Novel treatment regimens are required for castration-resistant prostate cancers (CRPCs) that become unresponsive to standard treatments, such as docetaxel and enzalutamide. Histone deacetylase (HDAC) inhibitors showed promising results in hematological malignancies, but they failed in solid tumors such as prostate cancer, despite the overexpression of HDACs in CRPC. Four HDAC inhibitors, vorinostat, pracinostat, panobinostat and romidepsin, underwent phase II clinical trials for prostate cancers; however, phase III trials were not recommended due to a majority of patients exhibiting either toxicity or disease progression. In this review, the pharmacodynamic reasons for the failure of HDAC inhibitors were assessed and placed in the context of the advancements in the understanding of CRPCs, HDACs and resistance mechanisms. The review focuses on three themes: evolution of androgen receptor-negative prostate cancers, development of resistance mechanisms and differential effects of HDACs. In conclusion, advancements can be made in this field by characterizing HDACs in prostate tumors more extensively, as this will allow more specific drugs catering to the specific HDAC subtypes to be designed.  There were ~1.3 million new cases of prostate cancer worldwide in 2018 and it is the second most commonly diagnosed cancer in men . The majOne of these strategies involves modulating histone acetylation and deacetylation mediated by histone acetyl transferases (HATs) and histone deacetylases (HDACs), respectively. HATs and HDACs affect both, histone and non-histone proteins . HistoneThe histone core proteins have lysine residues on their N-terminal tails . If thes+ as a co-factor . However, HDAC3 levels did not correlate with Gleason scores. The proliferative marker Ki67 also correlated positively with HDAC1 (p < 0.032), HDAC2 (p < 0.002) and HDAC3 expression (p < 0.001). HDAC2 was also significantly associated with relapse-free survival. Although statistically significant data were not obtained for HDAC1 and HDAC3, the results demonstrated that the PSA (Prostate-Specific Antigen)-relapse-free survival was decreased in HDAC-positive cells compared to -negative cells [Overexpression of HDACs is a recurring theme in different malignancies, including gastric, colon, prostate and breast cancers . In the ve cells . Howeverve cells . Similarve cells .n = 24) was lower compared to Weichert et al. as well as results from the Oncomine database. In addition, the patients in this study exhibited a tumor stage of T1 or T2 [In contrast, only one study has failed to show a difference in the expression of either HDAC1 or HDAC2 between cancerous and normal tissues . ExpressT1 or T2 , while iT1 or T2 . Thus, tAs HDAC expression has been shown to be associated with poor clinical outcome, HDAC inhibitors have been explored as a potential therapeutic option. The five classes of HDAC inhibitors include hydroxamic acids, cyclic tetrapeptides, short chain carboxylic acids, benzamides and keto-derivatives . These iDue to the role of HDAC inhibitors in inducing cell cycle arrest, apoptosis, autophagy, heat shock protein-90 (HSP90) inhibition and reactive oxygen species generation, this class of drugs has been trialed in cancers . In 200650 values of different classes of HDAC inhibitors. Hydroxamic acid derivatives, including SAHA, panobinostat, belinostat and pracinostat, inhibit different classes with different efficacies; similarly, different compounds from the benzamide class, including chidamide, entinostat and mocetinostat exhibit different affinities for different HDACs  of panobinostat was administered in clinical trials. Of the 35 patients, 25 reported at least one grade 3 adverse event, whereas grade 4 toxicities were seen in 4 patients. Disease progression occurred in 29/35 patients. Based on these findings, panobinostat was not recommended for further testing.Panobinostat (LBH589) was FDA-approved in 2015 for treatment of multiple myeloma in patients who had previously received bertezomib and an immunomodulatory agent . Various2). The study concluded that