Insulin, besides its pivotal role in energy metabolism, may also modulate neuronal processes through acting on insulin receptors (InsRs) expressed by neurons of both the central and the peripheral nervous system. Recently, the distribution and functional significance of InsRs localized on a subset of multifunctional primary sensory neurons (PSNs) have been revealed. Systematic investigations into the cellular electrophysiology, neurochemistry and morphological traits of InsR-expressing PSNs indicated complex functional interactions among specific ion channels, proteins and neuropeptides localized in these neurons. Quantitative immunohistochemical studies have revealed disparate localization of the InsRs in somatic and visceral PSNs with a dominance of InsR-positive neurons innervating visceral organs. These findings suggested that visceral spinal PSNs involved in nociceptive and inflammatory processes are more prone to the modulatory effects of insulin than somatic PSNs. Co-localization of the InsR and transient receptor potential vanilloid 1 (TRPV1) receptor with vasoactive neuropeptides calcitonin gene-related peptide and substance P bears of crucial importance in the pathogenesis of inflammatory pathologies affecting visceral organs, such as the pancreas and the urinary bladder. Recent studies have also revealed significant novel aspects of the neurotrophic propensities of insulin with respect to axonal growth, development and regeneration. Insulin is a major hormone regulating the energy metabolism of the organism. Besides its endocrine effects, insulin may modulate distinct neural functions through insulin receptors (InsRs) expressed in neurons of the central and peripheral nervous system. Neuronal localizations of insulin and InsRs have been first described in the central nervous system mostly in areas involved in the regulation of energy balance and food intake . Later sInsR, as a member of the receptor tyrosine kinase family, has a heterotetrameric structure with two extracellular α and two transmembrane glycoprotein β subunits which contain the tyrosine kinase domain. Insulin activates the InsR by binding to the α subunit of the receptor which induces the phosphorylation of tyrosine residues of intracellular proteins such as the insulin receptor substrate (IRS) proteins ,8. PhospIn recent years, electrophysiological, immunohistochemical and functional studies demonstrated the localization and functional significance of InsRs in primary sensory neurons (PSNs), in particular nociceptive PSNs expressing the transient receptor potential vanilloid type 1 (TRPV1) receptor ,23,24,25Nociceptive PSNs transmit impulses evoked by noxious mechanical, thermal and chemical stimuli . MorpholBandeiraea simplicifolia [Immunohistochemical studies have revealed that capsaicin-sensitive PSNs can be divided into two major categories: peptidergic and non-peptidergic neurons. These subpopulations of PSNs have different trophic factor sensitivities and neurochemical characteristics. TRPV1 receptor-expressing peptidergic PSNs contain CGRP, SP, vasoactive intestinal polypeptide, galanin and neurokinin A ,50,60,61icifolia ,69. Besiicifolia ,71,72. Aicifolia ,23,40,73Characterization of the chemical phenotypes of PSNs have revealed the sensitivity to insulin and the localization of InsRs in a sizeable subpopulation of neurons amounting to about 60 per cent of spinal PSNs of unidentified target identity ,22,23 FuEarly light and electron microscopic studies demonstrated the presence of InsR immunoreactivity in a population of small dorsal root ganglion (DRG) neurons and in Rexed’s laminae V and X of the spinal cord . At the Later, the expression pattern of InsR and the co-localization the InsR with the TRPV1 receptor, CGRP and IB4 have also been revealed in cultured rat DRG neurons. It has been demonstrated that about half of the DRG neurons showed InsR-immunopositivity and about 60%, 50% and 30% of InsR-expressing sensory neurons exhibited the TRPV1 receptor, CGRP and the IB4-binding glycoprotein, respectively . FurtherThe neurochemical phenotypes of InsR-expressing PSNs innervating different organs have also been revealed. It has been demonstrated that about one-quarter of somatic (cutaneous and muscle) and about half of visceral (urinary bladder and pancreatic) PSNs express the InsR . In addiThe phenotypes of pancreatic spinal and vagal PSNs have been analyzed in more detail. It has been demonstrated that about 50% of DRG and nodose ganglia (NG) neurons innervating the rat pancreas express the InsR . It has In summary, these quantitative immunohistochemical studies have revealed the disparate localization of InsRs in somatic and visceral PSNs with dominance of neurons innervating visceral organs. This suggests that visceral PSNs involved in nociceptive and inflammatory processes may be more prone to the modulatory effects of insulin than somatic PSNs. Additionally, these studies have demonstrated that majority of InsR-expressing PSNs also display TRPV1 receptor immunoreactivity. Importantly, these observations have revealed that the pancreas is innervated by a meshwork of InsR-expressing spinal and vagal PSNs which contain sensory neuropeptides and express the TRPV1 receptor. These morphometric data implicate that an interaction among insulin, InsR, TRPV1 receptor and sensory neuropeptides may play an important role in the development of pancreatic pathologies.2+ or Na+ channels; hence, cobalt accumulation specifically indicates changes in the conductance of plasma membrane TRPV1 channels upon agonist-induced activation [per se indicating the activation of the TRPV1 receptor. The insulin-induced cobalt accumulation was reduced by administration of both competitive and non-competitive antagonists of the TRPV1 receptor, capsazepine and ruthenium red, respectively, further suggesting the involvement of TRPV1 receptor in the action of insulin. Whole-cell voltage clamp experiments disclosed that acute administration of insulin induced inward currents in subsets of cultured PSNs of both the rat (18%) and the mouse (9%) in the concentration range of 300 nM-10 µM [Studies on cultured adult rat DRG neurons have revealed that insulin can sensitize the TRPV1 receptor toward the effect of the selective agonist capsaicin. It has been demonstrated that administration of insulin enhances the accumulation of cobalt ions in capsaicin-stimulated neurons . The cobtivation . The sentivation . FurthernM-10 µM ,79. All nM-10 µM . The senFurthermore, increased expression of TRPV1 receptor protein has been demonstrated following long-term administration of IGF-1 and insulin in TRPV1 receptor-expressing neuroblastoma cells. In addition, it has been disclosed that expression of TRPV1 receptor protein is decreased by inhibiting the MAPK and PI3K cascades. These findings suggested a crucial role of MAPK and PI3K signaling pathways in the regulation of the TRPV1 receptor by IGF-1 and insulin . In accoSimilarly, acute administration of NGF has been shown to induce translocation of intracellular TRPV1 receptors into the plasma membrane, underlying its sensitizing effect on the TRPV1 channel . It is wStudies utilizing calcium imaging demonstrated the activation of a subset of mouse NG neurons by insulin in a dose-dependent manner . All neuIt has been demonstrated that a TRPV1 receptor antagonist, capsazepine decreases the expression level of neurokinin-1 receptor, the receptor of SP, the pancreatic enzymes associated with pancreatitis, and the severity of histological changes in caerulein-induced experimental pancreatitis in mice. These findings suggested the importance of the TRPV1 receptor expressing SP-immunopositive fibers in pancreatic pathologies . FurtherThe TRPV1 receptor is also expressed in the endocrine pancreas, and it has also been demonstrated that activation of the TRPV1 receptor can modulate the glucose-induced insulin secretion of pancreatic islet β cells ,88. The Recent work has indicated that the TRPV1 receptor may play an important modulatory role in glucose-induced insulin secretion as well. It has been demonstrated that TRPV1 receptor knock out mice show significantly higher blood glucose levels, and a higher decrease in glucose induced insulin secretion compared with wild type mice . AdditioTaken together, these studies have demonstrated that insulin may potentiate the release of inflammatory neuropeptides by sensory nerves innervating the islet apparatus and the exocrine pancreas through direct and/or indirect modulation of TRPV1 receptors ,101,102.The role of capsaicin-sensitive sensory nerves in the function and pathologies of the urinary bladder is well established. Functional morphological studies have shown that a high proportion of sensory nerves which innervate the urinary bladder are mostly unmyelinated and capsaicin-sensitive ,103. ActOver the past few years, many reports have indicated that nociceptive sensory neurons are involved in the development of diabetic peripheral neuropathy and diabetic visceral hypo- or hypersensitivity ,119,120.Recent work using immunohistochemistry and western blot analysis has revealed that expression of TRPV1 receptor and CGRP decreased in spinal cord dorsal horn of in rats with streptozotocin (STZ) diabetes. It has also been demonstrated that expression of these markers were also reduced in peripheral nerves . In addiAdditionally, in the past decade, several lines of evidence have suggested that insulin has a direct role on PSNs and insulin signaling may be involved in the development of diabetic peripheral neuropathy ,126,127.It has also been revealed that insulin infusion causes a decrease in CGRP-mediated sensory neurogenic vasodilatation in rats . FurtherIn summary, these studies suggested a significant role of the TRPV1 receptor in the development of diabetic peripheral neuropathy and visceral hypo- or hypersensitivity. Although some of these experiments suggested that impairments in neuronal insulin signaling may contribute to the development of diabetic neuropathy, the role of interactions among insulin, InsRs and the TRPV1 receptors in these processes have not yet been evaluated in detail.Early studies have revealed that insulin has a potent neurite outgrowth-promoting effect on cultured adult rat sensory and sympathetic neurons . FurtherRecent observations confirmed and extended the findings of previous reports on the neurite growth promoting effect of insulin. It has been revealed that insulin modulated neurite outgrowth in a concentration dependent manner. In cultured DRG neurons insulin administered at low (nM) concentrations elicited a growth promoting effect, whereas at micromolar concentrations an inhibition of axon growth was observed . ImportaThe exact molecular background of insulin’s neurite outgrowth promoting effect has not yet been evaluated in detail. However, in the light of previous observations, it could be hypothesized that the interaction between insulin and the InsR by activating the PI3-Akt pathway ,141, thaAlthough, these studies suggested a crucial role of the InsR in neurite growth, it has also been demonstrated that the InsR forms a molecular complex with the TrkA in PC12 cells which show similarities with nociceptive sensory neurons . FurtherImportantly, in vivo studies have revealed that intrathecal  or systeIn vivo experiments have also indicated an increased insulin sensitivity of peptidergic PSNs. Thus, it has been revealed that administration of intrathecal insulin increased the expression of CGRP in regenerating nerve fibers and DRG neurons after nerve injury . AdditioSeveral reports have demonstrated that the density of CGRP-expressing perivascular sensory fibers and the CGRP content of DRGs decreased in mice with experimental insulin resistance ,118,119.Besides its fundamental significance in energy metabolism, a significant role of insulin in modulation of neural functions has emerged. InsRs are expressed mostly on nociceptive TRPV1 receptor expressing PSNs innervating somatic and visceral organs with predominance in the latter. InsRs are involved in mechanisms of organ pathologies, in particular inflammatory processes of both the exocrine and endocrine pancreas. Experimental evidence indicates a pivotal role of TRPV1 receptor expressing PSNs in the pathomechanism of acute pancreatitis. Pancreatic TRPV1 receptor- and InsR-expressing peptidergic afferent nerves are also implicated in the mechanism of insulitis and islet dysfunction contributing to the development of type 1 diabetes mellitus.


