictory reports on the role of these cells in the tumour microenvironment (TME). Some studies have suggested that MSCs promote tumourigenesis while others have suggested the contrary. To better evaluate the role of MSCs in the TME, it may be helpful to understand the role of MSCs in chronic wounds. Here, we discuss the role of MSCs in chronic wounds and extrapolate this to the TME. Chronic wounds are stuck in the inflammatory phase of wound healing, while in the case of the TME, both the inflammatory and proliferative phases are exploited. MSCs in chronic wounds promote a switch in macrophage phenotype from proinflammatory (M1) to anti-inflammatory (M2), thereby suppressing T, B, and natural killer cells, consequently promoting wound healing. In the case of the TME, MSCs are reported to promote tumorigenesis by suppressing T, B, and natural killer cells in addition to dendritic cells, cytotoxic T cells, and Th1-associated cytokines, thereby promoting tumour growth. Some studies have however suggested that MSCs inhibit tumourigenesis, depending on the source of the MSCs and the specific mediators involved. Therefore, the role of MSCs in the TME appears to be complex and may result in variable outcomes. Compelling evidence to suggest that MSCs are an effective treatment option against tumour progression is lacking. MSCs are positive for CD105, CD73, and CD90 and negative for CD45, CD34, and CD14 or 11b, CD79, or CD19, and HLA-DR [Sox2, Oct4, p53, and c-myc albeit at different levels [NRP, SYNP02, NPY, and CDH2, which may be related to synaptic transmission or neurogenesis, while UCB exclusively expresses osteoblast-specific factor and osteopontin [Mesenchymal stromal/stem cells (MSCs) are multipotent cells that can be differentiated into bone, muscle, cartilage, adipose, and other tissues of mesodermal origin , 2. MSCst levels . Perinatt levels . Some geeopontin . MSCs freopontin . In thisMSCs migrate to sites of injury where they interact both with the innate and adaptive immune systems leading to tissue repair and regeneration , 11. TheCancer has been described as a “wound that never heals” . SeveralAcute wound healing involves an acute inflammatory response comprised of four phases: haemostasis, inflammation, proliferation, and tissue remodelling , 26. ProMacrophages and keratinocytes secrete chemoattractant MCP-1, which recruits mast cells (MCs) to acute wound sites to release proinflammatory mediators including histamine, IL-6, IL-8, and VEGF . These pβ and secreting IL-10. The non-immune-mediated mechanism involves Tregs that secrete the epidermal growth factor-like growth factor amphiregulin. Amphiregulin stimulates cell differentiation in injured tissue leading to tissue homeostasis, restoration and wound healing [In acute wound healing, the transition from a pro- to an anti-inflammatory response depends on the presence of foxp3-expressing regulatory T cells (Treg) . Tregs c healing . A more Chronic wounds fail to heal when one or more of the wound-healing phases is disrupted  resultinCertain factors in chronic wounds such as iron overload may prolong the presence of M1 macrophages, which are responsible for inciting an inflammatory response, stalling resolution of the inflammatory phase, and impairing the function of M2 macrophages . StallinNK cells are rare or depleted at chronic wound sites both in the early and late stages , 34, sugLoots et al. found that chronic diabetic and venous ulcers had more B cells and a reduced CD4/CD8 ratio compared to acute wounds . Chronicα, IL-1β, and IL-6, as well as decreased growth factor activity [Chronic wound fluid contains increased levels of matrix metalloproteinases (MMPs) and proinflammatory cytokines such as TNFactivity , 41. Extactivity , 32.There is increasing evidence that acute and chronic musculoskeletal injuries including articular cartilage injury, osteoarthritis, and meniscus injury can be successfully treated with MSCs . We nextγ), interleukin (IL-β1), TNFα, IL-8, vascular endothelial growth factor-alpha (VEGF-A), insulin-like growth factor (IGF), erythropoietin (EPO), stromal cell-derived factor-1 (SDF-1), and keratinocyte growth factor (KGF) but lower amounts of TGF-β1 [α and IFN-γ, while simultaneously stimulating the production of anti-inflammatory cytokines IL-4 and IL-10, to restart the healing process [Numerous studies have investigated the efficacy of MSC therapy in chronic wounds including chronic ulcers and chronic bacterial infections , 55. MSCf TGF-β1 , 57. MSCf TGF-β1 . MSCs di process .β and PDGF), break down the extracellular matrix (ECM), and maintain the wound in an inflammatory state [in vitro and in vivo via soluble factors such as IL-6 produced by MSCs and not by cell-to-cell contact [Neutrophils in chronic wounds secrete proteases such as elastase and collagenase that destroy growth factors . PGE2 acts on T cells and blocks the secretion of TNF-α [Mice with chronic tissue injury treated with MSCs express an M2 phenotype, while untreated mice express an M1 phenotype . Zhang end TNF-α . When mind TNF-α . The MSCnd TNF-α . MSCs alresponse . MSCs haof TNF-α . MSCs prof TNF-α . High coof TNF-α . Similarof TNF-α .γ which induces MSC-derived suppressive factor indoleamine 2,3-dioxygenase (IDO), and in the presence of IL-2, NK proliferation is inhibited [β, PGE2, and human leukocyte antigen G5 (HLA-G5) [MSCs suppress NK cells in various ways including via inhibiting cytokine secretion, cytotoxicity, and proliferation. NK cells secrete IFN-nhibited . Other M(HLA-G5) . MSCs ca(HLA-G5) , 62. Con(HLA-G5) . OverallIn contrast to chronic wounds that are seemingly stuck in the proinflammatory phase of wound healing and are characterized mostly by an M1 macrophage-associated inflammatory response, the TME is characterised by being in an inflammatory phase of wound healing marked by the presence of an M2 macrophage-associated inflammatory response. In the TME, T cells and NK cells are key players, but MSCs have been shown to inhibit these cells in chronic wounds. To assess the potential role of MSCs in treating cancer, we need to understand how MSCs interact with the immune response in the TME.The TME is a complex system comprised of numerous cell types in addition to tumour cells, including endothelial cells, pericytes, myofibroblasts, fibroblasts, smooth muscle cells, neutrophils, MCs, T cells, B cells, dendritic cells (DC), macrophages, and NK cells . These cγ that is secreted by effector cells and that can induce macrophage cytotoxic activity [To better understand the effect of MSCs in the TME, this review will use carcinoma as an example to discuss the immune response to cancer. Carcinomas are the most frequent form of malignancy and account for about 90% of all human cancers , 67. Immactivity , 70.γ, TNFα, IL-2, and IL-12. These cytokines work together to activate CTLs, M1 macrophages, and NK cells, which increases the presentation of tumour antigens [β that inhibit T cell mediated cytotoxicity, cause T cell anergy, and enhance B cell function. Th2 cells further stimulate myeloid-derived suppressor cells (MDSCs) and tumour-associated macrophages (TAMs), which also secrete protumour cytokines  in the TME [CD4+ T helper cells can either inhibit or promote tumour cell survival. CD4+ T helper cells are either Th1 or Th2, with each secreting a specific set of cytokines , 70. DCsantigens –71. In r the TME . Elevate the TME . Establi the TME .γ. Bregs also encourage conversion of CD4+ into Treg and inhibit NK-mediated immunity, thus promoting tumour progression [Th2 suppress antitumour immunity and improve protumour humoral responses , 73. Pargression . In the gression . Neutropgression .In contrast, although specific lymphocytes are associated with either a pro- or anti-inflammatory response, some anti-inflammatory lymphocytes may inhibit cancer progression. For example, activated antitumour B cells differentiate into clones of plasma cells that produce antibodies specific to the antigens expressed on cancer cells. This leads to antibody-dependent cell-mediated cytotoxicity (ADCC) causing cell lysis via complement activation , 70. In γ production in tumour infiltrating T cells and NK cell activation [α, and different proteases [Tumours have other survival strategies including glucose metabolism and tumour acidity. Both pro- and anti-inflammatory cells compete for nutrients in the TME. Tumour cells consume large amounts of glucose which affects the metabolism of other cells in the TME , 76. Lactivation , 76. Cantivation . Tumour tivation . Tumour roteases . These froteases . MC-mediroteases .β which is a key mediator of angiogenesis in the TME [Apart from proinflammatory leukocytes that support tumour growth, elements of the coagulation pathway further exacerbate the already compromised TME. Tissue factor (TF), which initiates coagulation, is expressed by tumour cells and is increased in breast, glial, colon, and lung cancers. TF expression has been associated with increased vascular density, tumour grade, and a worse prognosis , 79. Pla the TME , 80. It  the TME , and und the TME . It is tMSCs migrate to tumour sites in breast, colon, ovarian, brain, hepatocellular, and lung carcinomas as well as melanoma , 81, 82.MSCs affect cytokine secretion, differentiation, the phenotype of mature and immature monocyte-derived DCs, as well as immunostimulatory activity such as the induction of T cell activation , 86. Addγ and TNF-α, while Th2 type cytokines were significantly increased [β1 which increases Tregs production [in vivo breast cancer study demonstrated that tumour-derived MSCs (T-MSCs) and TNF-α-activated MSCs increased neutrophil tumour infiltration. More neutrophils were also observed in the blood and primary tumour [α-activated MSCs mimicked T-MSCs by promoting tumour growth and breast cancer metastasis to the lung [Patel et al.  demonstrncreased . MSCs aloduction . An in vy tumour . Additiothe lung .There are other ways in which MSCs promote cancer progression. In a breast cancer study, the presence of MSCs in the TME acted as a stimulus to induce EMT . EMT proin vitro study showed that MSCs inhibit cell cycle progression in breast cancer cells and promote apoptosis by downregulating the Stat3 signalling pathway [in vitro study using the immortalized MSC line RCB2157 cocultured with MDA-MB-231 and T47D cells further demonstrated that MSCs are antimetastatic and antitumorigenic. In the first 24 hours, MSCs secreted MMPs that have a prometastatic effect. After 5 days in culture, MSCs secreted tissue inhibitors of metalloproteinase-1 and -2 (TIMP-1 and TIMP-2) which have an antimetastatic effect [in vivo and in vitro [An  pathway . This st pathway  demonstr pathway . An in vc effect . Similarin vitro –93. Sevein vitro –94.