dacinostat was well-tolerated [Romidepsin belongs to the cyclic tetrapeptide class of HDAC inhibitors . In 2009olerated . In addiolerated . HSP90 iolerated ,86.2 of romidepsin. Two patients achieved a radiological partial response (RECIST) and greater than 50% decline in PCA lasting more than 6 months. However, 22 patients in the study showed progression, and 11 patients showed stable disease. No grade 4 toxicities were reported; however, 11 patients had to be taken off the treatment regimen early. Based on these results, treatment using romidepsin was not recommended for CRPC.Efficacy studies using romidepsin in preclinical prostate cancer models have not been conducted. However, a phase II trial has been conducted with romidepsin . The stuPracinostat, another hydroxamic acid derivative, showed promising activity in in vitro and in vivo cancer models, including colorectal, ovarian and prostate ,87. It w6 cm/s) [As a single agent, HDAC inhibitors have shown poor activity in CRPC and other solid tumors ,89. Prev6 cm/s) . Among o6 cm/s) . However6 cm/s) ,92,93. I6 cm/s) ,83,88,94Recently, new insights have been shed on prostate cancer, bringing the centrality of AR, effects on different classes of HDACs by non-specific HDAC inhibitors and evolution of resistance mechanisms in response to HDAC inhibitors into question.AR is thought to play a central role in the progression of prostate cancers . Most stThese findings are also supported by another study which showed that serum PSA declines of >50% were observed in 56% of 140 patients with metastatic CRPC following treatment with the AR antagonist enzalutamide . The basInterestingly, there has been no correlation between AR and PSA, which may just be a trend seen in late stage prostate cancer . HoweverAlthough HDAC inhibitors induce apoptosis and cell cycle arrest in various tumors, they also lead to upregulation of epithelial to mesenchymal (EMT) transitioning proteins and confer protective effects in cancer cells via p21 inductions ,102,103.Various studies support these theories. Kong and colleagues (2012) showed that HDAC inhibitors caused acetylation of histone 3 residues in the promoter regions of EMT-related factors such as vimentin, zinc finger E-box-binding homeobox 1 (ZEB1), Slug and matrix metalloproteinase (MMP2). The Western blotting analysis also revealed that the levels of HDAC1 and HDAC2 were unchanged and interacted with the promoters of vimentin, Slug and ZEB1 following treatment with trichostatin A (TSA) and SAHA. These findings suggested that the activity of HDACs was repressed, but the amount of HDACs were unchanged . Using iOf the few studies examining changes in angiogenesis elicited by HDAC inhibitors, most of the studies have focused on modulating the expression levels of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor receptor (VEGFR)-2 and vascular endothelial growth factor (VEGF)-A in cancer cell lines ,106. MixVarious class I and II HDACs exert anti-angiogenic effects . KruppelThe studies discussed above demonstrate that HDACs can in fact exert anti-tumorigenic effects, as seen in studies discussed above. Resistance to HDAC inhibitors has been explored in preclinical models of various cancers, however, it has not been explored in the clinical setting . Four coP-gp, encoded by the gene ABCB1/MDR1, is an ATP-dependent efflux pump on the cell membrane which has a broad substrate specificity and expels drugs out of cells . In 198950 values obtained from cytotoxicity assays for romidepsin were proportional to the ABCB1/MDR1 mRNA level (r2 > 0.9) [The upregulation of P-gp is an impediment to using HDAC inhibitors in combination with other chemotherapeutic agents. Not only are the chemotherapeutic agents such as doxorubicin expelled, but certain HDAC inhibitors are also P-gp substrates . In fact2 > 0.9) . Xiao an2 > 0.9) . However2 > 0.9) . While c2 > 0.9) . Additio2 > 0.9) . As evid2 > 0.9) .50 values between the non-resistant, HL-60, and the resistant, HL-60/LR, cell line for LAQ824, SAHA and panobinostat, respectively. The doubling time of HL-60 cells was 24 