Protein synthesis is a highly complex process executed by well-organized translation machinery. Ribosomes, tRNAs and mRNAs are the principal components of this machinery whereas RNA binding proteins and ribosome interacting partners act as accessory factors. Angiogenin (ANG)—Ribonuclease inhibitor (RNH1) system is one such accessory part of the translation machinery that came into focus afresh due to its unconventional role in the translation. ANG is conventionally known for its ability to induce blood vessel formation and RNH1 as a “sentry” to protect RNAs from extracellular RNases. However, recent studies suggest them to be important in translation regulation. During cell homeostasis, ANG in the nucleus promotes rRNA transcription. While under stress, ANG translocates to the cytosol and cleaves tRNA into fragments which inhibit ribosome biogenesis and protein synthesis. RNH1, which intimately interacts with ANG to inhibit its ribonucleolytic activity, can also bind to the 40S ribosomes and control translation by yet to be known mechanisms. Here, we review recent advancement in the knowledge of translation regulation by the ANG-RNH1 system. We also gather information about this system in cell homeostasis as well as in pathological conditions such as cancer and ribosomopathies. Additionally, we discuss the future research directions and therapeutic potential of this system. Protein synthesis is an energy-expensive process, and the proteome of a cell is extremely dynamic. Every cell has to adjust its proteome according to the multiple endogenous and exogenous signals, including nutrients and stress. Thus, each cell-type has a different repertoire of proteins due to its diverse internal and external environment. Interestingly, the protein pool of a cell also changes with the phase of the organismal development and stage of a cell-cycle . For exaThe process of translation has been extensively studied using lower model organisms because of their genetic simplicity, amenability to genetic manipulations and conserved translation components. Interestingly, during evolution, from bacteria to mammals, a cell has minimally evolved its core translational machinery, however, the regulatory mechanisms to control protein translation are increasingly advanced . These iGata1 mRNA to regulate erythropoiesis [Historically, ribosomes have been viewed as a conserved molecular machine of fixed intrinsic components that uniformly translate given genetic code. However, recent studies suggest plasticity of ribosomal machinery with dynamic molecular composition, distinct properties and preference over mRNAs . Such riopoiesis . Furtheropoiesis , and sucopoiesis . Thus, tRibonucleases have been of interest for about a century in the fields of protein folding and function, molecular evolution, biochemistry and biomedical research. ANG, also known as RNase5, is a unique ribonuclease enzyme belonging to the Ribonuclease A superfamily, blessed with both angiogenic as well as weak ribonucleolytic activity. ANG is notable for its role in neovascularization, as a potent inducer of blood vessel growth . In contRNH1 is ubiquitously expressed in the human tissues and detected in cytoplasm, nucleus, mitochondria as well as on endoplasmic reticulum . It is oRNH1 is evolved in amniotes via gene duplication and conserved among mammals. For over a half-century, RNH1 has been extensively studied for its structure and functions using in vitro cell cultures, but no attempt was made to decipher in vivo functions of it. Our group was the first one to generate RNH1 knockout mice. Surprisingly, we found that complete RNH1 deletion is embryonically lethal and mice died in utero during embryonic stage E10 due to anemia . Thus, tRibonucleases (RNases) are ubiquitously expressed enzymes which are not only important as a mechanism of defense against pathogens but also the regulators of gene expression, cell survival, growth and differentiation. ANG is an exceptional RNase as it is expressed only in vertebrates, possesses weak ribonuclease activity and remarkable angiogenic potential. ANG has been widely studied in perspective of angiogenesis and implicated in the establishment, growth, and metastasis of tumors ,26. AparANG is actively secreted by multiple cell-types and organs of the human body. Once secreted, it enters neighboring cells through receptor mediated endocytosis and in the nucleus through its nuclear localization signal, RRRGL. Nuclear ANG subsequently accumulates in the nucleolus , where iProtein synthesis is an energy consuming process and eukaryotic cells need to save energy in response to environmental stress. Thus, cells under chronic stress activate integrated stress response programs which rewire transcription and translation to express genes required for the cell survival. One of the key regulatory mechanisms under stress is inhibition of global protein synthesis. One of the ways it is achieved is by tRNA hydrolysis. ANG in response to various stresses cleave mature cytoplasmic tRNAs within their anticodon loops to generate small RNAs called tRNA-derived stress-induced RNAs (tiRNAs) or tRNA halves (tRHs)  Figure B. TransfOverall, the above discussed translational regulatory mechanisms of ANG indicate the dual ability of ANG to promote as well as repress the global protein synthesis. Thus, ANG has emerged as a double-edged sword of translational regulation.Higher vertebrate specific expression of RNH1 forced molecular biologists to rely on in vitro cell culture experiments to study RNH1 functions, as genetically amenable lower model organisms lack RNH1 gene. Thus, the majority of studies focused on ribonuclease inhibitor activity of RNH1 based on its binding with nucleases or claimed it as an oxidation sensor based on its cysteine residues. The first strong in vivo evidence of RNH1 mediated translation regulation came from RNH1-knockout mice (RNH1-KO) . RNH1-KOMicroRNAs (miRNAs) are small non-coding RNAs which are important post-transcriptional regulators. They regulate the expression of genes by interacting with 3′UTR of target mRNAs to destabilize them or/and inhibiting their translation . miRNAs Another important target of RNH1 mediated miRNA control is the Mechanistic target of rapamycin (mTOR). RNH1 has been shown to regulate mRNA levels of mTOR via miRNAs in tamoxifen-resistant breast cancer cell-line MCF-7R . RNH1 enTranslation initiation and elongation are tightly controlled steps of protein synthesis that rapidly get blocked in response to diverse cellular stresses, such as Endoplasmic reticulum (ER) stress caused by misfolded proteins. These untranslated and translationally stalled mRNAs along with associated proteins, then aggregate in the cytoplasm as a specialized, membrane-less, transient structure called stress granules (SGs) . SG possTranslation attenuation is also important when unfolded or misfolded proteins accumulate in the ER lumen which leads to ER stress and activation of the unfolded protein response (UPR). This UPR reprogram the gene transcription and translation such that it restores protein homeostasis. One of the important ER transmembrane protein sensors is Inositol-requiring enzyme-1(IRE1) which is endoribonuclease that oligomerizes and autophosphorylates under ER stress. Interestingly, on the surface of ER, RNH1 inhibits endoribonuclease activity of IRE1 by direct physical interaction . IRE1 isIn summary, ANG and RNH1, altogether, interact with all the principal components of translation machinery  and regulate their activity to control translation.Controlled protein translation is extremely important for cell homeostasis and organismal physiology. Mutations in ribosomal protein genes, RIPs or RBP genes leads to disbalance in the ribosome levels and perturb translation causing various ribosomopathies. On the contrary, excess production of rRNAs or activation of positive modulators of translation trigger uncontrolled cell proliferation and cancer. As the ANG-RNH1 system is capable of activation as well as repression of translation, perturbation in this system can cause multiple disorders related to translation.Neurons are functional units of a brain which form a dense network along with astrocytes that physically and metabolically support neurons. These cells often undergo various genetic, molecular and oxidative stresses during their life. To survive under such stresses, these cells have developed specialized mechanisms. One such key mechanism is translation repression by tRNA cleavage. However, excessive tRNA cleavage is also deleterious to the cell. Thus, neuroepithelial progenitors in the developing brain have devised a mechanism to protect their tRNAs from aberrant and excessive ANG mediated tRNA cleavage. NSUN2 is methyltransferase that methylate tRNAs to reduce their affinity with ANG. NSUN2 is essential for brain development and loss-of-function mutations in the NSUN2 gene cause neurodevelopmental disorders including microcephaly, motor deficits and growth retardation in humans and mice . In the G93A, the common genetic determinant of ALS, increases their lifespan and motoneuron survival [ANG has been implicated in pathophysiology of several neurodegenerative disorders, including Parkinson’s disease (PD) , amyotrosurvival . Apart fsurvival . In summHematopoietic system (HS) is conceived and then maintained by the lifelong process of hematopoiesis, where hematopoietic stem cells (HSCs) proliferate and differentiate in a strict hierarchical manner to produce cells of the entire blood lineage. HSCs possess quiescence as a defining characteristic, while their progeny shows dramatic proliferative ability with extensive differentiation capacity . HSCs maAngiogenesis is a process that induces new capillary blood vessels formation and is central to the tumor growth and progression of cancer. ANG is a key angiogenic factor in cancer. It contributes to the tumor pathology by not only activating cells to induce neovascularization, but also promoting cell survival, proliferation, growth and/or migration of tumor cells. Elevated levels of ANG have been detected in a number of cancer cell lines and tumor tissues including hematopoietic, leukemic cells and the plasma of Acute Myeloid Leukemia (AML) and advanced myelodysplastic syndromes (MDS) patients . HoweverFor the past three decades significant efforts were made to understand the structure, biochemical properties and function of the ANG-RNH1 system. Although, multiple roles of the ANG-RNH1 system have been elucidated in a great detail, yet the functional relevance of this system in cell homeostasis and pathophysiology of diseases is poorly understood. Majority of information about the functions of ANG-RNH1 system is obtained from in vitro cell-culture studies. Thus, many questions still remain unanswered regarding the exact in vivo biological functions of ANG and RNH1 as a complex or individually. For example, does the ANG-RNH1 complex itself have any biological function? What is the role of ANG-RNH1 as a complex in translation? and what are the molecular mechanisms of RNH1 and ANG mediated specific translation? Since complete deletion of RNH1 in mice is lethal, we still do not completely know what the in vivo role of RNH1 in adult mice is. Further studies along with employment of new advanced technologies must be implemented to answer above questions and to harvest full therapeutic potential of ANG-RNH1 system in cancer, ribosomopathies, neurodevelopmental and neurodegenerative disorders.