γ. When IFN-γ is upregulated, MHC II expression is increased and accessory adhesion molecules such as intracellular adhesion molecule 2 (ICAM-2) and vascular cell adhesion molecule 1 (VCAM-1) are expressed [MSCs lack costimulatory molecules  and MHC II; however, these molecules can be induced when MSCs are exposed to IFN-xpressed , 63. Thixpressed . This maxpressed , therebyin vitro, MSCs are grown in 20% oxygen, and this may cause DNA damage and early senescence [in vivo administration of possibly compromised MSCs may not portray a true reflection of their influence on tumours under natural conditions in vivo. Similarly, in in vitro studies when MSCs were cocultured with a head and neck squamous cell carcinoma cell line (FaDu), cancer cell viability and proliferation decreased when incubated in conditioned media that was derived from MSCs grown under hypoxic conditions. Levels of anti-inflammatory cytokines such as IL-4, IL-5, and IL-10 are reduced, and IL-6 is increased in this conditioned medium [MSCs thrive in hypoxic (2-9% O2) conditions such as those found in the TME, and this environment therefore resembles their natural niche (1-5% O2). When expanded nescence , 96. Cond medium . This daThe effect of MSCs on the immune response in chronic wounds is similar to that observed in the TME in that they exhibit immunosuppressive characteristics , 87. MSCThis review has discussed the role of MSCs and their influence on the immune response in chronic wounds and the TME. Chronic wounds are in a continuous proinflammatory state due to the constant presence of immune cells whose function is impaired in the wound environment. These cells include neutrophils, macrophages, B cells, and T cells. There is a decrease in the number of lymphocytes while Treg increase and the transition from M1 to M2 is delayed. MSCs induce a transition to an M2 type inflammatory response and modulate effector T cell cytokines which leads to reduced proinflammatory cytokine production and an increase in anti-inflammatory cytokines. MSCs promote the arrest of T and B cells at G0/G1 in the cell cycle, thereby promoting T cell apoptosis and limiting antibody secretion by B cells. MSCs also suppress NK cell proliferation and cytokine secretion, which under normal circumstances would enable chronic wounds to heal.Stat3 pathway and (b) MSCs producing TIMP-1 and TIMP-2.Th1 via proinflammatory cytokines activate CTLs, DCs, and NK cells in the TME to attack tumour cells. In contrast, Th2 produce anti-inflammatory cytokines that inhibit T cell cytotoxicity and increase B cell function. Tregs inhibit CTLs, DCs, NK, and B cells while Bregs suppress CD8+ T cells and encourage conversion of CD4+ T cells to Tregs. All of the above support tumour progression and metastasis. The presence of MSCs in the TME encourages tumour progression via several mechanisms: (a) monocytes fail to differentiate into DCs, delaying the activation of T cells; (b) MSCs inhibit T cell proliferation and suppress NK and CTL responses; (c) MSCs induce Th2 cytokine production and decrease Th1 cytokine production; (d) MSCs induce EMT and differentiate into CAFs that secrete angiogenic growth factors. Together, these observations suggest that MSCs support cancer growth and metastasis, and this is based primarily on the immunosuppressive properties of MSCs. Conversely, other studies have demonstrated that MSCs can inhibit tumour growth and metastasis by (a) downregulating the Despite MSCs exhibiting similar immunosuppressive characteristics in both chronic wounds and the TME, several studies have observed that MSCs inhibit cancer progression. In chronic wounds, the application of MSCs is beneficial while in the TME, their application could be either beneficial or detrimental. Various factors such as immunosuppression, glucose metabolism, and Th2 associated leukocytes and cytokines may result in a net effect in which tumour progression outweighs inhibition. The use of MSCs in the clinical setting for therapeutic purposes unrelated to cancer should be approached with this in mind and requires long-term follow-up/monitoring as MSCs may promote cancer cell survival and stimulate the progression of a latent tumour.The TME is similar to chronic wounds in that the phases of healing, inflammation, and proliferation are either hijacked or stalled. In chronic wounds, the constant inflammatory state can be corrected by applying MSCs to downregulate T cells, NK cells, and B cells, resulting in enhanced wound healing. In the TME, different lymphocyte subtypes promote rather than inhibit cancer progression. Tumour cells also secrete immunosuppressive molecules and compete for nutrients. All these factors contribute to the complex nature of the TME. While a few studies have suggested that MSCs inhibit cancer progression, a greater number suggest that MSCs increase tumour progression and metastasis. Hence, based on the available literature on the role of MSCs in the TME, the use of MSCs for the treatment of cancer is not supported, as MSCs in the TME suppress inflammation and the immune response that is required for wound healing.


Autophagy is an intracellular catabolic program regulated by multiple external and internal cues. A large amount of evidence unraveled that cell-cycle regulators are crucial in its control. This review highlights the interplay between cell-cycle regulators, including cyclin-dependent kinase inhibitors, cyclin-dependent kinases, and E2F factors, in the control of autophagy all along the cell cycle. Beyond the intimate link between cell cycle and autophagy, this review opens therapeutic perspectives in modulating together these two aspects to block cancer progression.In the past decade, cell cycle regulators have extended their canonical role in cell cycle progression to the regulation of various cellular processes, including cellular metabolism. The regulation of metabolism is intimately connected with the function of autophagy, a catabolic process that promotes the efficient recycling of endogenous components from both extrinsic stress, e.g., nutrient deprivation, and intrinsic sub-lethal damage. Mediating cellular homeostasis and cytoprotection, autophagy is found to be dysregulated in numerous pathophysiological contexts, such as cancer. As an adaptative advantage, the upregulation of autophagy allows tumor cells to integrate stress signals, escaping multiple cell death mechanisms. Nevertheless, the precise role of autophagy during tumor development and progression remains highly context-dependent. Recently, multiple articles has suggested the importance of various cell cycle regulators in the modulation of autophagic processes. Here, we review the current clues indicating that cell-cycle regulators, including cyclin-dependent kinase inhibitors (CKIs), cyclin-dependent kinases (CDKs), and E2F transcription factors, are intrinsically linked to the regulation of autophagy. As an increasing number of studies highlight the importance of autophagy in cancer progression, we finally evoke new perspectives in therapeutic avenues that may include both cell cycle inhibitors and autophagy modulators to synergize antitumor efficacy. The cell cycle constitutes a fundamental biological process divided into four successive steps: G0/G1, S, G2, and M phases. Cell-cycle progression is tightly regulated by both extrinsic  and intrinsic  cues  Figure a. The acBeyond their canonical role, cell cycle regulators also control cellular metabolic functions, especially in the cancer context . ConversUpon the receipt of stress signals, autophagy is a catabolic process allowing the removal and recycling of metabolic intermediates. Autophagy is divided into two independent complementary mechanisms. First, selective autophagy through micro- or chaperone-mediated autophagy (CMA), and second, non-selective bulk autophagy, termed macroautophagy . Many adAt the molecular level, macroautophagy requires the creation of autophagosomes (the nucleation phase) and their subsequent fusion with lysosomes (the elongation phase) to ultimately form autolysosomes. The initiation step involves the formation of the ULK1/ATG1 complex, later directly phosphorylating a key component of the Class III PI3K complex, VPS34. The further association of this Class III PI3K complex, notably with UVRAG, ATG14, and Beclin-1, allows the proper nucleation of phagophores vesicles. The incorporation of organelles or targeted proteins are mediated by chaperones, including p62 b. In theAutophagy is controlled by multiple signaling cues that are notably integrated by the main intracellular sensor of cellular stress and growth factor stimuli, i.e., the mammalian target of rapamycin (mTOR). Via AKT or MAPK signaling, the activation of mTOR reduces macroautophagy by inhibiting the ULK1/ATG1 kinase. In contrast, the downregulation of mTOR by AMPK or p53 signaling activates ULK1/ATG1 kinase promoting autophagy. During the progression of the cell cycle, autophagy mostly occurs during interphase, in particular in the G1 and S phases ,21,22. IIn this review, we will describe how main cell-cycle regulators, particularly in focusing on cyclin-dependent kinase inhibitors (CKIs), CDKs-cyclins complexes, and E2F transcription factors, impact key steps of the autophagic process. In addition, how autophagy