h, whereas HL-60/LR cells had a doubling time of 12 h [Changes in HDAC levels are another proposed mechanism for resistance. In a study conducted by Fiskus and colleagues (2008), HDACs 1, 2 and 4 were upregulated, while HDAC6 was downregulated in an HDAC inhibitor-resistant human leukemia cell line, HL-60/LR . This ag of 12 h . This wa of 12 h . MYST2, PHF17, PHF15, ING5, BRPF3, BRPF3, MUST4, ING3, EPC1, YEATS4, CSRP2BP, TAF6L and CREBBP) in response to SAHA and VPA [Acetylation of histone and non-histone proteins is regulated by a balance between HATs and HDACs . A study and VPA . Surpris and VPA . Similar and VPA . Functio and VPA . Surpris and VPA . No expl and VPA . Histone and VPA . Further and VPA . High bap21/Cdkn1a deletion were crossed with TRAMP mice. Two groups, p21−/− TRAMP and p21+/−/TRAMP, were compared to TRAMP mice. Dorsolateral prostates were histopathologically analyzed to assess the different stages of prostate tumorigenesis. In the p21−/− group, no incidence of moderately different (MD) tumors were seen, whereas 1/13 p21+/−/TRAMP mice showed MD tumors. In comparison, 5/15, 6/15 and 3/15 TRAMP mice showed well differentiated, MD and poorly differentiated prostate adenocarcinoma lesions [P21 upregulation leads to a decrease in cyclin D1, which is essential for middle G1 phase transitioning . Given t lesions . P21 is  lesions . This co lesions . A pro-m lesions .Following HDAC inhibition, p21 levels have been upregulated in various cancers ,128. A p50 values with regards to specific HDAC class inhibition is lacking in most reports. In studies where specific HDAC class inhibitions have been measured, IC50 values across studies can vary as much as 100-fold [Currently, two layers of information are lacking in studies conducted on HDAC inhibitors in cancer. Firstly, the expression profiles of different HDACs in prostate cancer models are absent. Although attempts to study expression profiles have been made, the data are contradictory and incomplete. This is important because different HDACs exhibit opposing pro- and anti-tumorigenic functions. The effect produced by the different HDACs may also lead to the upregulation of different resistance mechanisms. Recently, studies have also shown that HDACs deacetylate non-histone proteins, which in turn can lead to more varied effects ,131. Sec100-fold ,133. The100-fold . Example100-fold . An alteAnother piece of the puzzle that is missing in terms of understanding the role of HDACs in prostate cancer is the involvement of the AR. Recently, the emergence of an AR- prostate cancer has been reported ,97. Regu


Throughout the last decades, dendritic cell (DC)-based anti-tumor vaccines have proven to be a safe therapeutic approach, although with inconsistent clinical results. The functional limitations of ex vivo monocyte-derived dendritic cells (MoDCs) commonly used in these therapies are one of the pointed explanations for their lack of robustness. Therefore, a great effort has been made to identify DC subsets with superior features for the establishment of effective anti-tumor responses and to apply them in therapeutic approaches. Among characterized human DC subpopulations, conventional type 1 DCs (cDC1) have emerged as a highly desirable tool for empowering anti-tumor immunity. This DC subset excels in its capacity to prime antigen-specific cytotoxic T cells and to activate natural killer (NK) and natural killer T (NKT) cells, which are critical factors for an effective anti-tumor immune response. Here, we sought to revise the immunobiology of cDC1 from their ontogeny to their development, regulation and heterogeneity. We also address the role of this functionally thrilling DC subset in anti-tumor immune responses and the most recent efforts to apply it in cancer immunotherapy.  