Specific and adequate nutrition during pregnancy and early life is an important factor in avoiding non-communicable diseases such as obesity, type 2 diabetes, cardiovascular disease, cancers, and chronic allergic diseases. Although epidemiologic and experimental studies have shown that nutrition is important at all stages of life, it is especially important in prenatal and the first few years of life. During the last decade, there has been a growing interest in the potential role of epigenetic mechanisms in the increasing health problems associated with allergic disease. Epigenetics involves several mechanisms including DNA methylation, histone modifications, and microRNAs which can modify the expression of genes. In this study, we focus on the effects of maternal nutrition during pregnancy, the effects of the bioactive components in human and bovine milk, and the environmental factors that can affect early life , and which contribute to the epigenetic mechanisms underlying the persistent programming of immune functions and allergic diseases. This knowledge will help to improve approaches to nutrition in early life and help prevent allergies in the future. There is increasing evidence to suggest that maternal diet during pregnancy, breastfeeding, early life nutrition, and early life malnutrition can have sustained effects on immunological outcomes, such as respiratory allergies, and metabolic outcomes such as type 2 diabetes and obesity. Nutritional programming during gestation might permanently affect the immunological competence and nutritional status in early life in utero by gestational week 28 (Epigenetic mechanisms may play an important role in these effects. It has even been suggested that early life nutrition forms the basis for susceptibility to a plethora of chronic age-related non-communicable diseases (NCD), like respiratory allergies –9. Thus, week 28 . Increas week 28 –14. In t week 28 . Breastf week 28 . Besides week 28 . In anal week 28 –20. Stud week 28 . In addi week 28 –29. EpigEpigenetic mechanisms  and tranBox 1Epigenetics refers to systems that control gene expression in a heritable fashion without changing the genomic sequences. The epigenome is much more flexible than the genome and shows different phenotype variations that are influenced by environmental factors and dietary habits. Epigenetic mechanisms include DNA methylation, histone modifications, and RNA interference by microRNAs (miRNAs) (See in this Box figure). Epigenetic mechanisms thus contribute to the regulation of gene expression at the level of transcription by DNA methylation and by modifying chromatin accessibility through posttranslational modifications of histones, and after transcription by mRNA silencing. These epigenetic mechanisms can regulate gene expression by modifying the accessibility of the DNA to transcription enzymes without altering the DNA nucleotide sequence, influencing stability of mRNA or translation efficiency, and others –32. The Epigenetic changes have been strongly associated with allergies and asthma and might thereby serve as biomarkers. The role of epigenetic mechanisms, particularly DNA methylation, in allergic diseases is at the interface of gene regulation, environmental stimuli, and developmental processes, thereby determining the pathogenesis of asthma and allergy. Alterations of the DNA methylation status in the genes specific for a different subset of T helper (Th) cells that are considered to be a good example of how epigenetic modulation can influence the development of asthma and other allergic diseases.The differentiation of naïve CD4+ T cells into Th subpopulations is strictly regulated, with changes in epigenetic marks at main lineage-determining loci encoding transcription factors like GATA3, RORγt, TBX21, and Foxp3 playing a pivotal role. These changes affect the differentiation into mature Th subpopulations, such as Th1, Th2 (and Th9), regulatory T cells (Treg cells), and Th17 , 47, 48.In addition to DNA methylation, histone modifications are also important in guiding T-cell differentiation. T-bet and GATA3 transcription factors control lineage-specific histone acetylation of IFN-γ and IL-4 loci during Th1/Th2 differentiation. Rapid methylation of H3K9 and H3K27 residues (repressive marks) at the IFN-γ locus was associated with differentiating toward Th1 cells, while demethylation of H3K9 and methylation of H3K27 was associated with Th2 differentiation . EpithelDemethylation of the IL-4 promoter leads to allergic sensitization . Th1 difBox 2The differentiation of naïve CD4+ T cells upon antigen exposure into effector T helper (Th) subsets  or induced regulatory T (iTreg) cells relies on epigenetic regulation and the establishment of cell-fate programs , 41. DNAThe WHO recommends exclusive breastfeeding for infants during the first 6 months of life, and that it should be given alongside complementary feeding up until children are 2 years old . If mothThere is increasing evidence to suggest that the maternal diet during pregnancy and breastfeeding can have sustained effects on immunological outcomes in the infant and even have ramifications for their health later in life. The maternal diet can modify some immune supporting micronutrients in breast milk, such as the fat-soluble vitamins A and D, as well as the water-soluble B vitamins, and polyunsaturated fatty acids (PUFA), but maternal diet does not influence other components such as iron and zinc . AlthougLong-chain PUFA (LCPUFA) induce inflammation by modulating inflammatory mediators like prostaglandins and immunomodulatory factors like IL-10 and TSLP . ConsumpLactobacillus, Bifidobacterium, or Escherichia species, which are commonly found in a normal microbiota. Prebiotics are mostly dietary fibers that are non-digestible food ingredients and beneficially affect the host's health by selectively stimulating the growth and/or activity of some genera of microorganisms in the colon, generally lactobacilli and bifidobacteria.Probiotics are living microorganisms which, when administered in adequate amounts, confer a health benefit to the host. They generally exist of Intestinal microbiota strongly influence the maturation of the immune system  and partper se  and fructo-oligosaccharides (FOS), and have widely been used in infant nutrition to mimic the bifidogenic- and SCFA-inducing effect of HMO. There is some evidence that prebiotic oligosaccharides in infant nutrition may prevent eczema in infants –112. It Both human milk and bovine milk contain many other bioactive components that can modulate immune function [reviewed in , 105–107Several epigenome wide-association studies on allergies have been performed, as reviewed elsewhere . These sAs already mentioned, the mechanisms underlying the anti-allergic effects of human milk are most probably complex, as human milk contains not only nutritional substances but also functional molecules including polysaccharides, cytokines, proteins, and other components forming a real biological system which can modulate and shape the innate and adaptive immune responses of the infant in very early life , 145. IfEpigenetic modulation of the Foxp3 gene by farm milk was demonstrated in an animal model. In this study, exposure to raw, unprocessed, cow's milk for 8 days, increased histone acetylation of Foxp3 in splenocyte-derived CD4+ T cells compared to processed milk exposure . In the There is evidence that the epigenome is affected by the farming environment. The first indication for a potential role of epigenetic regulation in the protective “farm effect” was provided by Slaats et al. who demonstrated that DNA methylation of the promoter region of CD14 in placentas of mothers living on a farm was lower compared to mothers not living on a farm . These lin vitro . miRNAs represent short noncoding RNA molecules that control 40–60% of the total gene expression by inducing mRNA degradation and/or post-transcriptional inhibition of translation. As a result, specific miRNA can silence selective gene expression. The expression of a single gene can be regulated by several miRNAs, and likewise, a single miRNA can regulate over 100 genes , 149. Thin vitro . Based oin vitro . In addiin vitro , and recigations , 160. Thin vivo evidence that the induction of SCFA by sialyllactose when ingested in bovine milk, but sialyllactose has been reported to induce SCFA production in in vitro fecal microbiota cultures  ligands, inducing IL-22 and antibacterial peptide production , SCFA caThis review summarizes current knowledge on the potential effects of human and bovine milk on neonatal immunity and epigenetic programming and its possible consequences on the development of allergies in early childhood and beyond see .Breast milk is the food of choice for newborns and infants. When breast milk is not sufficiently available, cow's milk based formula is the best alternative, and thus cow's milk has become an integral part of early life diet.Several epidemiological studies that have shown that exposure to a farm environment as well as to raw/unprocessed cow's milk in the prenatal period and early childhood is associated with protection against the development of asthma and other allergies later in life. Many cow's milk components have been shown to have similar effects on human immune cells as their breast milk counterparts.Some of the molecular pathways that may explain the association between the consumption of raw milk asthma and allergy may be linked to epigenetics. Epigenetic mechanisms like DNA methylation, but also histone modifications, and non-classical epigenetics represented by miRNA may all contribute to the effects induced by raw cow's milk.However, milk and dairy products are subject to industrial processing to ensure microbiological safety. As a result, milk proteins can be denatured, and lose their functional activity. In addition, glycation of milk proteins is thought to increase the risk of developing cow's milk allergy, illustrating that preserving milk proteins and preventing glycation may be important innovations to help prevent allergies.Based on what is currently known on immunological and epigenetic effects that can be exerted by human and different types of bovine milk, future research should focus on enhancing the functional  activity of milk components in early life nutrition, and on establishing epigenetic markers of immunological responses to milk. These could be especially important for diagnostic purposes and assessing the risk of developing CMA. Knowledge gathered during studies on the epigenetic effects of milk can be used in the future to drive the development of preventive or therapeutic anti-allergic strategies based on components that affect epigenetic mechanisms.Finally, the continuation of epidemiologic and mechanistic studies on the effects of the components of breast and bovine milk on human immune function and health will increase our knowledge and help in finding potential applications that may help prevent allergies in the neonatal period.All authors contributed to the writing of the manuscript.BE and JG are partly employed by Nutricia Research. RN is employed by FrieslandCampina. The remaining 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.