impacts cell-cycle regulators, notably modulating their quantity all along the cell cycle, will be addressed. Finally, the idea of a therapeutic avenue combining both cell-cycle and autophagy modulators during tumorigenesis will be examined.Cell-cycle regulation integrates both external and internal stimuli to sustain cellular viability. Multiple regulators of the cell cycle, including (among others) CKI, CDKs, and E2F transcription factors, were found to regulate autophagic processes. Kip1, p21CIP1, p57CIP2, and p16INK4A  [CIP1/WAF1.Among the CIP/KIP family, p21S phases    through CIP2. Only one study reported a link between p57CIP2 and autophagy. In contrast to p27KIP1 and p21CIP1/WAF1, p57CIP2 accumulation decreases autophagy in hepatocarcinoma cells upon EGFR-targeted therapy, through upregulation of the PI3K/AKT/mTOR axis [CIP2 may impact autophagy is unknown. Taken together, these results demonstrate that the CIP/KIP family of CKI, specially p27KIP1 and p21CIP1/WAF1, has an overall pro-autophagic role  are direct targets of CKIs and, when associated with distinct cyclins, constitute the main effector kinases regulating key processes during all phases of the cell cycle. They include two distinct subfamilies: cell-cycle-associated CDKs , which directly participate in the cell-cycle progression; and transcription-associated CDKs , which directly participate in the regulation of gene transcription. Importantly, most of the studies highlighted the role of cell-cycle-associated CDKs in the control of autophagy.CDK1 is a kinase associated with cyclin A in the G2 phase and with cyclin B during the mitosis phases of the cell cycle a. CDK1 hKIP1 through miR-221 demonstrates the functional involvement of CDK2 in inhibiting autophagy in cardiomyocytes. Mechanistically, the inhibition of CDK2 restores p27KIP1 and mTORC1 mediates autophagy. Interestingly, like CDK1, CDK2 also promotes the lysosomal degradation of HIF-1α at the G1/S transition phase, allowing cell-cycle progression under hypoxic conditions [CDK2 is another key kinase associated with cyclin A or E and is mostly involved in late G1/S interphase and in G2 a. CDK2 hnditions . AltogetCDK4/6 associate with cyclin D to phosphorylate pRB and release E2F transcription factors during the G1 phase a. BeyondCDK4/6 inhibitors (CDK4/6i) also induce cellular senescence in cancer cells  promotinOn the one hand, CyclinD-CDK4/6 negatively regulates TSC2 through phosphorylation of Ser1217 and Ser1452, subsequently activating mTOR ; on the Only a few studies reported a role in autophagic process for non-canonical CDKs, in particular, the roles of CDK5 and CDK11. Not typically activated by cyclins, CDK5 phosphorylates pRB, ultimately resulting in the progression of the cell cycle . CDK5 waCollectively, and beyond the canonical cell-cycle-associated CDKs , these studies also suggest a relative and neglected role of non-canonical CDKs in the modulation of autophagic process.Mitophagy is a selective autophagy process, involving sequestration of damaged dysfunctional mitochondria to maintain global mitochondrial homeostasis . MitochoULK1/ATG1, ATG5, DRAM, and MAP1LC3B/LC3B, and enhances basal autophagy. In contrast, decreasing endogenous E2F1 expression using short hairpin RNAs (shRNAs) inhibits DNA-damage-induced autophagy [ATG9A, MAP1LC3B/LC3B, UV radiation resistance associated gene (UVRAG), and GABA type A receptor-associated protein (GABARAP) as transcriptionally regulated by E2F1. Although the exact mechanisms of how SiNPs activate autophagy are not clear, this study suggests that signaling through the CDK7-CDK4 axis potentiates SiNP-induced autophagy by phosphorylating pRB, activating E2F1 or FOXO3, and enhancing mRNA expression levels of several autophagy regulators and ATG genes [The adenoviral early region 2 binding factors (E2F) family of transcription factors, which is comprised of at least eight members (E2F1-8), are the downstream effectors of CDKs a. Their utophagy . In addiutophagy . Furtherutophagy . MoreoveTG genes .Autophagy can be induced by the tumor suppressor transforming growth factor-β (TGFβ) in various cancer cell lines . To be pATG5 and MAP1LC3B/LC3B. Moreover, a direct binding of E2F1 to the MAP1LC3B promoter in adipose tissue explants has been demonstrated by chromatin-immunoprecipitation experiments. In cellular models, E2F1 overexpression in HEK293 cells triggered the promoter activity of autophagy-related genes and the autophagy flux and sensitized cells to tumor necrosis factor (TNF)-induced hyperactivation of ATG12, MAP1LC3B, and DRAM1 promoter activity. Conversely, in mouse embryonic fibroblast-derived adipocytes from E2f1 knockout mice (E2f1−/−), autophagic gene expression and autophagic flux are downregulated in both basal and stimulated conditions [In addition to cancer, autophagy is associated with obesity; autophagy-related gene expression is upregulated in visceral fat in human obesity . Interesnditions .However, the literature connecting pRB-E2F and autophagy is complex, as suppression of autophagy by E2Fs has also been reported. A study by Jiang et al. demonstrated that pRB restoration in a panel of pRB-deficient cells positively regulates autophagosome formation, maturation, and autophagy flux. In addition, they showed that increased pRB activity or lack of E2F1 trigger autophagy, and pRB-E2F interaction, is required for pRB-mediated autophagy. Moreover, excessive E2F1 activity blocked pRB-induced autophagy. One possible mechanism for how E2F1 mediates suppression of autophagy is the activation of Bcl-2 . Bcl-2 hAnother suppressive function of the pRB-E2F pathway in the regulation of autophagy has been reported in ovarian cancer. Autophagy is one mechanism by which dormant, drug-resistant ovarian cancer cells survive in nutrient-poor environments. A frequently downregulated tumor suppressor gene in ovarian cancer, DIRAS3, has been illustrated to facilitate autophagy induced by amino acid deprivation in the presence or absence of serum. In fact, nutrient-depleted conditions lead to transcriptional upregulation of DIRAS3 by decreased binding of E2F1 and E2F4 and increased binding of CEBPα to the DIRAS3 promoter . This meIt is now evident that E2F1 transactivates more than 2000 genes and that the transcriptional regulation is highly contextual . TherefoCollectively, previous works explicitly documented a prominent role of the pRB-E2F1 pathway in the regulation of autophagy c. NotablKIP1 [While cell-cycle regulators interact with autophagic key players and modify autophagic processes, there is increasing evidence of a feedback loop of autophagy components that could influence the activity of cell-cycle regulators. Previously, mTOR inhibition through rapamycin has demonstrated the role of this complex in the regulation of the cell cycle, notably via the degradation of cyclin D1 and upregulation of p27KIP1 ,93. AddiKIP1 ,95,96, iCIP1 can be targeted by autophagy [CIP1 levels [CIP1 expression [KIP1 [INK4A [INK4A in liver [The protein p21utophagy ,98,99. U1 levels ,98,99. Opression . In addion [KIP1 . Moreove1 [INK4A ,46. Indein liver . Interesin liver . CIP1, p27KIP1, and p16INK4A levels, further impacting cell-cycle progression. While most of these studies were conducted in a non-tumoral context, these data are also in line with a pro-tumoral role of autophagy via the degradation of tumor suppressor proteins, such as CKIs  and its derivative hydroxychloroquine (HCQ) are FDA-approved modulators of autophagy that inhibit lysosomal acidification and thereby impair autophagosome maturation. The effects of HCQ are explored in phase I and II clinical trials for various cancers. However, its use as a monotherapy does not provide satisfactory results ,117.. For instance, palbociclib treatment leads to autophagy activation in breast cancer cells, which prevents palbociclib-induced senescence. However, combined usage of the CDK4/6 inhibitor palbociclib and the autophagy inhibitor HCQ results in growth arrest, accumulation of reactive oxygen species, and cellular senescence in cancers with an intact G1/S transition. In addition, this study identified that the Rb-positive and low-molecular-weight isoforms of cyclin E (cytoplasmic)-negative are reliable prognostic biomarkers in ER-positive breast cancer patients, and predictive of preclinical sensitivity to the combination of these drugs [Several preclinical studies have consistently shown that CDK4/6 inhibitors lead to autophagy activation in different cancer cells ,118,119.se drugs . The synse drugs ,120. In pancreatic ductal adenocarcinoma (PDAC), the co-encapsulate CDK4/6 inhibitor palbociclib and the autophagy inhibitor HCQ also generate synergistic antitumor effects in subcutaneous and orthotopic PDAC models . FurtherThese promising preclinical results led to several clinical trials  to asseIn constant interactions, cell-cycle regulators and modulators of autophagy are intrinsically related, facilitating the synchronization of the cell cycle and catabolism during cell-cycle progression. These observations explain most of the previous observations that link cellular senescence, stable cell-cycle arrest, and autophagy pathways. In addition to CKIs, CDKs, and cyclins, other cell-cycle regulators were found to participate in autophagy, as Aurora Kinase A ,127 or pThe cell cycle and autophagy are two critical processes during tumorigenesis. In the tumorigenesis steps, cell-cycle and autophagy fluxes are coordinated  during tumor progression and tumor therapeutic interventions . During Beyond the temporal modulation of autophagy during tumorigenesis , the impOverall, combining autophagy and cell-cycle modulators opens new therapeutic perspectives to initiate additional cell death mechanisms and synergetic antitumoral effects. Altogether, these studies reinforce the importance of timing, duration, and selective efficiency of drug administration, which are three main parameters to optimize for autophagy-/cell-cycle-related therapeutic interventions in the future.