The manipulation and education of the immune system for targeting and eliminating cancer cells has been viewed as a crucial goal of cancer therapy for decades ,2,3. TheDCs have been clinically used for three decades, with more than 300 completed or ongoing registered clinical trials conducted to test their application for boosting anti-tumor immunity . DCs are+ monocytes (MoDCs), cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) [Currently, there are four approaches for exploring DCs in cancer immunotherapies: (1) non-targeted protein and nucleic acid-based vaccines; (2) antigens targeting endogenous DCs; (3) ex vivo generated DCs matured and loaded with tumor antigens; and (4) biomaterial-based platforms for the in situ recruitment and reprogramming of endogenous DCs ,16. Amon4 (IL-4) . Althoug4 (IL-4) . The com4 (IL-4) ,18. In f4 (IL-4) ,22,23,24+ T cells towards the Th1 subset, to cross-present tumor antigens to CD8+ T cells and to both interact with and activate NK cells [+ T cells, causing their differentiation into antigen-specific CTLs [In the past two decades, the increasing knowledge on DCs and tumor biology has demonstrated that DCs protective role is highly dependent on their ability to effectively polarize CD4NK cells ,25. CTL-fic CTLs ,27. Thenfic CTLs . Hence, fic CTLs ,30. It ific CTLs ,32. + T cells trigger the activation and activity of tumor-infiltrating macrophages and are necessary to the adequate establishment of long-term memory CD8+ T cells [Efficient anti-tumor immunity also relies on the DC production of IL-12p70, which potentiates Th1 polarization and the capacity of this effector T cell population to secrete Interferon gamma (IFN-γ). Th1 lymphocytes directly eliminate tumor cells and, through the production of IL-21 and IL-2, support the differentiation and expansion of antigen-specific CTLs . Moreove T cells ,35,36. + and CD8+ T cells, indirectly improving anti-tumor immunity [Finally, it has been shown that NK cells actively participate in tumor immunosurveillance ,38,39,40immunity . Furtherimmunity , mature immunity . Therefore, the criteria that DCs should fulfil to produce a potent anti-tumor vaccine are challenging and include enhanced cross-presentation, the production of IL-12p70, the expression of high levels of co-stimulatory molecules and a strong capacity to interact with NK cells. + CLEC9A+ classical/myeloid DCs (cDC1), CD1c (BDCA1)+ classical/myeloid DCs (cDC2s) and CLEC4C (BDCA2)+ CD123+ plasmacytoid DCs (pDCs) [DCs are a class of bone-marrow-derived cells present in blood, epithelial, interstitial and lymphoid tissues, originated from lympho-myeloid hematopoiesis through a series of differentiation processes ,46. Humas (pDCs) ,47,48,49+XCR1+ DCs (the homologues of murine CD8α+ DCs), besides being highly proficient in executing cross-presentation, produce large amounts of IL-12 and IL-15. Therefore, they excel in promoting the proper activation of NK and natural killer T (NKT) cells and priming CTLs [+ XCR1+ DCs or enhance their functions constitute a very promising and thrilling goal in the field of cancer therapy. Lymphoid organ-resident cDC1, cDC2 and pDCs were shown to possess a comparable capacity to cross-present soluble antigens, while only cDC1 and cDC2 effectively cross-present dead cell-derived material . Notablying CTLs ,51,52,53In this review, we summarize the actual knowledge and most recent advances in the understanding of cDC1 biology and their clinical applicability in the context of cancer immunotherapy. Ontogeny studies on murine models are beginning to unravel the development of DC subsets; however, the translation of these discoveries to human biology is not always straightforward . Human D+ progenitors [+ progenitors [cDC1 population development is under the control of a set of key transcription factors, namely IRF8, BATF3, GATA2, PU.1, GFI1 and Id2 ,56,60,61genitors ,56,63. Fgenitors ; by blocgenitors . These dgenitors . + DCs with their murine counterpart CD8+/CD103+ DCs, many other markers have been recognized to allow for a more accurate identification of this specific population. The cell surface expression of the C-type-lectin CLEC9A  and the presence of the adhesion molecule CADM1 (NECL2) and the protein BTLA (CD272) substantially increase the preciseness of the identification [+ DCs can also be characterized by the expression of the chemokine receptor XCR1 [cDC1 are approximately ten times less abundant than cDC2, being present in the blood, lymph nodes, tonsils, spleen, bone marrow and non-lymphoid tissues, such as the skin, lung, intestine and liver ,46,49. Tfication ,46,49,64fication ,46,49,64fication ,46. Betwtor XCR1 ,64,65.