Much interest has been directed towards stem cells, both in basic and translational research, to understand basic stem cell biology and to develop new therapies for many disorders. In general, stem cells can be cultured with relative ease, however, most common culture methods for stem cells employ 2D techniques using plastic. These cultures do not well represent the stem cell niches in the body, which are delicate microenvironments composed of not only stem cells, but also supporting stromal cells, extracellular matrix, and growth factors. Therefore, researchers and clinicians have been seeking optimal stem cell preparations for basic research and clinical applications, and these might be attainable through 3D culture of stem cells. The 3D cultures recapitulate the in vivo cell-to-cell and cell-to-matrix interactions more effectively, and the cells in 3D cultures exhibit many unique and desirable characteristics. The culture of stem cells in 3D may employ various matrices or scaffolds, in addition to the cells, to support the complex structures. The goal of this Special Issue is to bring together recent research on 3D cultures of various stem cells to increase the basic understanding of stem cells and culture techniques, and also highlight stem cell preparations for possible novel therapeutic applications. Stem cells are cells that demonstrate the abilities to self-renew and differentiate. Many types of stem cells can be isolated from embryonic or adult tissues, varying in their potency from pluripotent to unipotent depending on the stem cell type. Many cells can be found throughout the human body, often localized in niches that provide a nurturing microenvironment for the stem cells while directing their proliferation and differentiation. Stem cells are typically cultured in 2D tissue culture plastic for ease and maximal expansion, as large numbers are often needed for translational research and therapies. The 2D cultures do not, however, represent the natural environment of stem cells in the body well. Many beneficial properties of stem cells might be lowered or even lost in 2D cultures. Therefore, the use of 3D culture techniques has become more common in basic and translational research. The 3D cultures often provide more complete cell-to-cell and cell-to-matrix interactions, mimicking the natural environment in which the stem cells reside better than the traditional 2D cultures. Furthermore, many desired cellular characteristics are maintained or even promoted in 3D cultures, further supporting their use in basic and translational research. In this Special Issue, recent advances in the 3D cultures of stem cells are highlighted, bringing together a collection of articles presenting various stem cell types and their characteristics in 3D environments.This Special Issue includes articles highlighting the effective use of induced pluripotent stem cells (iPSCs) to develop cardiac microtissue , neurospA very relevant problem in cardiology is cardiac fibrosis. In the work by Blyszczuk et al., a model of fibrotic cardiac microtissue was generated using iPSC-derived cardiomyocytes and cardiac fibroblasts by treatment with transforming growth factor β1 (TGF- β1) or by use of cardiac fibroblasts from heart failure patients [A 3D in vitro model utilizing iPSCs was generated by Kobolak et al. to study neurotoxicity . In thisThe work by Cutarelli et al. employed iPSC-derived cortical progenitors and scaffolds to mimic the radially oriented cortical radial glia fibers that play an important role in the development of the cerebral cortex . The autAnother in vitro 3D model was generated by Ciardulli et al. for the study of MSCs and their tenogenic differentiation . This woTo circumvent some of the challenges with the 2D culture expansion of ESCs, McKee et al. developed a 3D scaffold to mimic in vivo stem cell niches . The autTo aid in the study of intestinal disorders, Kramer et al. developed intestinal organoids from stem cells obtained from various parts of the intestine . These iAnother study utilizing intestinal stem cell organoids explored the cellular origin of carcinogenesis . In thisA study by Tan et al. utilized ESCs to generate ureteric bud progenitor cells . The autThis Special Issue includes articles highlighting recent discoveries in 3D stem cell cultures using MSCs , ESCs 55,8, iPSC


Efficient inflammation resolution is important not only for the termination of the inflammatory response but also for the restoration of tissue integrity. An integral process to resolution of inflammation is the phagocytosis of dying cells by macrophages, known as efferocytosis. This function is mediated by a complex and well-orchestrated network of interactions amongst specialized phagocytic receptors, bridging molecules, as well as “find-me” and “eat-me” signals. Efferocytosis serves not only as a waste disposal mechanism (clearance of the apoptotic cells) but also promotes a pro-resolving phenotype in efferocytic macrophages and thereby termination of inflammation. Alterations in cellular metabolism are critical for shaping the phenotype and function of efferocytic macrophages, thus, representing an important determinant of macrophage plasticity. Impaired efferocytosis can result in inflammation-associated pathologies or autoimmunity. The present mini review summarizes current knowledge regarding the mechanisms regulating macrophage efferocytosis during clearance of inflammation. Specific recognition and engulfment of “foreign” material or pathogens by host cells, designated as phagocytosis, is an essential process modulating the immune response and tissue homeostasis , 2. BesiA central player in sterile inflammation or inflammation associated with infection are neutrophils recruited to the inflamed site , 5. RecrEmerging evidence suggests that macrophage function is regulated by alterations in their cellular metabolism in response to environmental cues within the inflammatory milieu . For insThe engulfment of dying cells by efferocytic macrophages requires the recognition of the former by the latter and the formation of the engulfment synapse, which is regulated by a network of “find-me,” “eat-me” and bridging molecules, “don’t eat-me” signals and specialized phagocytic receptors  Figure . NeutropAs alluded to above, bridging molecules are often key to efficient interactions between apoptotic neutrophils and macrophages . Milk faPhagocytic receptors on macrophages involved in the regulation of efferocytosis include also the PS receptors of the T-cell membrane protein (Tim) family, such as TIM-1 and TIM-4 , 54. TheThe presence of “don’t eat-me” signals further adds to the complexity of the regulation of apoptotic cell clearance. Specifically, surface expression of CD47  prevents phagocytosis by macrophages , 60. Binin vivo models of inflammation triggers resolution of inflammation in a manner dependent on TGFβ upregulation  that oxidizes polyunsaturated fatty acids and generates bioactive lipid metabolites leading to the biosynthesis of pro-resolving lipid mediators , 76. SpePlasminogen and its cleavage product plasmin not only regulate the initiation but also the resolution phase of inflammation. Treatment of mice with plasminogen/plasmin resulted in recruitment of pro-resolving macrophages and in upregulation of TGFβ. Administration of plasminogen/plasmin at the peak of inflammation was associated with increased neutrophil apoptosis and efferocytosis; the pro-resolving effect of plasminogen was mediated by annexin A1 . In accoMoreover, IFN-β from macrophages was recently identified as a factor promoting resolution of inflammation. IFN-β levels were higher in the resolution phase of pneumonia and peritoneal inflammation. Activation of IFN-β signaling via STAT3 enhances apoptosis of neutrophils and their subsequent efferocytic clearance, resulting in a pro-resolving reprograming of macrophages .The impact of cellular metabolism on macrophage function and plasticity has gained much attention recently , 89–91. Moreover, macrophage tissue specificity may be associated with differential metabolic activity. For example, resident peritoneal macrophage survival depends on the transcription factor GATA6 that is regulated by the vitamin A metabolite retinoic acid , 94, whiRegulation of macrophage metabolic activity in the context of efferocytosis  is of maSlc2a1) and monocarboxylate transporter 1 (encoded by Slc16a1) promoting lactate release. Enhanced glycolysis in efferocytic macrophages promoted actin polymerization and continued uptake of apoptotic cells. On the other hand, lactate from efferocytic macrophages contributed to establishment of an anti-inflammatory phenotype of the surrounding tissue  deficient mice  reductase, leads to increased apoptotic cell clearance . AdminisMacrophage efferocytosis is a major player in resolution of inflammation. Efferocytosis not only paves the way toward the timely termination of the inflammatory response, but also promotes restoration of tissue homeostasis. In this context, alterations in macrophage cellular metabolism are important regulators of efferocytosis. At the same time, metabolic reprograming in efferocytic macrophages induced by digested apoptotic material substantially regulates the function and plasticity of efferocytic macrophages. Given the relevance of efferocytosis as a mechanism against chronic inflammatory disease, a better mechanistic understanding of the pathways that orchestrate the mutual interaction between clearance of dying cells and metabolic alterations in macrophages is required. This knowledge will provide a scaffold for designing therapeutic approaches to improve macrophage function in inflammation resolution and harness macrophage efferocytosis for the treatment of pathologies associated with chronic inflammation or autoimmunity.IK, GH, and TC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HIV-1 eradication strategies aim to achieve viral remission in the absence of antiretroviral therapy (ART). The development of an HIV-1 cure remains challenging due to the latent reservoir (LR): long-lived CD4 T cells that harbor transcriptionally silent HIV-1 provirus. The LR is stable despite years of suppressive ART and is the source of rebound viremia following therapy interruption. Cure strategies such as “shock and kill” aim to eliminate or reduce the LR by reversing latency, exposing the infected cells to clearance via the immune response or the viral cytopathic effect. Alternative strategies include therapeutic vaccination, which aims to prime the immune response to facilitate control of the virus in the absence of ART. Despite promising advances, these strategies have been unable to significantly reduce the LR or increase the time to viral rebound but have provided invaluable insight in the field of HIV-1 eradication. The development and assessment of an HIV-1 cure requires robust assays that can measure the LR with sufficient sensitivity to detect changes that may occur following treatment. The viral outgrowth assay (VOA) is considered the gold standard method for LR quantification due to its ability to distinguish intact and defective provirus. However, the VOA is time consuming and resource intensive, therefore several alternative assays have been developed to bridge the gap between practicality and accuracy. Whilst a cure for HIV-1 infection remains elusive, recent advances in our understanding of the LR and methods for its eradication have offered renewed hope regarding achieving ART free viral remission. Infection with human immunodeficiency virus type-1 (HIV-1) requires life-long adherence to antiretroviral therapy (ART) due to the presence of latently infected cells that are central to viral persistence and rebound viremia following ART interruption  are routinely used to measure the LR but are labor and resource intensive and invariably underestimate the size of the replication competent reservoir , to LR maintenance is a topic of continued debate , transitional (TTM), effector memory (TEM), and memory stem (TSCM) cells, although the exact contribution of each cell type to the replication competent reservoir is still to be determined , Th17 cells and follicular T helper cells (Tfh) are now being characterized in more detail to determine which cells are the primary contributor to HIV-1 latency. Treg cells modulate the immune response through regulation of T cell proliferation and differentiation whilst Th17 cells are critical to maintaining mucosal immunity via secretion of IL-17 and