GALNT2, which encodes the GalNAc-transferase 2 involved in the first step of O-linked glycosylation, has attracted great attention as a possible player in many highly prevalent human metabolic diseases, including atherogenic dyslipidemia, type 2 diabetes and obesity, all clustered on the common ground of insulin resistance. Data available both in human and animal models point to GALNT2 as a molecule that shapes the risk of the aforementioned abnormalities affecting diverse protein functions, which eventually cause clinically distinct phenotypes . Pathways linking GALNT2 to dyslipidemia and insulin resistance have been partly identified, while those for type 2 diabetes and obesity are yet to be understood. Here, we will provide a brief overview on the present knowledge on GALNT2 function and dysfunction and propose novel insights on the complex pathogenesis of the aforementioned metabolic diseases, which all impose a heavy burden for patients, their families and the entire society.O-linked glycosylation, the greatest form of post-translational modifications, plays a key role in regulating the majority of physiological processes. It is, therefore, not surprising that abnormal O-linked glycosylation has been related to several human diseases. Recently,  GALNT2 encodes the polypeptide N-acetyl-galactosaminyl-transferase 2  [O-linked glycosylation, the greatest diverse form of post-translational modifications, affects the folding, stability, processing and trafficking of most secreted proteins , thus pllNAc-T2) ,4, whichlNAc-T2) ,6,7 in tlNAc-T2) ,12,13,14lNAc-T2) ,16 and olNAc-T2) ,18. AfteInsulin resistance is a complex and highly prevalent metabolic disorder, which is mainly due to the inability of multiple insulin target sites, including but not limited to skeletal muscle, liver, and adipose tissue, to respond to insulin. It is widely accepted that insulin resistance represents a common pathogenic ground for many additional disorders and highly prevalent diseases, comprising atherogenic dyslipidemia , type 2 Over the last few decades, several possible pathophysiological mechanisms leading to insulin-resistant individuals eventually developing the aforementioned metabolic and vascular disorders have been addressed. Just as an example, many studies have demonstrated that when pancreatic beta-cells fail to provide compensatory insulin secretion (chronic compensatory hyperinsulinemia response) ,27, insuThe intertwined risk factors shaping the risk of developing insulin resistance also draw a complex scenario that has yet to be fully clarified. In general, it is widely accepted that over both genetic predisposition and environmental factors, obesity and adipose tissue dysfunction are major movers of insulin resistance ,31. It iENPP1) [TRIB3) [PTPN1), serine phosphorylation of IRS1  and 5′ dephosphorylation of PIP3  [The first step of insulin action involves its binding to the insulin receptor at the plasma membrane, which consists of two extracellular α and two transmembrane β subunits ,39,40. FENPP1) ,44,45,46 [TRIB3) , tyrosin, SHIP2) ,49. In s, SHIP2) ,39,48,49, SHIP2) ,51, the , SHIP2) ,53. OverGALNT2, located on chromosome 1q42.13 [GALNT2 likely shares a common ancestor with the other GALNT genes [ 1q42.13 . As indi 1q42.13 , GALNT2 NT genes  and exhiNT genes ,56.GALNT2 is expressed in nearly all cell types and tissues [GALNT2/ppGalNAc-T2 is considered by far the major contributor of the whole O-glycoproteome [ppGalNAc-T2 participates in the initiation step of O-linked glycosylation ,58 by co tissues  and cont tissues ,68,69,70proteome .GALNT2/ppGalNAc-T2 expression and/or function may contribute to shaping the risk of several metabolic abnormalities characterized by energy substrate imbalance, systemic low-grade inflammation and vasculature damages.Only a few ppGalNAc-T2-targets have been identified so far. The most studied are angiopoietin-like protein 3 (ANGPTL3) ,72,73, aGALNT2 as a mediator of insulin sensitivity comes from our studies on human liver cells (HepG2) as well as on mouse fibroblasts (3T3L1) during their differentiation into mature adipocytes [GALNT2 expression in HepG2 cells impairs insulin signaling and action, as indicated by the reduced ability to stimulate insulin receptor, IRS-1 and protein kinase beta Akt2 phosphorylation [GALNT2 over-expression significantly improves insulin signaling and, in turn, stimulates adipocyte maturation and enlargement [GALNT2 affects insulin sensitivity is not yet known. In this regard, it is of note that in both HepG2 and 3T3L1 cells, the expression of GALNT2 is inversely correlated with that of ENPP1, a well-established negative modulator of insulin signaling [ENPP1 down-regulation is likely to mediate, at least partly, the insulin-sensitizing effect of GALNT2. Whether ENPP1 is a primary ppGalNAc-T2 target or its modulation is secondary to upstream O-glycosylation events is not yet known. Taken as a whole, our data on two different insulin responsive cells do suggest that GALNT2 is a positive modulator of insulin sensitivity.Evidence on the role of ipocytes ,84. The rylation  and to drylation . Coherenignaling ,44,45,46GALNT2 variability is associated with HOMA-insulin resistance index in women with polycystic ovary syndrome [A role of GALNT2 in modulating insulin sensitivity is also suggested by preliminary genetic studies indicating that syndrome .As said before, three main players in lipid metabolism have been validated as non-redundant substrates of ppGalNAc-T2. These are ANGPTL3 ,72,73 anGALNT2 loss of function (LOF), ranging from humans  to genetically modified nonhuman primates and mice, affect HDL-C consistently across species [GALNT2-LOF also increased TG levels in monkeys and mice but not in humans [GALNT2 modulates lipid metabolism in a species-specific manner.Extreme conditions of  species . NotablyGALNT2 affects lipid metabolism. In detail, data from genome-wide studies highlight that the common rs4846914 SNP at the GALNT2 locus, which is associated with lower GALNT2 expression in human liver [GALNT2 expression levels are correlated directly with HDL-C and inversely with TG serum levels [GALNT2 mRNA levels remain significantly associated with TG but no longer with HDL-C in conditional analyses including both lipid fractions, thus suggesting the association with HDL-C is mainly mediated by the very much expected strong and inverse correlation between HDL-C and TG levels [GALNT2 promoter is hyper-methylated, a proxy of gene down-regulation, in patients with CAD and low HDL-C levels [Additionally, fine tuning of an liver ,87. Alonm levels . Of noteG levels . FinallyC levels ,89.GALNT2 exerts pleiotropic effects [Overall, the available data depict a complex scenario compatible with the hypothesis that  effects  on diffe effects .GALNT2 levels are reduced in obese patients with type 2 diabetes as compared to obese patients with no diabetes and even more to non-obese control individuals [GALNT2 down-regulation in liver of Goto-Kakizaki diabetic rats as compared to their normoglycemic counterparts [GALNT2 down-regulation causing hyperglycemia or, vice versa, is hyperglycemia causing GALNT2 down-regulation?) is clearly indicated by data in human cultured monocytes, where a high glucose concentration causes GALNT2 down-regulation [GALNT2 expression and insulin signaling [GALNT2 down-regulation is part of the mechanism underlying hyperglycemia-induced insulin resistance [Studies in human PWBC show that ividuals . These hterparts . The biogulation . These dignaling ,84 make sistance .Adig, Retn, Nr1h3, Ucp1, Slc2a4, Lipe, Fgf2 and Tcs22d3) [GALNT2 expression is increased in subcutaneous adipose tissue from obese individuals [GALNT2 expression changes contributing to the increase in adiposity. This hypothesis is further supported by models of GALNT2-LOF mutations in both cattle [Given the well-established role of insulin sensitivity in promoting fat mass expansion, it is not surprising that GALNT2 over-expression in pre-adipocytes, by improving insulin signaling, stimulates adipocyte maturation  and leadTcs22d3) . Of noteividuals , thus mah cattle  and rodeh cattle  which, ih cattle ,86. GALNT2 changes affect differently specific protein functions in an organ-specific manner, thus causing clinically distinct phenotypes (pleiotropism) [Taken altogether, the data that we have reported here are quite consistent with the contribution of GALNT2 to several highly prevalent metabolic abnormalities sharing the common ground of insulin resistance, namely atherogenic dyslipidemia, type 2 diabetes and obesity. Unfortunately, they leave open the question on which mechanisms underlie some of the effects of GALNT2 on the aforementioned diseases. Pathways through which GALNT2 impairs lipid metabolism and insulin resistance have been partly identified, while those connecting GALNT2 to hyperglycemia/type 2 diabetes and obesity are only scarcely understood. With this caveat in mind, it is possible to envision a scenario in which GALNT2 operates through various mechanisms . The simtropism) . A cleartropism) ,10,13,14tropism) ,86,92, ctropism) ,86.Overall, we must admit that, unfortunately, no single scenario is available within a solid framework for the many effects exerted by GALNT2 on the cluster of metabolic abnormalities, all sharing the common ground of insulin resistance. Several data indicate that they may actually represent different effects with different mechanisms , although it is possible that, for at least some of them, a pathogenic link mediated by abnormal insulin signaling and insulin resistance does actually exist.A further lack of knowledge concerns the possibility of modulating GALNT2 with the drugs currently in use for other purposes . To the best of our knowledge, there are no in vivo or in vitro studies that have specifically investigated the effect of any drug on GALNT2 expression or ppGalNAc-T2 enzyme activity. Given the potential role of GALNT2 on the aforementioned severe and highly prevalent disorders, studies addressing the potential role of drugs already available today on GALNT2 would certainly be useful and are, therefore, timely.In all, several studies both in humans and animal models point to GALNT2 as a contributing molecule to highly prevalent metabolic abnormalities, including atherogenic dyslipidemia, type 2 diabetes and obesity, which all impose a heavy burden for patients, their families and society as a whole. Conversely, our knowledge of the intimate mechanisms through which GALNT2 exerts these effects is still poor. A full understanding of GALNT2 function and dysfunction is, therefore, mandatory and will certainly improve our comprehension of the aforementioned metabolic diseases. Such knowledge gain is a prerequisite for discovering new pathogenic targets to be addressed with new therapies, so as to hopefully ameliorate the strategies currently available to treat patients with related insulin-resistance abnormalities.