+ CADM1+ CD141+ lineage in the blood was also reported, splitting this population between XCR1− CXCR4hi and XCR1+ DCs [− cells, this DC subset proliferated and acquired XCR1 expression, indicating that these cells are immediate precursors of XCR1+ cDC1 [+ DCs: a pre-cross-presentation phase and a subsequent cross-presenting stage, in which cDC1 acquire the capacity to cross-present antigens due to the GM-CSF-mediated expression of XCR1  are the most effective human cross-presenting cells and thus potent CTL inducers [+ DCs [+ T cells at a steady-state, being enhanced during infectious and inflammatory responses [+ T cells with CD141+XCR1+ DCs, which amplifies their activation state [+ T lymphocytes abundantly secrete XCL1 after in vivo antigen recognition through CD8+ DC presentation, increasing the number of antigen-specific CD8+ T cells and their capacity to secrete IFN-γ. In contrast, the absence of XCL1 was shown to impair cytotoxic responses to antigens cross-presented by CD8+ DCs [+ DC maturation, which in turn provides support for T cell survival and functioning [In light of our current knowledge on DC immunobiology, cDC1  produced by pDCs improve the maturation and cross-presentation by CD141+XCR1+ DCs, thus enhancing the development of the CD8+ T cell response [+XCR1+ DCs are needed for the optimum expansion of memory CTLs in response to most pathogens. Moreover, the reactivation of memory CTLs relies on their interactions with CD141+XCR1+ DCs, an event that is triggered by the DC production of IL-12 and CXCL9 in response to NK cell-derived IFN-γ [CD141fections . During response . In the ed IFN-γ . Batf3–/– mice, have revealed that these cells may play a central role [+ DC as responsible for priming CTL responses [The relevance of cDC1 subsets in tumor immune surveillance has not yet been fully established; however, collected data from experiments with cDC1-deficient animal models, such as ral role . In factral role ,77,78,79esponses ,81. + cDC1 precursors that were recruited to the sentinel lymph nodes by type I IFN and afterward maturated under the combined effect of CpG and GM-CSF. The presence of in vivo CpG/GM-CSF-induced CD141+DCs in sentinel lymph nodes was correlated with an increased cross-presenting capacity, T cell infiltration and patient survival [Although cDC1 are a scarce immune population in the tumor microenvironment, their abundance positively correlates with patient survival across several cancers and is an indicator of the responsiveness to therapy with immune checkpoint inhibitors ,78,79,82survival . Concordsurvival .+ T cells predominantly occurring in tumor-draining lymph nodes, cDC1 also play an important role in orchestrating local anti-tumor immunity [+ effector cells, such as T cells, NK cells and innate lymphoid cells (ILC1)   [85,86,8survival ,92. Thissurvival . Althougsurvival .+XCR1+ DCs in peripheral blood has hindered their broader study and clinical exploitation. CD141+ DCs represent only ~0.03% of human PBMCs, meaning that their isolation would not result in a sufficient quantity for their use as a therapeutic vaccine [+ DC populations. For these reasons, anti-tumor vaccines based on naturally circulating pDCs and cDC2 have already been directly assayed in several clinical trials with encouraging results , while there has been no registered trial for cDC1 until the present (February 2020) [Given the functional specificities of cDC1, strategies aimed at their mobilization to the tumor microenvironment, as well as their expansion and activation, are viewed as highly promising approaches for boosting anti-tumor immunity and improving the success of cancer immunotherapies . However vaccine . Furtherry 2020) ,97,98,99ry 2020) ,101.