the balance of these two cell subsets is therefore critical in providing effective immune function , resident within the B cell follicle of LN have recently been identified as a major host of the replication competent viral reservoir  and gut associated lymphoid tissue (GALT), as these sites are enriched in activated CD4 T cells  are potential hosts of the LR, and despite being infected at a lower frequency, may play an important role in viral persistence  to induce viral gene expression and productive infection in latently infected cells, exposing those cells to immune clearance or the viral cytopathic effect with the aim of reducing the size of the LR and limiting viral rebound . A majorin vivo, however, two recent studies have demonstrated potent and persistent latency reversal in mouse and SIV models in multiple tissues as well as peripheral blood: one utilized a LRA that activates the non-canonical NF-κB pathway  and histone methyltransferases inhibitors (HMTi), which induce HIV-1 expression by reversing epigenetic silencing  has led to increased hope of a HIV-1 cure by targeting various host or viral genes to induce host resistance, enforce viral latency or silence integrated provirus. Gene editing approaches have the advantage of highly specific gene targeting, so unlike LRAs, can produce the desired outcome without global physiological impact. Nevertheless, off-target effects have been observed in a number of studies and may affect the safety of these methods  makers such as PD1 and CTLA-4, are a major reservoir of replication competent provirus  in cancer therapy, their potential to treat HIV-1 is the subject of ongoing research. CAR-T cells are autologous T cells genetically engineered to express disease specific antibodies linked to an intracellular T cell receptor domain; therefore, when re-administered to the patient can direct the CTL response to cells expressing the disease epitope Wagner, . As suchAs discussed earlier, myeloid cells such as macrophages are known to support virus replication and may represent an additional barrier to HIV-1 cure. The use of “shock and kill” may not be effective against these cellular reservoir as they are refractory to CTL mediated immune clearance and the viral cytopathic effect . Prominent assays have targeted various, highly conserved regions in the HIV-1 genome including As discussed above, LTR based DNA assays can distinguish different HIV-1 DNA forms. During HIV-1 replication, linear unintegrated cDNA accumulates in the cell as well as abortive DNA forms such as 1-LTR and 2-LTR circular DNA, which are products of recombination events and interaction with host DNA repair mechanisms .To exclude unintegrated DNA forms from quantification, it is possible to amplify specifically integrated provirus by targeting an endogenous n genome . GeneralAs discussed above, the selection of an appropriate calibration standard is required for quantification of HIV-1 DNA, however, quantification relative to a standard is inherently biased. Amplification efficiencies between the standard and the sample must be equal to limit bias when quantifying relative to a standard curve  relies on measurement of tat/rev transcripts from cells plated in limiting dilution, following activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin  and flow cytometry to measure CA RNA or capsid p24 protein  of samples that are positive for two out of four regions to confirm replication competence  has demonstrated the use of a multiplexed ddPCR approach to measure the size of the intact LR based on the presence of regions that are frequently mutated in defective genomes . In thismpetence . In compmpetence . NeverthPreviously, full-length sequencing of proviral DNA has provided invaluable insight into the composition of the LR (Ho et al., The use of antiretroviral therapy has succeeded in reducing HIV-1 mortality but cannot eliminate the virus due to the persistent and stable LR. The global disease burden, equating to ~36 million infected individuals of which ~22 million have access to ART, warrants the continued search for a therapeutic approach that can either eliminate the virus or induce sustained viral remission in the absence of therapy (Sung et al., in vivo, but has been unable to lead to a meaningful increase in the time to viral rebound; suggesting improvement is required to aid the “killing” of infected cells. Alternative approaches, such as therapeutic vaccination, aim to prime the immune response to HIV-1 infection with the rationale that upon treatment interruption, immune mediated control of the virus will be improved. Several new technologies and approaches, such as immune checkpoint inhibitors, gene editing and CAR-T cells may offer an alternative method for cure, though currently their assessment in clinical trials is limited. An added complication in the search for an HIV-1 cure is the difficulty in accurately measuring the success of such trials. The inherent variability of the HIV-1 genome, the low frequency of latently infected cells as well as the abundance of defective provirus contribute to the complexity of LR quantification.Currently, several approaches for HIV-1 cure have been proposed and trialed to varying degrees of success. One of the most prominent cure strategies, “shock and kill,” has demonstrated virus reactivation Rather than an improvement in the current strategies leading to a cure, it is likely that synergistic combinations of different approaches, such as the use of LRAs following therapeutic vaccination, will lead to more drastic reductions in the LR and may aid the ultimate goal of long term ART free viral remission.JT conceptualized and outlined the manuscript and wrote the first draft. AR, WP, and GP contributed to editing the manuscript. All authors approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors GP.


Hyperfunctioning of the parathyroid was detected in 19 patients. Our data showed a significant positive association among the sestamibi uptake and the proliferation index histologically evaluated both in terms of the number of Ki67 positive cells and mitosis. According to these data, lesions with a higher valuer of L/N (lesion to nonlesion ratio) frequently showed several vessels in tumor areas and histological evidence of vascular invasion. It is noteworthy that among patients with negative scintigraphy, 2 patients showed a neoplastic lesion after surgery . However, it is important to highlight that these lesions displayed very low proliferation indexes, which was evaluated in terms of number of both mitosis and Ki67-positive cells, some/rare vessels in the main lesion, and no evidence of vascular invasion. In conclusion, data obtained on patients with positive or negative scintigraphy support the hypothesis that sestamibi can be a tracer that is capable of predicting some biological characteristics of parathyroid tumors such as angiogenesis, proliferation indexes, and the invasion of surrounding tissues or vessels.The aim of this study was to evaluate the possible association among sestamibi uptake and the main histopathological characteristics of parathyroid lesions related to aggressiveness such as the proliferation index (Ki67 expression and mitosis), angiogenesis (number of vessels), and vascular invasion in hyperparathyroidism patients. To this end, 26 patients affected by primary hyperparathyroidism subjected to both scintigraphy with [ Recent data indicate a continuous increase in hyperparathyroidism (PHP) incidence . It affeMost cases of primary PHP are related to the presence of 1) solitary parathyroid adenoma (85% of cases), 2) glandular hyperplasia (10%), 3) multiple adenomas 4%), and (4) parathyroid carcinoma (<1%) Sestamibi SPECT to identify some biological characteristics of human carcinomas, i.e., breast cancers Sestamibi SPECT and histological data (both morphology and immunohistochemistry) from 26 PHP patients have been collected.Starting from these considerations, the aim of this study was to evaluate the possible association among sestamibi uptake and the main histopathological characteristics of parathyroid lesions related to aggressiveness such as proliferation index (Ki67 expression and mitosis), angiogenesis (number of vessels), and vascular invasion in PHP patients. To this end, Tc-SPECT with Sestamibi and a parathyroid bioptic procedure from January 2018 to December 2019. For each of them, histological diagnosis and immunohistochemical investigations were performed.Exclusion criteria: a second cancer and neoadjuvant hormonal or radiation therapy prior to surgery. According to these criteria, we retrospectively enrolled 26 consecutive patients with parathyroid dysfunction , who underwent both [Early acquisition was performed at 15 min after the intravenous injection of 740 MBq Tc-sestamibi  according to the recommendations of the European Association of Nuclear Medicine [99mTc]Sestamibi SPECT was performed before biopsy in 15 patients and after biopsy in 11 patients. When [99mTc]Sestamibi SPECT was performed after biopsy, the minimum interval between biopsy and imaging was 7 days in an effort to avoid the effects of post-biopsy inflammation as much as possible.All 26 patients had biopsy. [99mTc]Sestamibi SPECT, two investigators classified positive and negative findings. Lesions with no demonstrable uptake and those with diffuse heterogeneous or minimal patchy uptake were considered negative, whereas lesions with scattered patchy uptake, partially focal uptake, or any other focal uptake were regarded as positive. Irregular-shaped regions of interest (ROIs) were used to encase the lesions. The evaluation of the lesion to nonlesion ratio (L/N) was estimated according to our previous study [99mTc]Sestamibi SPECT before biopsy (n = 15), the [99mTc]Sestamibi SPECT-guided biopsy procedure was performed. Semiquantitative analysis of the [99mTc]Sestamibi was performed.For qualitative analysis of Sestamibi SPECT analyses showed sestamibi uptake in 19 patients  (Sestamibi is the main radiotracer employed in parathyroid scintigraphy, since this molecule remains longer in the mitochondria of the parathyroid rather than thyroid, where it is washed out quickly Sestamibi and surgery/bioptic procedure were retrospectively enrolled. Hyperfunctioning of the parathyroid was detected in 19 patients.To this end, 26 PHP patients subjected to both scintigraphy with Sestamibi could underestimate the presence of parathyroid lesions with a greater morphological aspect of neoplasia but low aggressiveness. Nichols et al. demonstrated that most frequently, false negative scintigraphy with [99mTc]Sestamibi occurs in the presence of parathyroid lesions contiguous with the upper or lower poles of the thyroid gland [99mTc]Sestamibi and histopathological characteristics of parathyroid lesions. In this study, for the first time, a relationship between sestamibi uptake and the histopathological characteristics of parathyroid tumors was shown. Both data obtained on patients with positive and negative scintigraphy support the hypothesis that sestamibi can be a tracer that is capable of predicting some biological characteristics of parathyroid tumors such as angiogenesis, proliferation indexes, and the invasion of surrounding tissues or vessels. The possibility of detecting these characteristics by in vivo analysis opens new perspectives in the management of PHP patients. Indeed, our data, if confirmed on a large cohort of patients, could be used to develop diagnostic protocols that are capable of stratifying PHP patients according to prognostic and predictive information generally provided by histological and immunohistochemical analysis. In addition, our approach can be used for other diseases, thus expanding the diagnostic “equipment” available to nuclear physicians.It is noteworthy that among patients with negative scintigraphy, 2 patients showed a neoplastic lesion after surgery . However, it is important to highlight that these lesions displayed very low proliferation indexes, which were evaluated in terms of the number of both mitosis and Ki67 positive cells, some/rare vessels in the main lesion, and no evidence of vascular invasion. Thus, our data seem to indicate that scintigraphy with [id gland . However99mTc]Sestamibi analysis and the severity of disease [99mTc]Sestamibi SPECT in PHP patients. In general, the association between nuclear medicine and anatomic pathology data could provide the scientific rationale for developing new in vivo diagnostic methods that are capable of predicting prognosis or response to therapy for human cancers.The capability of sestamibi to identify malignant lesions by SPECT analysis has been shown for several human diseases such as hearth injury, breast cancer renal carcinomas, and PHP ,31,32,33 disease ,31,32,33 disease ,35. Ther