Computed free energies, based on optimized surface hydration structures utilizing a harmonic approximation, typically  disagree with experimental free energies. To remedy the harmonic approximation, we utilize single-point electronic structure calculations on cluster geometries sampled from an AIMD (ab initio molecular dynamics) simulation stream. This rough-landscape procedure is broadly satisfactory and suggests unfavorable ligand crowding as the physical effect addressed. Nevertheless, this procedure can break down when physical cluster theories. With ramified structures, the central equation for the present rough-landscape approach can acquire some inconsistency. Extension of these physical cluster theories in the direction of QCT should remedy that issue, and should be the next step in this research direction.With a longer-term goal of addressing the comparative behavior of the aqueous halides F Hofmeister devised a ranked list of salts to classify their influence on protein precipitation and swelling ,2. Laterry (QCT) ,13,14,15ry (QCT) . Becausetheories ,20,21,22theories ,24.Common to all these theories is the study of associative equilibriaHere X ≡ FEquation  then dirThe computational study of clusters benefits from a good experimental basis . Widely Kn(0)  under asKn(0) . A challKn(0) ,29,30. SKn(0) ,31, harmperiment , we see crepancy , making clusters .ab initio molecular dynamics) simulations of The essential feature of the computational approach here is the deployment of standard electronic structure computations for cluster geometries sampled from AIMD  thtrometry . Figure trometry , the firStructures of the Wr F−(aq) ,49. StruIt might be guessed that these structural characteristics would be simpler for clusters  for whicider W3X , in cont W3X  makes tEquation ) improveMolecular dynamics trajectories of the isolated ing CP2K ,58. The ing CP2K  functioning CP2K ) in the ing CP2K . Moleculing CP2K  basis seOur initial assessment of cluster free energies  applied ussian09  with theussian09 ,65 functussian09  for F− aasis set ,67, and ing QZVP  and DEF2ing QZVP  basis seTlnKn(0)  was evalEquation —was assid H2O5I− , optimizFor the rough landscape treatment, structures from To confirm our results, and to investigate the functional dependence of the cluster free energy, additional AIMD trajectories for  the PBE  functionEF2TZVPD  basis seEF2TZVPD .Classic statistical thermodynamics ,15,71 anV is the system volume and configurational integrals in the traditional classical-limit analysis [n W ligands. Each V, so the ratio Equation  is baseEquation . This isn) in gas-phase mass spectrometry [n varies, the energy combination of Equation (The free energies we arrive at correspond to water addition reactions (Equation ). They atrometry . BecauseEquation  is not aFor Equation  correctlperiment  or theorThe analysis above is based on the classical limit formula for the partition function The rough-landscape analysis (Equation ) is surpphysical cluster theories. With ramified structures, the central equation (Equation (The tested rough-landscape remedy  to the hEquation ) for the


In order to evaluate the maize volatiles that can influence consumers’ acceptance of broas, twelve broas were prepared from twelve maize varieties , following a traditional recipe. All maize flours and broas were analyzed by HS-SPME-GC-MS (headspace solid-phase microextraction) and broas were appraised by a consumer sensory panel. In addition, the major soluble phenolics and total carotenoids contents were quantitated in order to evaluate their influence as precursors or inhibitors of volatile compounds. Results showed that the major volatiles detected in maize flours and broas were aldehydes and alcohols, derived from lipid oxidation, and some ketones derived from carotenoids’ oxidation. Both lipid and carotenoids’ oxidation reactions appeared to be inhibited by soluble phenolics. In contrast, phenolic compounds appeared to increase browning reactions during bread making and, consequently, the production of pyranones. Traditional samples, especially those with higher contents in pyranones and lower contents in aldehydes, were preferred by the consumer sensory panel. These findings suggest that, without awareness, consumers prefer broas prepared from traditional maize flours with higher contents in health-promoting phenolic compounds, reinforcing the importance of preserving these valuable genetic resources. In Portugal, maize has been used for centuries to produce an ethnic bread called  For centuries, natural and human selection adapted varieties to different environments, creating a diverse maize germplasm . Portuguze bread , which pze bread . This tyourdough ,3. Afterourdough . Neverth program . In factoduction .Quality criteria important to consumers’ acceptance of breads are related to both their rheological (texture) and organoleptic  properties ,5,6. FlaIn general, lipid oxidation products are associated with off-flavors ,18 and aApart from lipid oxidation, aldehydes can also be formed inside the yeast cells, by the degradation of flour amino acids via the Ehrlich pathway, and originate the corresponding alcohols, acids and esters ,17,20. DIn addition to color development, browning reactions produce compounds that contribute to the flavor of bakery products . In partbroas may be especially affected by their phenolic composition. Additionally, these compounds exhibit antioxidant properties, contributing to the prevention of non-communicable diseases [Some studies have reported that variations in wheat flour odor directly affect bread flavor ,27. Morediseases  that arebroas’ volatile composition in order to identify volatiles that may influence consumer’s choice, and to (2) shed light on the characteristics of traditional maize varieties responsible for their better suitability for broas production. In particular, the influence of (2a) soluble phenolic compounds, (2b) total carotenoids content and (2c) volatiles from maize flours on broas’ volatile composition was evaluated. This knowledge is important to both the baking industry and maize breeders, as cereals’ composition contribution to odor can possibly become a future quality parameter in breeding [Several studies have been published on the identification of key volatiles responsible for odor quality in maize-based foods, such as popcorn and cornflakes ,35,36,37breeding .Eleven traditional or participatory improved open pollinated Portuguese maize varieties  and a commercial hybrid maize flour—Nacional Type 175 (F12)—were studied . The trabroas (B1 to B12) were prepared in a bakery following a traditional recipe. The procedure was previously described [Twelve escribed . The ingbroas preparation were acquired already milled and analyzed in the same conditions.The commercial rye and wheat flours used for 8-C20 solution (40 mg L−1 in hexane) was purchased from Fluka . Ultra-pure water (18.2 MΩ.cm−1) was obtained from a Millipore-Direct equipment Q3 UV system . GC vials were sealed with a silicone/teflon-lined septum and screw cap .The different SPME fibers used  were purchased from Supelco . The alkane standard mixture CThe analysis of volatile compounds was carried out using a GCMS-QP2010 Plus mass spectrometer  and an AOC-5000 autosampler . The compounds were separated using a Varian Factor Four DB-5MS column .The commercial maize flour was used to optimize the HS-SPME extraction of volatile compounds from cereal flours. Different ratios of flour:water were studied , as well as different types of fibers , temperatures  and times  of extraction, in triplicate. Additionally, sample stability in the autosampler was evaluated for 32 h. The selected conditions were also applied to the commercial wheat and rye flours. broas, 4 g of sample  was smashed manually and placed in a 20 mL vial. A 50/30 µm DVB/CARBOXEN/PDMS fiber was selected to concentrate the volatile compounds. Samples were extracted for 40 min at 60 °C and 250 rpm. Before extraction, the fiber was conditioned in the GC injector at 270 °C for 30 min, in order to remove any contaminants. Fiber blanks analyses were performed using the same SPME and chromatographic conditions. Samples were prepared in duplicate. After optimization, the following conditions were used for the volatile analysis of cereal flours : flour (2 g) was placed in a 20 mL GC-vial, deionized water (5 mL) was added and the mixture was vortexed for 2 min before HS-SPME-GC-MS analysis. For −1 to a final temperature of 230 °C, which was kept for 5 min . The column carrier gas was helium at a constant flow rate of 2.1 mL min−1. The mass spectrometer was operated in the electron impact mode (EI) at 70 eV, scanning from m/z 30 to 300, at a scan rate of 555 scan s−1, and the ion source temperature was set at 250 °C. Desorption time was 5 min at 260 °C (injector temperature). The extracted volatiles were injected in the GC column in split mode (ratio 1:2). The oven temperature was kept at 35 °C for 5 min, followed by an increase of 5 °C minAs previously reported , the idebroas were extracted with 50% aqueous ethanol, and the major soluble phenolic compounds  were quantitated by HPLC-DAD (high-performance liquid chromatography coupled to a diode array detector), following the procedures previously described [Phenolic compounds of maize flours and escribed ,41.The total carotenoids content was spectrophotometrically measured at 450 nm according to the AACC method 14-60.01 . Results were expressed in micrograms of lutein equivalent per gram of sample, as the main carotenoid found in maize, as previously described .broas was conducted using a hedonic quantitative response scale  in a test room  with a consumer panel of 52 assessors. The test was performed using a numeric category scale from 1 (extremely unpleasant) to 8 (extremely pleasant) for all the attributes. Further details on this hedonic test have been described elsewhere [A sensory evaluation  of lsewhere .n-alkanes (C8-C20) injected under the same chromatographic conditions, and results were compared with data from the literature. A positive identification was considered when the experimental spectra shared at least an 80% similarity with spectra from the software libraries and the LRI deviation was less than 1%, when comparing to the LRI reported in the literature. The software LabSolutions GCMS solution Release 2.53SU1  was used to analyze the data and to calculate the areas of the peak chromatograms. The identification of volatile compounds was performed by comparison of the mass spectra obtained for each compound with the mass spectra from the software library (NIST 27 and WILEY 229 and 147). The linear retention index (LRI) was calculated for each compound using the retention times of a homologous series of ® version 21 was used to: (1) evaluate the differences among the results obtained during the optimization of the experimental conditions (ANOVA and Student’s t test), and to (2) measure the degree of association between broas volatile compounds and (2a) maize flours’ volatiles, (2b) maize flours and broas’ phenolic compounds, (2c) maize flours and broas’ total carotenoids content and (2d) scores obtained in broas’ sensory analysis. A non-parametric test (Spearman correlation) was used to measure the degree of association between variables, since they did not meet the assumptions for a parametric analysis . The software SPSSbroas, and to study their interdependent relations, a hierarchical cluster analysis was performed using R software. Since this analysis requires a number of variables lower than the number of samples, it was necessary to decrease the number of variables. Therefore, after variables’ standardization, a PCA  was performed, in order to group the variables according to their correlations. The number of principal components considered was based on the Kaiser criteria and a cluster analysis was then performed. The distance was measured by the Euclidean distance, and Ward’s method was used to form the clusters, allowing the formation of clusters with the lowest relative standard deviations among the groups. The optimal number of clusters was estimated using two different methods, namely the elbow point and silhouette methods. A statistical significance test (v.test) was performed in order to identify the variables which significantly contributed to clusters’ differentiation.In order to identify the main differences among the 12 maize and p > 0.5). Taking into account the time required for exposure of the fiber and chromatographic analysis, a time of exposure of 40 min was selected. The repeatability of the whole procedure (n = 6) was determined, and the relative standard deviation (RSD) was 17%, similar to values previously reported [The analysis of volatile components was performed by headspace solid-phase microextraction (HS-SPME) followed by gas chromatography coupled to mass spectrometry (GC-MS). The commercial maize flour was used to optimize the conditions of analysis in order to achieve a higher extraction efficiency of the volatiles. The total chromatogram areas were measured, focusing on the aldehydes’ content, since they have been described as mainly responsible for the aroma of cereal flours . Differereported . The stareported ,46,47,48broas were analyzed using the optimized conditions. Illustrative chromatographic profiles of a maize flour (F1), corresponding broa (B1) and wheat and rye flours are presented in broas by comparison of their mass spectra with those from the software library and LRI from the literature. Their characteristic flavors, LRI, similarity indexes and retention times (RTs) are described in All twelve maize flours and corresponding Z) and 5-undecene (Z), having identical similarity indexes. The presence of the ion at m/z 29, also present in other aldehydes, suggests that this compound is 6-nonenal. In turn, the mass spectrum of peak 50 suggests a structure corresponding to pantolactone -furanone), with a molecular ion at m/z 130 and three major fragments at m/z 43, 57 and 71. However, it was not possible to confirm the identification of this compound, since the LRI values found in the literature were not similar (>1% deviation) to the LRI obtained in the present work. The chromatographic profiles suggested that several different compounds could be co-eluting. For peak 99, two compounds can be proposed: geranylacetone and nerylacetone, with close similarity indexes. This peak was tentatively identified as geranylacetone, since it has been widely described in several maize-based products [Aldehydes were the most abundant volatiles detected in maize flours, corresponding to 30% of the total chromatogram area, which is in accordance with results previously reported ,40,42. Aproducts ,35,36.broas were mainly aldehydes , alcohols (16%) and hydrocarbons (14%). Ketones (7%), furans (5%), pyrans (3%), lactones (3%), esters (3%), acids (3%) and pyrazines (1%) were also present. All these compounds have been described in other baked cereal products [m/z 88, and other fragments at m/z 43, 60, 70, 99 and 115. A structure corresponding to ethyl hexanoate, with a similarity index of 85%, was suggested. This compound has been reported in baked cereal products [m/z 124, a base peak at m/z 67 and fragments at m/z 39, 41, 55, 95 and 109. This compound has been described in the crust of whole-meal wheat bread and other maize-based foods [m/z 110 and a base peak at m/z 95; however, it was not possible to confirm this identification since no LRI value was described in the literature. For peak 51, two compounds were suggested: 3,5-octadien-2-one  and 1-octyn-3-ol, as they presented similar spectra. However, the compound 3,5-octadien-2-one was selected, due to the presence of the base peak at m/z 95 and the molecular ion at m/z 124. Compound 74 was tentatively identified as 2-hexyl-1-octanol, due to the high-spectrum similarity index (90%), and it has been described in other cereals, such as rice [The families of compounds identified in products ,20. As iocarbons %. Ketoneed foods . A tenta as rice ,58.broa, these samples were analyzed in the same conditions as maize flours. The chromatographic profiles corresponding to these flours are presented in E,E) (84) were detected in broas and rye and wheat flours, but not in maize flours. Other compounds, such as 2(3H)-furanone-5-heptyldihydro (peak H) and linalyl acetate (peak F), were detected only in wheat and rye flours. 2,4-Decadienal  (84) was particularly abundant in the commercial wheat flour. Rye was distinguished by the presence of acids, such as octanoic (peak C) and nonanoic (G) acids, and terpenes, such as α-terpinolene (A), camphor (B), 4-terpineol (D) and α-terpineol (E).Since rye and wheat flours were also included in the recipe of The majority of the compounds identified in maize flours were aldehydes , alcohols , hydrocarbons and 2-pentylfuran, which have been described in cereal flours as secondary products of lipid oxidation of unsaturated fatty acids ,20,37,44In order to evaluate the differences among maize flours and to study possible relations among the different volatiles, a cluster analysis was performed and the Spearman coefficients among the main volatiles were determined. Twenty-nine compounds, described in p < 0.05) of other aldehydes , 2-nonenal (E), hexanal, heptanal, octanal, 2-heptenal (Z), 2-undecenal, decanal and 6-nonenal), alcohols  and 3-ethyl-2-methyl-1,3-hexadiene, 2-pentylfuran, limonene, trimethyldodecane, 2-heptanone and pantolactone. The mentioned aldehydes showed very strong and positive correlations among them (E) and 2-nonenal (E), two by-products of linoleic acid oxidation, one of the most abundant lipids in maize kernel [E) and 2-nonenal (E). Additionally, F12 was acquired already milled, and it may have been kept at room temperatures for a longer period than the traditional maize flours, contributing to its higher amounts in lipid oxidation products [p-coumaric acids, diferuloyl putrescine, coumaroyl feruloyl putrescine, dicoumaroyl spermidine and bis-diferuloyl putrescine) were quantitated  not only with diferuloyl putrescine, but also with total carotenoids’ content. Hence, phenolic compounds in traditional maize flours and, to a lesser extent, carotenoids, may have inhibited lipid oxidation reactions, and contributed to their longer preservation. In fact, ferulic acid has been approved in certain countries as a food additive to prevent lipid oxidation [The commercial sample was distinguished from the traditional varieties due to the presence of benzaldehyde and higher amounts   . Therefo < 0.05) . In addi < 0.05) , corrobolic acid .Finally, cluster 3, constituted by the sample F1, was distinguished by higher contents in compounds derived from lipid oxidation, similar to the commercial hybrid (F12). This sample was a synthetic open pollinated maize variety developed as an experimental higher-quality cultivar with increased precocity, obtained through the crossing of 12 maize populations . As previously discussed for the commercial sample, the differences observed may be at least partially explained by their different genetic origin.broas were mainly derived from lipid oxidation (Candida spp. [Z) and 2,4-decadienal  were detected in broas, but not in maize flours. Both aldehydes have been reported as primary odorants to contribute to the flavor of wheat bread [broas preparation, it could also have been derived from the lipid oxidation of linoleic acid [The volatile compounds present in xidation  and origxidation  and non-xidation . It is kida spp. . 4-Hepteat bread , and 2,4at bread . They haat bread . Since 2eic acid  present broas despite being only detected in the commercial maize flour. It may have been produced by auto-oxidation of 2,4-decadienal [broas, but not in maize flours, as acids (acetic and hexanoic acids), esters , ketones  and furanones . These compounds may have been originated by yeast and/or bacteria during fermentation [broa. In spite of being volatilized during baking [p-Vinylguaiacol was also detected in broas, but not in maize flours. It has been described as an important odorant in other breads and baked products, derived from ferulic acid decarboxylation [Benzaldehyde was detected in all cadienal  or formecadienal ,25. Otheentation ,17,20,22g baking , acetic g baking ,25. Otheg baking ,25,71, ag baking . p-Vinylxylation ,28,29. Oxylation ,20, or pxylation .broas and already described in wheat bread and other maize-based foods are highlighted in The contribution of a compound to the final bread aroma depends not only on its concentration, but also on its odorant power, which is determined by its odor threshold, or odor activity value (OAV) . TherefoBroas’ crumb and crust samples were analyzed separately in the same conditions. The crust was characterized by a higher content in aldehydes, furans, pyrrolines and acids, while the crumb presented a higher content of alcohols and esters (data not shown). These results were expected, since different compounds are mainly present in the crumb, where temperatures during bread making are in general less than 100 °C, and others are formed in the crust, at temperatures of around 230–250 °C [0–250 °C . The mai0–250 °C  and ferm0–250 °C ,12,16,720–250 °C  increaseAs for maize flours, the associations among different samples and the correlations among volatiles were studied. All eighty-seven compounds were considered for these analyses . After sBroas were differentiated mainly by their content in volatiles from lipid oxidation and formed during baking. In particular, clusters 3 and 4 were characterized by higher amounts in volatiles from lipid oxidation. As observed for maize flours, the broa prepared from the commercial maize variety (B12) was discriminated from all the others, belonging to cluster 4  [broas B6, B7 and B8 belonged to cluster 1, characterized by higher contents in compounds resulting from the baking process, particularly maltol, 1-furfurylpyrrole, 2-acetylpyrrole and 5-methylfurfural.luster 4 . This sa 4  . Finallybroas’ volatiles were also evaluated (p < 0.05) were also detected among compounds formed during baking, such as methylpyrazine, 5-methylfurfural, furfurylpyrrole and 2-furanmethanol, and among volatiles which may have been originated during fermentation, namely hexanoic acid, benzyl alcohol, ethyl hexanoate, 1-hexyl acetate, ethyl octanoate, γ-nonalactone, 3-octen-2-one and 2-methyl-3-octanone. Positive correlations were also commonly found among these volatiles and several alcohols, such as isopentanol, 1-pentanol, 1-hexanol and 1-heptanol, suggesting that these compounds have also been originated by bacteria or yeast fermentation. Conversely, significant negative correlations were found among some volatiles derived from fermentation, such as 2-methyl-1-butanol, phenylethyl alcohol and 2-methyl-3-octanone, with several lipid oxidation products, such as aldehydes , heptanal, benzaldehyde, nonanal and decanal) and hydrocarbons . Other authors have reported similar negative correlations in other food products [p-vinylguaiacol can significantly reduce the generation of Maillard-type aroma compounds, but only a modest negative correlation was found between this compound and methylpyrazine in broas , whereas no correlations were found between this compound and other Maillard derivatives  and maize flours with higher contents in heptanal originated broas with higher contents in 3-ethyl-2-methyl-1,3-hexadiene .The volatile composition of maize kernels plays a major role in overall end-product quality . Thereforrelated . In genebroas were quantitated  were found between broas’ diferuloyl putrescine and coumaroyl feruloyl putrescine contents and alkanes  and alcohols . Negative correlations were also obtained between phenolic compounds and 1-nonanol and 1-octanol. Therefore, phenolic compounds seamed to inhibit lipid oxidation reactions not only in maize flours, as previously discussed, but also in broas and possibly during bread making, as described for other breads [p < 0.05) were found between both free ferulic acid and coumaroyl feruloyl putrescine in maize flours and several lipid oxidation aldehydes [broas, such as hexanal, 4-heptenal, heptanal, nonanal, decanal and benzaldehyde. Some studies have reported that phenolics, including ferulic acid, can actually increase the generation of lipid oxidation volatiles [Significant negative correlations were found between phenolic compounds from maize flours and r breads ,33,73,76ldehydes ,20,44 inolatiles , possiblolatiles ,73. Convolatiles  have demolatiles  and by dolatiles ,73,77. HNegative correlations were also found between phenolic compounds and some volatiles originated by yeast and/or bacteria during fermentation, such as ethyl hexanoate, ethyl octanoate, 1-hexyl acetate, benzyl alcohol, 2-methyl-1-butanol and γ-nonalactone ,17,20,22On the contrary, significant positive correlations were found among phenolic compounds and furan derivatives from browning reactions  act as antioxidants, inhibiting lipid oxidation reactions in both maize flours  and broabroas , (2) inhbroas  and (3) broas . As prevBroas produced from maize flours with a higher content in carotenoids showed higher amounts of geranylacetone and α-ionone  . Both of these compounds can result from the degradation of phytoene and ζ-carotene [ < 0.01) . Thus, molatiles . For inscarotene ,61.N-caprolactone, 2-phenylacetaldehyde, acetoin (3-hydroxy-2-butanone), hexanoic acid and indole [Significant negative correlations were found between carotenoids’ content and some compounds formed