Deficient glucose transport and glucose disposal are key pathologies leading to impaired glucose tolerance and risk of type 2 diabetes.  The cloning and identification of the family of facilitative glucose transporters have helped to identify that underlying mechanisms behind impaired glucose disposal, particularly in muscle and adipose tissue.  There is much more than just transporter protein concentration that is needed to regulate whole body glucose uptake and disposal.  The purpose of this review is to discuss recent findings in whole body glucose disposal.  We hypothesize that impaired glucose uptake and disposal is a consequence of mismatched energy input and energy output.  Decreasing the former while increasing the latter is key to normalizing glucose homeostasis. While it is clear that both insulin-dependent GLUT4 trafficking and total cellular GLUT4 pool size play a critical role in glucose disposal, other glucose transporters and other factors influence glucose uptake in tissues including interstitial glucose concentration and intracellular glucose metabolism. The contributions of transporter pool size, rate of glucose delivery to the tissues, competition with other energy substrates, and ATP turnover are the key issues that must be resolved to understand how to effectively prevent and treat type 2 diabetes.One of the central metabolic features of type 2 diabetes in humans is decreased glucose disposal. This process is rate limited by the capacity for glucose transport into the peripheral and splanchnic tissues. The prevailing hypothesis for limited glucose transport and disposal in type 2 diabetes has centered on the function of the insulin-dependent glucose transporter GLUT4. This transporter, expressed at the highest levels in adipose tissue, skeletal muscle, and cardiac muscle, is recruited to the cell surface when activated by rising insulin levels following consumption of a meal. This hypothesis is supported in large part by studies performed using whole animal and tissue-specific transgenic manipulation of GLUT4 expression and2. Glucose uptake in these tissues occurs by facilitative diffusion. Because glucose is rapidly phosphorylated once it enters the cell, the rate of glucose transport is dictated by the extracellular glucose concentration as well as the number of transport proteins found on the cell surface. In the case of visceral adipose tissue and skeletal muscle, the extracellular  glucose concentration is dictated by blood flow into the tissue. It has long been known that insulin action increases vasodilation, presumably to deliver nutrients and insulin to tissues  into the femoral artery during the clamp to constrict the vasculature. Indeed, restriction of blood flow reduced glucose uptake. These data point out that both insulin-dependent and exercise-dependent increases in glucose uptake are regulated by both cell permeability and interstitial glucose concentration. The key question is whether glucose disposal in people with prediabetes or type 2 diabetes is limited exclusively by impairment in insulin-dependent GLUT4 translocation or insulin-dependent tissue perfusion. A partial answer to this question is found in the work of Henstridgeet al., where overweight and obese men with type 2 diabetes were given a nitric oxide (NO) donor, sodium nitroprusside, or verapamil to increase leg blood flow. While both drugs increased leg blood flow, only sodium nitroprusside increased glucose uptake in the treated muscle5. The authors interpret this data to mean that increased blood flow alone is not sufficient to increase glucose uptake when membrane permeability (i.e. GLUT4 plasma membrane levels) is low. Furthermore, the authors raised the possibility that NO signaling was responsible for exercise-dependent GLUT4 translocation, a finding that has since been supported in both cultured myotubes and mouse skeletal muscle7.In human subjects with prediabetes and type 2 diabetes, impaired glucose uptake in skeletal muscle, visceral adipose tissue, and the brain appear to be the best predictors of insulin resistanceet al., inhibition of blood flow by vasoconstriction in conscious, sedentary mice resulted in an increased transendothelial insulin efflux and, in turn, increased glucose clearance8. The authors of this study demonstrated that vasoconstriction via inhibition of NO synthase (NOS) resulted in increased glucose clearance in response to an insulin tolerance test. While the results of these two studies are conflicting, it is important to note that the McConell study utilized a euglycemic/hyperinsulinemic clamp protocol that measures steady-state insulin action. The Williams study used an insulin bolus, which better represents insulin kinetics. It is likely that the initial increase in transendothelial insulin efflux increased glucose uptake in the first 15 minutes of NOS inhibition, compensating for decreased perfusion of the tissues.In contrast to the human study by McConell9. Mice were fed a 45% fat diet for 6 days to increase fatty acid availability and then given a single injection of etomoxir to inhibit CPT-1. Within 4 hours, plasma glucose levels decreased and glucose oxidation in peripheral tissues increased. The authors observed that free fatty acid levels became elevated with the acute etomoxir treatment, and they became concerned that the lipidemic environment would be enhanced with long-term treatment. This prediction proved to be correct. Prolonged inhibition of fatty acid oxidation led to decreased glycemic control, hepatic insulin resistance, and fatty liver. Thus, the substrate competition hypothesis failed to provide a useful therapeutic hypothesis for the treatment of insulin resistance. More importantly, the prolonged inhibition of fatty acid oxidation led to decreased glucose clearance, further supporting the idea that a high-lipid environment can impair glucose transport and glucose disposal. While long-term clinical trials of etomoxir in human subjects have not been carried out, a short-term clinical trial showed that treatment of subjects with type 2 diabetes with etomoxir for 3 days increased glucose oxidative metabolism under basal conditions but had no effect under hyperinsulinemic clamp conditions10. This study suggested that inhibition of fatty acid oxidation would not improve insulin sensitivity in patients with type 2 diabetes.A predisposing factor for developing insulin-resistant glucose transport is obesity and the lipid overload associated with that condition. Simply put, a high-lipid environment results in decreased glucose disposal. This line of thinking led to the notion that inhibiting fatty acid oxidation would increase glucose oxidation and glucose disposal, thus curing insulin resistance. This substrate competition model has been tested in mice using pharmacologic inhibition of carnitine palmitoyltransferase-1 (CPT-1) activity11. Carnitine supplementation clearly increased plasma carnitine levels and synthesis of acetylcarnitine levels in the muscle, indicating that carnitine concentration is rate limiting for fatty acid oxidation. These carnitine-dependent increases in fatty acid metabolism did not improve insulin sensitivity or change carbohydrate oxidation. Thus, substrate switching alone is not sufficient to improve glucose transport and glucose disposal in glucose-intolerant volunteers.If increasing glucose oxidation does not improve metabolic homeostasis in a high-lipid environment, can increased lipid oxidation solve the problem? This question was approached by supplementing carnitine in individuals with impaired glucose tolerance and normal glucose tolerance12. As expected, the lipid infusion resulted in a significant decrease in glucose disposal in the sedentary subjects. On the other hand, the glucose disposal rate and insulin sensitivity were less affected by lipid infusion in the endurance-trained athletes. Unlike sedentary controls, lipid infusion did not decrease rates of glycogen synthesis in the skeletal muscle of the endurance athletes. The latter finding suggests that glucose transport and glucose metabolism remained intact during lipid infusion in trained athletes. This conclusion was supported by the observation that GLUT4 concentration in muscle biopsy taken before and after lipid infusion was unchanged in the trained athletes while GLUT4 levels were significantly decreased by lipid infusion in the untrained participants. While a GLUT4 half-life of about 50 hours has been measured in cultured 3T3-L1 adipocytes, the direct measurement of GLUT4 half-lifein vivo or in muscle has not been made. Therefore, the lipid-induced decline in GLUT4 protein content over a 6-hour period in this study suggests that increased turnover may be responsible for the decline in GLUT4. Importantly, this study provokes questions about the metabolic regulation of GLUT4 expression. Further work is required to understand the mechanisms that regulate GLUT4 protein synthesis and degradation in a physiologic setting.Some light has been shed on this problem in a study comparing rates of glucose disposal during lipid infusion in endurance-trained athletes and sedentary men14. These rats were selected for their high-capacity running (HCR) and are compared to similar rats that were concurrently selected for their low-capacity running (LCR). Noticeably, the high-fat-fed HCR rats, even when maintained under sedentary conditions, showed increased glucose clearance and insulin sensitivity under euglycemic/hyperinsulinemic clamp conditions. Glucose clearance was largely accounted for by muscle glucose uptake and glycogen synthesis14. In our lab, we have shown that transgenic overexpression of GLUT4 protein under the control of the humanGLUT4 gene promoter also enhanced glucose disposal under high-fat feeding conditions15. In our model, glucose uptake by muscle and adipose tissue resulted in enhanced production of alanine, a potential gluconeogenic substrate for the liver. Pyruvate tolerance in these animals was enhanced, further supporting the notion that the glucose–alanine cycle was enhanced in the GLUT4 transgenic mice16. In our model, the enhanced glucose–alanine cycle is perhaps serving as a futile, energy-consuming cycle that is increasing ATP turnover, at least in the liver. The HCR rats demonstrated a thermogenic phenotype and increased metabolic rate17.It is well established that exercise is an important lifestyle intervention for enhancing both glucose disposal and insulin sensitivity. One of the key features of exercise is increased energy expenditure by working muscle, including the heart. Work by multiple laboratories demonstrated that glucose clearance in exercise-trained rodents was correlated with GLUT4 protein expression in skeletal muscle  cells that were systematically transduced with at least one human cDNA encoding each step of the glycolytic pathway beginning with the glucose transporters and ending with lactate transporters19. This approach showed that glycolytic flux could be increased independently by overexpression of proteins regulating at four key steps. These steps include glucose transporters, hexokinase, PFK-1, PFKFB3, and lactate transporters. The output for this study was glycolytic flux rather than glucose transportper se. One might conclude that an increase in glycolytic flux would require an increase in glucose transport, unless increased glycolytic flux was diverting glucose-6-phosphate away from other pathways such as glycogen synthesis or the pentose phosphate pathway. While it is not likely that glycogen synthesis in the cultured cells is an active pathway, it is possible that the pentose phosphate pathway was active. Further experiments are required to determine if the four key steps that regulated glycolytic flux also regulate the rate of glucose transport. This is an intriguing possibility because understanding how glycolysis is regulated may provide important insight into the defects that underlie decreased glucose disposal in type 2 diabetes.The hypothesis that glucose transport can be regulated by glucose metabolism has been systematically testedAmong the insulin-sensitive tissues, the heart is unique because it has continual energy requirements and it never fatigues yet it lacks