during bread making (fermentation and/or baking), namely γ-d indole ,21,25, sN-caprolactone present in broas and pantolactone in maize flours  showed the lowest mean scores for all the evaluated attributes . In addition, the majority of the negative comments were attributed to this sample, including ‘dry texture’, ‘weak typical flavor’, ‘wheat bread flavor’, ‘with a weak maize flavor’ and ‘no history’. As previously discussed, the commercial broa was distinguished from all the traditional broas (p < 0.01) with ‘taste and aroma’ scores. This aldehyde contributes to fatty, nutty, waxy and green notes [broas. Hence, it may contribute to aroma characteristics in broas that are not typical of this type of bread, giving rise to some of the negative comments described above. It can also be responsible for a bitter taste [broa.As previously reported , the senal broas , essentien notes ,51. Thisen notes ,17,20. Hen notes , and thuer taste , which wbroa B8 scored higher for the majority of the sensorial attributes  and was often described by the consumer panel as ‘the tastiest’ and ‘with a good maize flavor’. This sample belonged to cluster 1, together with B6 and B7, characterized by higher contents in compounds resulting from the baking process associated with positive notes [broas’ sweet aroma. Significant positive correlations were found between ‘taste and aroma’ scores and pyranones content . Despite the relatively high odor threshold of 9 mg/kg (water), maltol was very abundant in the B8 sample, which showed the highest maltol content of all broas . Similarly, B1 belonged to cluster 3, together with B2, B5, B9 and B10, characterized by higher amounts in lipid oxidation products, such as hexanal and heptanal, which were negatively correlated with ‘taste and aroma’ scores.The ve notes ,20,21,24ve notes , extrudeve notes  and tortve notes , suggestll broas . It has e flours  due to iE) and 2-undecenal, frequently described as negative contributors to the aroma of breads [B11, followed by B3, showed the highest ‘taste and aroma’ scores. These samples and B4, which showed the fourth highest scores, all belonged to cluster 2 , charactf breads .broas prepared from the traditional and commercial hybrid maize varieties and sustain the previous knowledge of producers regarding the better-quality aspects of traditional maize varieties for broas’ production [broa of traditional maize is softer, sweeter and can be preserved for longer periods than broas produced with hybrid varieties [broas sweeter taste, and lower contents in aldehydes suggest that lower lipid oxidation reactions were occurring in traditional samples, which can not only explain their longer preservation, but also contribute to their more pleasant flavor.These results explain some differences among oduction . In partarieties . Indeed,broas with higher carotenoids’ content, as previously discussed. To the best of our knowledge, 2,3-dihydrobenzofuran has not been described neither in other maize-based foods nor cereal breads and was not detected in maize flours, so it was originated during the bread making process, similar to other furan derivatives [Positive correlations were found between ‘smell and odor’ scores and 2,3-dihydrobenzofuran, which was more abundant in ivatives ,15,16. Tivatives ,83 and iivatives .broas’ yellowish color by the panel of consumers. The ‘global appreciation’ of broas was greatly influenced not only by ‘flavor and aroma’, but also by ‘texture’, which also showed a very strong positive correlation between them  and color scores, which can be explained by the preference for een them . Broas wentation ,84. Brieentation , while sbroas. Therefore, their absence can account for the different sensory characteristics of broas [broas [broas can also contribute to their characteristic aroma.Most of the volatiles commonly described as great contributors for other cereal breads and maize-based foods, such as 2-acetylpyrroline, 2-(methylimino)-3-butanone and 2,3-butanedione ,85, wereof broas , when coof broas ,26,36. Hs [broas . Furthers [broas . 2-Acetybroas. Other volatile compounds present in broas were esters, furans, furfurals and pyranones, derived from the bread making process (fermentation and baking). The differences in broas’ volatile composition were mainly due to lipid oxidation and browning reactions, which were influenced by soluble phenolic compounds. In particular, ferulic and p-coumaric acids and hydroxycinnamic acid amides inhibited lipid oxidation and fermentation reactions and promoted browning reactions. Broas less appreciated by consumers, especially the broa obtained from the commercial flour, showed higher lipid oxidation compounds, such as 2-octenal, hexanal and heptanal, and lower contents in pyranones, such as maltol and 3-hydroxy-2,3-dihydromaltol, in addition to lower phenolic compounds. Results showed that maize flours with higher levels in phenolic compounds give raise to broas with better sensory characteristics, especially related to ‘taste and aroma’. In conclusion, the selection of maize varieties richer in health-promoting phenolic compounds originate broas more appreciated by the consumers. These findings are relevant not only for the industry, contributing to the selection of the best maize varieties for higher-quality bread making, but also for maize breeders, who can add these quality traits to routine breeding of maize for broas’ production. These results sustain the previous knowledge of producers regarding the better-quality aspects of traditional maize varieties for broas’ production, and can contribute to the valorization of these varieties, which should be preserved as a safeguard against an unpredictable future.The main volatile compounds present in maize flours were aldehydes and alcohols derived from lipid oxidation reactions, which were present in higher amounts in the commercial maize variety. Ketones from carotenoids’ oxidation, such as α-ionone and geranylacetone, were present in higher amounts in yellow maize varieties. The presence of higher lipid and carotenoids’ oxidation volatiles in maize flours directly contributed to higher amounts of similar volatiles in


It has been suggested that pulmonary arterial hypertension (PAH) could be a potential sequela of coronavirus disease 2019 (COVID-19) in particular in those with hypertension; however, development of PAH after the course of COVID-19 in normotensive individuals are rarely reported. Here, we report a patient who developed PAH two months post-COVID-19. The patient was a 55-year-old female and normotensive, tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), developed mild respiratory distress syndrome and necessitated continuous positive airway pressure during the treatment in the hospital. After two months discharged from the hospital with RT-PCR negative for SARS-CoV-2, the patient presented with exertional dyspnea, dry cough, fatigue and episodes of syncope during exertion. Based on clinical presentation, electrocardiography, computed tomography, and transthoracic echocardiography assessment, PAH diagnosis was made. To our knowledge, this is a rare PAH case and this highlights the possible of PAH as sequala that might present in post COVID-19 patients. COVID-1Pulmonary arterial hypertension (PAH) is a chronic fatal disease that requires immediate and proper medical treatment since it could cause cardiopulmonary collapse ,10. Hear2A 55-year-old female, with no previous PAH comorbidities, initially presented with fever, shortness of breath, dry cough, chest pain, nausea, and generalized body aches for seven days. On presentation in the hospital, the blood pressure was 100/70 mmHg, pulse was 105 bpm, respiratory rate of 22/min, and oxygen saturation was 77%. On examination, there were bilateral coarse crackles in both lower lung fields. Based on the history and examination findings, a COVID-19 was suspected. The patient was admitted to the COVID-19 isolation unit and was started on oxygen by a simple face mask (5 L). The patient was started on intravenous (IV) azithromycin (500 mg/day) and ceftriaxone (75 mg/kg/day IV) along with enoxaparin sodium (40 mg for each 12 hours subcutaneously), dexamethasone (6 mg/day IV), vitamin C and Zinc. The baseline lab testing and a chest X-ray (CXR) were ordered. Moreover, a nasopharyngeal swab was collected for SARS-CoV-2 RT-PCR.3 with polymorphonuclear neutrophils 90% and lymphocyte 7%, LDH 900U/L, serum ferritin 1100 ng/mL, CRP 12 mg/L, PT 14 sec, INR 1.1, aPTT 32 sec, ALT 57 U/L, AST 78 U/L, serum bilirubin total 2 mg/dl, serum creatinine 1.1 mg/dL, and blood urea 40 mg/dL. Her CXR demonstrated bilateral infiltrates. RT-PCR result confirmed as SARS-CoV-2-positive. Arterial blood gas analysis was consistent with hypoxemia and respiratory alkalosis . Over the course of treatment, the patient developed mild respiratory distress syndrome (PaO2/FiO2 250) and necessitated continuous positive airway pressure (CPAP). The patient remained in the COVID-19 isolation unit for 18 days. On discharge, patient had improved clinically with oxygen saturation was 92% without oxygen and SARS-CoV-2 RT-PCR was negative.Her initial lab reports showed hemoglobin 11 g/dL, WBCs 17000/mm2O; left parasternal heave, and wide splitting of the second heart sound was noted. All the relevant blood tests were ordered along with a CXR, electrocardiography (ECG), and a SARS-CoV-2 RT-PCR. The ECG suggested right axis deviation, and the blood test results are presented in Two months after discharge, she visited the outpatient department with complaints of exertional dyspnea, dry cough, and fatigue for one month. She also reported an episode of exertional syncope. Her vitals revealed blood pressure of 140/90 mmHg, pulse rate 90 bpm, respiratory rate of 18/min, and oxygen saturation of 88% without any oxygen support. On examination, her jugular venous pressure raised 13 cm of HTo further investigate the cause of patient problem, transthoracic echocardiography was ordered which showed right ventricular systolic blood pressure (RVSP) 59 mmHg, tricuspid regurgitant jet velocity (TRV) 4.5 m/s, pulmonary artery diameter was 31 mm with normal left side heart parameters. Based on echocardiographic findings and clinical presentation of the patient, the diagnosis of PAH group 3 was made in accordance with the European Society of Cardiology (ESC) and European Respiratory Society (ERS). The patient was commenced on epoprostenol infusion , montelukast 10 mg/day, oxygen therapy, and spironolactone 100 mg/day. In addition, all routine vaccinations including pneumococcal and influenza vaccines offered to the patient, and a follow-up visit was scheduled with a multidisciplinary team consisting of a pulmonologist, pulmonary rehabilitation team, and a cardiologist. Partial improvement was recorded in the subsequent visit .3In this report, we aimed to highlight the possible relation of COVID-19 and PAH. Two months after recovering from COVID-19, the patient developed PAH with exertional dyspnea, dry cough, and fatigue as well as an episode of exertional syncope. One of the possible explanations is COVID-19 could cause pulmonary vasoconstriction that increased the pulmonary vascular resistance (PVR) . The patIt has been suggested that having history of hypertension increases the chances of PAH in patients with COVID-19 infection . TherefoBesides the above-mentioned limitation, PAH in case was diagnosed using a transthoracic echocardiography. The golden standard test to diagnose PAH is using right heart catheterization where PAH is defined with mean pulmonary artery pressure ≥25 mmHg, pulmonary artery wedge pressure <15 mmHg and pulmonary vascular resistance >3 units. However, transthoracic echocardiography results had enough parameters to suggested that the patient likely had PAH. Although direct association of SARS-CoV-2 infection and PAH is unable to be elucidated in this patient, this case highlights and informs the possible another sequala of COVID-19.4To our knowledge, this is a rare case of PAH that has not been previously reported. It is uncertain that either PAH is a risk factor of severe SARS-CoV-2 infection or vice versa. Further study is warranted to improve our understanding on the relationship between PAH and COVID-19 and therefore to be able to predict and to prevent PAH in COVID-19 patients.Written informed consent for publication was sought from the patient.This study received no external funding.AWK conceived and designed the study. AWK, IU, KSF, MJT were responsible for data collection and acquisition of data. AWK, IU, KSF, MJT, HH analyzed and/or interpreted the data. IU, HH wrote the initial manuscript. IU, HH critically revised the manuscript. All authors have read the final manuscript.The authors declare no conflicts of interest.