the capacity to store nutrients. Thus, the heart must be able to metabolize available circulating nutrients to meet these unyielding energetic demands. In healthy individuals, the heart primarily relies on fatty acids and, secondarily, glucose. However, like other insulin-sensitive tissues, GLUT4 is trafficked to the plasma membrane (sarcolemma) in response to insulin. Thus, after a meal, the cardiac uptake of glucose and glucose oxidation are increased.20. This is also true in humans who have type 2 diabetes and left ventricular dysfunction21. However, the decrease in cardiac glucose uptake likely depends upon the duration of disease. In a more recent study, glucose uptake rates in several tissues were assessed by using 18F-FDG PET and MRI in conjunction with a hyperinsulinemic clamp. In this small cohort study performed on control, prediabetic, and type 2 diabetic patients, glucose uptake was decreased in the liver, skeletal muscle, and adipose of type 2 diabetics but unchanged in the heart. This suggests that cardiac glucose uptake is maintained, at least at rest and under the conditions of the hyperinsulinemic clamp, as compared to other tissues. Whether or not cardiac glucose uptake is maintained during increased workload, or how it proceeds with the progression of the disease, must be further explored.It is well established that diabetes decreases the capacity of the heart to oxidize glucose and its reliance on fatty acid oxidation dramatically increases. This is referred to as metabolic inflexibility, and it is believed to be a significant component of diabetic cardiomyopathy. The underlying mechanisms of metabolic inflexibility are thus an area of intense research. Studies by William Stanley and others in the 1990’s demonstrated cardiac GLUT1 (the primary insulin-insensitive glucose transporter) and GLUT4 protein expression and glucose uptake are decreased in various animal models of diabetes22. Older work also reported that there is increased myocardial glucose uptake during exercise and increased workload, but this is not necessarily mediated by increased myocardial GLUT4 content as seen in exercised skeletal muscle24. However, the effect of exercise on cardiac glucose uptake and glycolysis depends upon exercise duration. Chronic exercise training results in decreased cardiac glycolysis during training but is elevated approximately twofold 24 hours afterwards25. This increase in glycolysis in the post-exercise phase is essential for cardiac growth. What is not clear, though, is how diabetes affects these adaptive processes in the heart. For example, if glucose uptake is increased in the heart by catecholamines, but downstream glycolysis is impaired, the beneficial effects may be compromised.Cardiac GLUT4 translocation and glucose uptake can also be stimulated in an insulin-independent manner in response to catecholamines. This is important for physiological and pathophysiological adaptations. For example, studies using a cardiac-specific GLUT4 knockout model demonstrated that GLUT4 is required for adaptations to hemodynamic stress resulting from swimming exercise or transverse aortic constrictionet al. examined the role of TXNIP in the diabetic heart and used a proteomics approach to identify interacting partners26. They found that TXNIP associated with GLUT1, and experiments performed in cell culture revealed overexpression of TXNIP decreased glucose uptake. Reciprocally, mouse embryonic fibroblasts isolated from TXNIP knockout mice displayed enhanced glucose uptake. This work suggests that unfettered glucose uptake via GLUT1 transporters may be limited in cardiomyocytes exposed to hyperglycemia by the increased expression of TXNIP.In addition to decreased GLUT4, additional mechanisms are also in place to limit basal cardiomyocyte glucose uptake by GLUT1 when confronted by hyperglycemia. Thioredoxin-interacting protein (TXNIP) is an inhibitor of the antioxidant enzyme thioredoxin, and its gene expression is highly responsive to glucose. A recent study by Myers25. This constitutive increase in glycolysis must be coupled with increased glucose uptake. In wild-type mice and in humans, cardiac PFK-2 is activated via phosphorylation by the insulin signaling cascade. This facilitates the metabolism of glucose, taken up by GLUT4, through glycolysis. In our own work, we have found that PFK-2 content is regulated by insulin signaling and that it is constitutively decreased in mouse models of diabetes27. Regulation of PFK-2 content by insulin may serve as a mechanism to decrease cardiac glucose uptake and metabolism during fasting, but, in the context of diabetes, a chronic decrease in PFK-2 may contribute to metabolic inflexibility. Furthermore, changes in PFK-1 or PFK-2 activity may have effects on how glucose that is taken up is then metabolized. For example, the Hill group has recently shown in a metabolic tracer study in cardiomyocytes that the activity of PFK-1 is a key determinant in the fate of glucose entering into glycolysis or ancillary pathways, such as the pentose phosphate, hexosamine biosynthesis, and glycerolipid synthesis pathways28. It has also been postulated that dysregulation of key glycolytic steps that affect PFK-1 or PFK-2 facilitate the production of glycogen, which abnormally accumulates in the diabetic heart  in the heart, there is an increased rate of glycolysis in the absence of insulin31. Like GLUT4, total GLUT8 protein expression is decreased in mice with obesity-induced insulin resistance31. SGLT2 inhibitors have gained ample attention because, in addition to their ability to decrease blood glucose levels, they also have a positive impact on heart failure. SGLT2 is not expressed in the heart, and the positive effects of its inhibitors may be mediated by the systemic lowering of blood glucose levels or direct effects on cardiomyocytes via mechanisms that are still under investigation32. A recent study has reported that the heart does express SGLT1 abundantly, though33. SGLT1 transports glucose by an active transport mechanism using the Na+ gradient. However, its functional role in the heart is independent of glucose transport and its importance to normal physiology and disease states, such as diabetes, must be further evaluated.In addition to the canonical GLUT1 and GLUT4 facilitative glucose transporters, cardiomyocytes also express other members of the GLUT family of proteins as well as members of the sodium glucose cotransporters (SGLTs) (reviewed inThe clearance of glucose from the blood plasma following a meal is important for overall metabolic health. Because the central nervous system is highly dependent on glucose for an energy source, multiple layers of regulation are in place to maintain a steady level of glucose in the blood plasma during both the fed and the fasted state. When this process breaks down, causing prolonged insulin resistance, the risk of developing type 2 diabetes and subsequent complications is heightened. Restoration of normal glucose disposal is the therapeutic goal for both insulin resistance and type 2 diabetes. It is likely that long-term success in reaching this therapeutic goal will rely on attacking the root cause of the glucose transport defect. Thus, if nutrient overload is at the heart of the development of insulin resistance and impaired glucose uptake, it stands to reason that matching energy input with energy output is the appropriate course to follow. Energy output might be enhanced by several approaches including increased physical activity, increased thermogenesis, or even enhancing the activity of futile metabolic cycles. Each approach leads to increased ATP turnover, helping to match energy output to energy input.


To better adapt transiently or lastingly to stimuli from the surrounding environment, the chromatin states in plant cells vary to allow the cells to fine-tune their transcriptional profiles. Modifications of chromatin states involve a wide range of post-transcriptional histone modifications, histone variants, DNA methylation, and activity of non-coding RNAs, which can epigenetically determine specific transcriptional outputs. Recent advances in the area of ‘-omics’ of major crops have facilitated identification of epigenetic marks and their effect on plant response to environmental stresses. As most epigenetic mechanisms are known from studies in model plants, we summarize in this review recent epigenetic studies that may be important for improvement of crop adaptation and resilience to environmental changes, ultimately leading to the generation of stable climate-smart crops. This has paved the way for exploitation of epigenetic variation in crop breeding. Intense breeding has narrowed crop genetic diversity. Epigenetic diversity could emerge as new source of phenotypic variations to improve crop adaptation to a changing environment and generate stable climate-smart crops. Plant response to abiotic stresses is complex and involves multiple mechanisms, activated and controlled by massive changes in gene expression and nuclear organization .Epigenetics is a fascinating field of genetics, completely meddling with classical knowledge of the interaction between the hereditary material (DNA/genes) and the phenotype, and puzzling scientists for decades. According to the definition of Arthur Riggs and his colleagues, epigenetics is ‘the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence’ . MoreoveAbiotic stresses negatively impact plant growth and development, with severe effects on crop yield leading to huge economic losses. Since most epigenetics studies have been carried out in model plants, particularly Arabidopsis  that are targeted to corresponding genomic loci by ARGONAUTE (AGO) family members, which in turn are methylated via DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2). A second pathway requires CHROMOMETHYLASE 2 (CMT2) through interaction with DECREASE IN DNA METHYLATION1 (DDM1) in histone H1-enriched chromatic regions  contexts through distinct pathways. METHYLTRANSFERASE 1 (MET1) and CHROMOMETHYLASE 3 (CMT3) are plant enzymes responsible for the maintenance of CG and CHG methylation, respectively  gene, and found that the FGT1 protein binds directly to a specific class of heat-inducible genes and ensures that the heat-inducible genes are always accessible and active by modifying the way the DNA containing these genes is packaged. Their findings could lead to new approaches in crop breeding programmes for enhancing the resistance to abiotic stress, as knowledge of stability and heritability features of epigenetic marks and epigenetic regulatory mechanisms is crucial for breeding applications , which is a principal cereal crop cultivated worldwide. It has recently been found that histone marks, in particular H3K4me3 and H3K27me3, may function as memory marks for stress-responsive genes and transcription factors in maize . Moreover, in maize seedlings, application of a short-term heat stress induces dynamic H3K4me2 and H3K9ac alterations in promoter regions, which were associated with heat stress factor (Hsf) and rRNA gene up-regulation, accompanied by perturbations of cell membranes and increase in reactive oxygen species  is one of the most important crops grown for human consumption. It exceeds any other cereal, including rice and maize, in total world growing area and total world food supply, with total growing area of 214.79 million ha in 2018  were found in shoots and roots of wheat genotypes differing in their sensitivity to this stress  and microRNA (miRNA), small regulatory RNAs, have been shown to be involved in wheat drought stress response (Hordeum vulgare L.) is a major cereal grown in temperate climates globally, with total production of 141.75 million tons in 2018 (HvCKX2.1) in the caryopsis exposed to terminal drought  is another of the world’s leading food crops, with rice, wheat, and maize together accounting for about 51% of human caloric intake  and DIHYDROFLAVONOL-4-REDUCTASE (DFR) mostly associated with developmental processes , Medicago truncatula, and Lotus japonicus due to their economic significance or their importance as genomic models (Phaseolus vulgaris (common bean), Cicer arietinum (chickpea), and Vigna unguiculata (cowpea)  and ALCOHOL DEHYDROGENASE 1 (ADH1), respectively, which contribute to an adjustment to water deficit conditions. Another shared mechanism between legume species that regulates plant responses to water deficit is miR1514a-mediated regulation of a NAC transcription factor transcript