Epidemiological studies aimed at defining the association of thyroid diseases with extra-thyroidal malignancies (EM) have aroused considerable interest in the possibility of revealing common genetic and environmental factors underlying disease etiology and progression. Over the years, multiple lines of evidence indicated a significant relationship between thyroid carcinomas and other primary EM, especially breast cancer. For the latter, a prominent association was also found with benign thyroid diseases. In particular, a meta-analysis revealed an increased risk of breast cancer in patients with autoimmune thyroiditis, and our recent work demonstrated that the odds ratio (OR) for breast cancer was raised in both thyroid autoantibody-positive and -negative patients. However, the OR was significantly lower for thyroid autoantibody-positive patients compared to the negative ones. This is in agreement with findings showing that the development of thyroid autoimmunity in cancer patients receiving immunotherapy is associated with better outcome and supports clinical evidence that breast cancer patients with thyroid autoimmunity have longer disease-free interval and overall survival. These results seem to suggest that factors other than oncologic treatments may play a role in the initiation and progression of a second primary malignancy. The molecular links between thyroid autoimmunity and breast cancer remain, however, unidentified, and different hypotheses have been proposed. Here, we will review the epidemiological, clinical, and experimental data relating thyroid diseases and breast cancer, as well as the possible hormonal and molecular mechanisms underlying such associations. Some of them indicated a 30–42% increased risk of primary EM following 131I exposure, but others did not recognize such correlation. Similarly, studies aimed at clarifying whether anticancer treatments of EM, in particular external beam radiations, may cause subsequent primary thyroid cancers have produced conflicting results. On the other hand, a significant association between benign TD and BC was also shown to occur, which seems to suggest that factors other than oncologic treatment may play a role in the initiation and progression of second malignancies [The association of either benign or malignant thyroid diseases (TD) with extra-thyroidal malignancies (EM) has been highlighted in several epidemiological studies, and whether a causal relationship exists between them has been a matter of debate over the last decades. Primarily, such connections have generated much interest around the possibility of identifying common genetic and environmental factors responsible for the etiology and progression of these diseases. In particular, a number of different reports have described the association between thyroid cancers and other primary EM, including breast cancer (BC) ,2,3,4. Tgnancies ,6,7,8. IIn the present manuscript, we will review the epidemiological, experimental, and clinical evidence relating benign TD and BC, as well as the potential molecular mechanisms underlying their associations. In particular, approaching the possible role(s) of thyroid gland dysfunctions in promoting breast cancer, we will take into consideration the effects of deregulated thyrotropin (TSH), thyroid hormone (TH), and prolactin (PRL) levels, as well as the effects of thyroid-specific autoantibodies on BC progression. 3) plasma levels in early and advanced BC patients compared to control women [4) treatment did not differ between control women and BC patients, but the latter had a higher TSH mean value and a lower T3 mean value, despite no variations being observed in T4 levels [4 peripheral conversion [The first clinical evidence of TD and BC association was reported in 1896, when Beatson described the beneficial effects of bilateral oophorectomy and thyroid extract administration in metastatic BC patients ,13,14,15ol women ,39,40. Aol women . Howeverol women ,43. Inteol women . Actuall4 levels . Such a nversion . In the nversion . In thisnversion . In 1976nversion . These anversion .In conclusion, from all the information acquired so far, it appears that alterations of the hypothalamus–pituitary–thyroid axis could affect BC progression in several ways, as schematically depicted in During pregnancy, increased estrogen levels stimulate lactotroph cell proliferation, leading to increased PRL secretion by the pituitary. The released hormone, along with estradiol, progesterone, placental lactogen, insulin, and cortisol, stimulates mammary gland (MG) growth. In this period, the ability of PRL to induce milk synthesis (lactogenic effect) is inhibited by the high plasma concentration of estrogen. After childbirth, the estrogen level falls, allowing the lactogenic effect of PRL to take place. During lactation, short-term peaks in PRL secretion occur after nipple stimulation by the suckling infant through a neuro-humoral axis ,48. At tDeregulated thyroid functions could affect PRL action in promoting BC by at least two different ways. The first one, as mentioned above, implies TRH overstimulation of pituitary PRL secretion in hypothyroidism conditions ,31,32,33Besides its well-known and characterized role in the regulation of thyroid gland development and thyrocytes differentiation and proliferation, TSH is thought to modulate the function of several extrathyroidal tissues. In fact, a growing number of experimental findings evidenced the expression of TSH receptor (TSH-R) in several non-thyroid cells, including murine and human normal and BC tissues ,65,66,674) to reduce or suppress serum TSH levels in patients at low or high risk for DTC recurrences, respectively [In thyrocytes, upon ligand binding, TSH-R interacts with Gs proteins leading to the stimulation of adenylate cyclase (cAMP) formation and protein kinase A (PKA) activation. It also interacts with Gq proteins, resulting in phospholipase C (PLC) stimulation, intracellular Ca++ influx, and increased protein kinase C (PKC) activity. In addition, TSH-R can signal through alternate pathways including p38, p42/44 MAPK, and phosphoinositide 3-kinase (PI3K) . Howeverectively . All togectively . In thesectively . Govindaraj and colleagues observed the expression of TSH-R mRNA and protein in normal and cancer breast cells, which was significantly higher in tumor tissues compared to normal breast tissues . The obsTo date, a major gap in this knowledge is represented by the lack of in vitro studies on the function(s) of TSH-R in BC cells, which could shed light on the signal transduction pathways actually involved in the proliferative stimulus in this cell type.4) and 3,5,3′-L-triiodothyronine (T3). Once secreted into the blood, THs are carried by three major transport proteins: thyroxine-binding globulin (TBG), thyroxine-binding prealbumin (TBPA or transthyretin), and albumin. The thyroid gland produces about 100 nmol of T4 and 5 nmol of T3 every day to maintain a serum total T4 concentration of about 103 nmol/L and a total T3 concentration of about 1.8 nmol/L [4 (about 19 pmol/L) and 0.4% of total T3 (about 4.3 pmol/L) circulate in free form and are responsible for the hormonal effects on target tissues [4 is deiodinated by deiodinases 1 (D1) or 2 (D2) to form T3, which binds TH nuclear receptor (THR) with greater affinity compared to T4 [3 [3-mediated transcriptional regulation [3 [Thyroid hormones are major regulators of growth and development as well as of a number of homeostatic functions in adults, including energy and heat production . Thyroid8 nmol/L . Only 0. tissues . The fre tissues ,82. The  tissues . Once ined to T4 . THRs beed to T4 ,85. Two ed to T4 ,87,88. Fto T4 [3 ,90. Compgulation ,90. The 4 and T3 may attach to specific regions present in the integrin αvβ3 [3 at physiological serum concentrations and leads to the intracellular activation of the PI3K, while S2 binds T4 and, to a lesser extent, T3, inducing the intracellular activation of extracellular signal-regulated kinases (ERK) 1 and 2 [The second mechanism of THs action, by which THs may elicit rapid cellular responses, is initiated at the plasma membrane, where both Trin αvβ3 ,82,91. I 1 and 2 ,82,92. 3 have been identified in the mammary gland and are thought to modulate, upon ligand attachment, ductal branching, alveolar budding, and lobules enlargement [THs have been suggested to play a role, along with other hormones  in normal breast growth and development ,94. In pargement ,96,97,98argement ,100.4 maximally stimulates αvβ3 at physiological free-T4 concentrations, while supraphysiological free-T3 concentrations are required to induce cell proliferation via this receptor [4 and replacement by T3) extended patient’s survival [The relevance of ERs overexpression during BC progression is well recognized and is such that the most commonly used drugs, i.e., tamoxifen, fulvestrant, and aromatase inhibitors, are aimed at reducing estrogen levels or blocking ER signaling ,102. Thereceptor ,142,143.survival ,144. ThiNonetheless, it should be taken into account that, while αvβ3 receptors are thought to mediate most of the tumor-promoting effects of THs in BC cells, nuclear THRs appear to play oncosuppressive functions in BC as well as in other solid tumors . The expIn conclusion, the imbalance of expression and/or activation between membrane and nuclear TH receptors may have detrimental consequences on BC progression. Studies aimed at defining the association between BC and benign TD, in particular AITD, have produced conflicting results causing a long-lasting debate ,163,164.The impact of thyroid axis dysfunctions on BC progression has been a matter of debate for more than a century, and still today many controversies exist. The available information strongly suggests that TD may affect BC progression in several ways, through (i) altered plasma levels of TSH and THs or production of specific thyroid autoantibodies; (ii) dysregulation of PRL secretion due to hypothyroidism; (iii) alterations in THs responsiveness of BC cells. Thus, different hormonal and molecular players should be taken into consideration in every single patient, when analyzing the association between TD and BC. This knowledge will likely shed light on the potential pathogenic links between TD and BC, possibly allowing a more personalized clinical management of these patients.