through phased siRNA (phasiRNA) production. Results by Phaseolus vulgaris demonstrate that the terminal drought-resistant cultivar PS targets a NAC transcription factor mRNA, through miR1514a induction, leading to NAC 700 transcript cleavage and phasiRNA production. Similarly, miR1514a modulation of a NAC transcription factor transcript that triggers phasiRNA formation in response to drought has been found in M. truncatula and in G. max as well ), with known ability to ameliorate plant growth and development, in controlling the regulation of miRNAs and their target genes and therefore in improving drought tolerance in chickpea , which can help scientists elucidate the adaptive responses of chickpea plants to drought stress through RA-mediated regulation of expression of miRNAs  is the second most important oilseed crop of the world, with total world production of 75 million tonnes in 2018  is one of the world’s most cultivated vegetable species. One of the first tomato epimutants characterized was the colourless non-ripening (Cnr) mutation, which inhibits ripening of tomato fruits  genes have been studied in this respect  was down-regulated by RNAi  had an increased level of DNA methylation. Furthermore, the seeds from the drought-stressed plants showed faster shoot emergence due to an abundance of cytokinin ribosides  is involved in the persistent expression of high temperature-responsive genes, as well as hyper-induction of such genes during repeated heat stress treatments , epigenomic selection (Based on the information described in this review, there are several possibilities that should be taken into consideration to improve crop stress tolerance. The identification of epialleles and epigenetic regulatory systems that have functional impacts on agronomic traits can lead to a range of different approaches for epigenetic breeding of crop plants, such as the use of mutant lines (Wang election , as wellelection .MSH1 system to induce epigenetic variation, and developed epi-lines by crossing between wild type and msh1-acquired soybean memory lines, with a wide variation for multiple yield-related traits in both greenhouse and field trials. Furthermore, obtained epitypes had low epitype–environment interaction, indicating higher yield stability and a lower effect of environmental constraints. The authors provided evidence that novel epigenetic variation induced by MSH1 suppression, following crossing and F2 segregation, can be inherited for at least three generations and bred for crop improvement with a few rounds of selection to enhance and stabilize crop yield. MSH1-based epigenetic variation could be of use in plant breeding for enhanced yield and yield stability and that environmentally induced epigenetic variation can result in heritable phenotypic plasticity, which may play a major role in adaptation to changes in the environment (A few approaches have been described for exploitation of epigenetic variation for crop improvement. A breeding strategy was suggested by ironment .MSH1-based epigenetic variation could be of use in plant breeding was provided by MSH1 in tomato cv ‘Rutgers’ and its effect on the developmental reprogramming of the plant. Crossing these transgene-null, developmentally altered plants to the isogenic cv ‘Rutgers’ wild type resulted in progeny lines that show enhanced, heritable growth vigour under both greenhouse and field conditions. This boosted vigour was also graft transmissible and could be partially reversed by treatment with the methylation inhibitor 5-azacytidine, implying the involvement of mobile, epigenetic factors and DNA methylation changes in the underlying molecular mechanism.Further evidence that In another study, Combined with classical genetic studies, newly available sequencing technologies are facilitating the study of epigenetic phenomena at the whole genome level in a way that was unthinkable only a few years ago. The application of epigenome profiling and engineering could generate new avenues for using the full potential of epigenetics in crop improvement. This, along with the new epigenome editing tools, could enable the creation of novel epiallelic variants by alteration of DNA methylation or other chromatin modifications and crop improvement through epigenome engineering .Genome-wide mapping of epigenetic marks and epigenetic target identification are currently two major strategies in many important crops: these will offer breeders new tools to increase and manipulate epigenomic variability for selecting novel climate-smart crop varieties that are more resilient to environmental changes. Genome-wide mapping of epigenetic marks led to the development of epigenomics, an emerging field that is expanding our ability to explain observed phenotypic variation through the identification of multiple cellular products such as RNAs, protein–DNA interactions, chromatin modifications, and chromatin accessibility (Different techniques to modify the epigenome globally or at target sites can be used for induction of epigenetic modifications useful for crop improvement. In recent years, the emergence of CRISPR/Cas9 and dCas technology has provided new routes in the epigenetic field. This new tool enables targeted manipulation of epigenetic characters and could be used to specifically modify plant phenotype or to elucidate the relationship between the epigenome and transcriptional control . The recAll these new technological advances have facilitated exploitation of epigenetic variation in crop breeding and acceleration and more efficient creation of climate-smart crop varieties. For optimal use of these new tools in plant breeding, further studies are needed for specific traits and crops in order to gain more knowledge on the association of stress-induced gene expression changes with alterations in DNA methylation and histone modifications, the mode of inheritance of these modifications, and their adaptive value .In the future, breeders will certainly have to pay more attention to crop epialleles and their potential role in adaptation to changes in environment. Furthermore, defining the molecular basis of transgenerational epigenetic inheritance could ultimately lead to development of epialleles designed for specific environmental conditions through targeted epigenetic modifications in genes of interest. However, more work on a greater range of plant species is needed in order to gain a more comprehensive understanding of the mechanisms inducing and stabilizing epigenetic variation in crops. This will require a combined and multidisciplinary effort of researchers involved in different areas of plant science and better integration of epigenomic data obtained in different crops.


Inflammatory lung disease results in a high global burden of death and disability. There are no effective treatments for the most severe forms of many inflammatory lung diseases, such as chronic obstructive pulmonary disease, emphysema, corticosteroid-resistant asthma, and coronavirus disease 2019; hence, new treatment options are required. Here, we review the role of oxidative imbalance in the development of difficult-to-treat inflammatory lung diseases. The inflammation-induced overproduction of reactive oxygen species (ROS) means that endogenous antioxidants may not be sufficient to prevent oxidative damage, resulting in an oxidative imbalance in the lung. In turn, intracellular signaling events trigger the production of proinflammatory mediators that perpetuate and aggravate the inflammatory response and may lead to tissue damage. The production of high levels of ROS in inflammatory lung diseases can induce the phosphorylation of mitogen-activated protein kinases, the inactivation of phosphoinositide 3-kinase (PI3K) signaling and histone deacetylase 2, a decrease in glucocorticoid binding to its receptor, and thus resistance to glucocorticoid treatment. Hence, antioxidant treatment might be a therapeutic option for inflammatory lung diseases. Preclinical studies have shown that antioxidants  are effective in the treatment of inflammatory lung diseases, although the clinical evidence of efficacy is weaker. Despite the high level of evidence for the efficacy of antioxidants in the treatment of inflammatory lung diseases, the discovery and clinical investigation of safer, more efficacious compounds are now a priority. Fo. Foα . M. M2O2) ty by SOD  and possy by SOD . It has y by SOD . Althougy by SOD .GPx activity is significantly reduced in subjects with asthma or COPD that indicates its prominent role in lung antioxidant defense  106]. I. I106]. κB and reduced oxidative stress and the development of emphysema in guinea pigs exposed to cigarette smoke [Myeloperoxidase is produced in neutrophils and macrophages. It has a damaging effect not only on bacteria but also on tissue. Thus, the selective, irreversible myeloperoxidase inhibitor 2-thioxanthine inactivated NF-te smoke .Antioxidant enzyme defense systems  are directly regulated by Nrf2. Thus, owing to its antioxidant effect, Nrf2 is a potential therapeutic target in lung disease . Sulforaα-tocopherol), resveratrol, and flavonoids) have been suggested as antioxidant treatments [in vitro and in vivo model of inflammation induced by bleomycin, lipopolysaccharide, and cigarette smoke, among others [Dietary antioxidants (including vitamin C (ascorbic acid), vitamin E -isoprostanes) in enhancing the inflammation through the activation of stress kinases  and thus increased activity of transcriptional factors such as NF-diseases .α/β oral inhibitor losmapimod has also been investigated: it decreased the number of moderate-to-severe COPD exacerbations in patients with blood eosinophil counts ≤ 2% [αMAPK inhibitor can reinstate corticosteroid sensitivity in alveolar macrophages obtained from patients with asthma [The association between corticosteroid resistance and PI3K inhibition was discussed above. Hence, treatment with a combination of a PI3K inhibitor and a corticosteroid should be a practical means of resolving inflammation in COPD. p38 MAPK inhibitors are capable of suppressing the release of proinflammatory mediators from alveolar macrophages and other immune/inflammatory cells taken from patients with COPD . The duants ≤ 2% . Furthernts ≤ 2% . Moreoveh asthma , 127. Neh asthma .in vitro and in vivo models [In the setting of chronic inflammatory lung disease, oxidative stress activates kinases and redox-sensitive transcription factors and modulates epigenetic chromatin modifications—resulting in changes in gene transcription. Recent studies have focused on identifying genes that undergo epigenetic modifications. In patients with asthma, microarray profiling of genes expressed in peripheral blood mononuclear cells can predict glucocorticoid sensitivity . Novel mo models –134.in vitro and in vivo models but few have been clinically effective—perhaps because the oxidative stress (but not disease onset or progression) was affected in the preclinical studies. Nevertheless, today's knowledge of mechanisms of ROS regulation might lead to the pharmacological manipulation of antioxidants and the development of novel, truly effective drugs. The antioxidant treatment approach might provide a ray of hope in the otherwise difficult setting of COPD, asthma, and IPF. However, much work remains to be done.Another point to bear in mind is that although many studies have found that the accumulation of oxidative damage in cellular macromolecules is immensely toxic, the ROS produced by normal cell metabolism are vital for cellular homeostasis—especially for immune competence and the activation of several signal transduction pathways. Lastly, several different approaches to antioxidant treatment of lung disease have been explored A growing body of evidence shows that oxidative imbalance has several pivotal roles in the pathophysiology of inflammatory lung diseases. Elevated ROS levels directly or indirectly affect a variety of receptors, other signaling molecules, proteins, and ion levels. The depletion of antioxidants and the accumulation of ROS reduce the cell's ability to mount an effective antioxidant response and thus contribute to the development of inflammatory lung and airway diseases. Therefore, a better understanding of the mechanisms through which the ROS affect intracellular homeostasis, cell signaling, and thus the onset and/or aggravation of inflammatory lung diseases may aid in the identification of new molecular pathways and the development of innovative, effective therapeutic strategies.