+ and CD4+ T cells, resulting in an 30% extent of survival [4 cells as opposed to 105–3 × 106 cells) [Nonetheless, pre-clinical studies have been carried out in order to explore the use of cDC1 in prophylactic and therapeutic vaccines. The vaccination of mice with syngeneic spleen cDC1, ex vivo-loaded with tumor lysates, caused a long-lasting effect in preventing the growth of B16-ovalbumin (OVA) melanoma cells engrafted 30 days after vaccination. Moreover, the progression of tumors in melanoma-bearing mice was halted as a consequence of the increased infiltration of CD8survival . The comsurvival . Similar6 cells) .+CLEC9A+ DCs could be generated from in vitro expanded cord blood CD34+ precursors by culturing them for 12 days in RPMI supplemented with 2-mercaptoetanol, 10% heat-inactivated fetal calf serum (FCS), SCF, GM-CSF, IL-4 and Flt3L [+CLEC9A+ DCs obtained was low (3–6%), but these cells were nevertheless shown to present a molecular signature characteristic of blood cDC1, such as the expression of Necl2, CD207, BATF3 and IRF8. Additionally, they respond to TLR3 and TLR8 ligands and efficiently internalize material from dead cells, cross-presenting the processed antigens to CD8+ T cells [+XCR1+ DCs, such as the expression of CD141, XCR1, CLEC9A and the adhesion receptor CADM1, as well as the expression of several genes involved in crosstalk with NK and T cells. Moreover, XCR1+ CD34-derived DCs were shown to have the ability to respond to TLR3 ligands and superiorly cross-present cellular antigens when compared to their XCR1− counterparts or MoDCs [+ cells for 3 weeks in serum-free medium, supplemented with Flt3L, SCF, TPO, IL-6 and the aryl hydrocarbon receptor antagonist StemRegenin 1, was shown to produce a mixed population of functional cDC1, cDC2 and pDCs [+ DCs express XCR1 and CLEC9A, indicating that their identity is undoubtedly distinct from endogenous cDC1.To overcome the limitation imposed by the low abundance of human primary cDC1, great efforts have been made to optimize novel protocols for their ex vivo differentiation. Poulin et al. reported, for the first time, that functional CD141nd Flt3L . The per T cells . These p T cells ,48,104. or MoDCs . In anotand pDCs . Notablyand pDCs . MA- andand pDCs . Of note+ XCR1+ DCs have been successfully obtained from HLA-A*020(+) iPSCs, with these cells being capable of performing cross-presentation and generating antigen-specific CTL responses [Another strategy consists on the ex vivo differentiation of DCs from induced pluripotent stem cells (iPSC). CD141esponses . As theyesponses . These c+ DCs, in the absence of exogenous adjuvant, protected mice against OVA-expressing melanoma cells in prophylactic and therapeutic settings [+ DCs cross-presentation capacity and to enhance CTL responses, promoting tumor elimination, both in prophylactic and therapeutic contexts [+ and CD8+ T cell proliferation and antibody responses, hampering tumor growth while increasing survival in breast cancer models [While many obstacles still stand in the way of ex vivo manipulation of cDC1 for clinical purposes, in vivo targeting and modulation of these cells is viewed as an approach with plenty of translational potential. Most of these strategies rely on the selective expression of cell surface markers, such as CLEC9A or XCR1, enabling their specific targeting with mAbs ,110,122.settings . Furthersettings ,116,119.contexts ,115,119.contexts . In an a+XCR1+ DCs, enhancing their CD8+ T cell priming capacity and thus boosting anti-tumor immune responses [+ DCs in secondary lymphoid organs [Besides direct targeting approaches, endogenous cDC1 populations could be indirectly modulated by other cell-based immunotherapies. In accordance with this, the administration of IFNα-producing iPSC-derived proliferating myeloid cells (pMCs) to mice strongly activated host CD141esponses . Furtheresponses . On the d organs . Due to + XCR1+ DCs. Two phase I clinical trials (NCT00062855 and NCT01713439) studied the safety and dosage of an injection of neuroblastoma cancer cells that were genetically engineered to express XCL1 and IL-2, with the first using autologous cancer cells and the second using the allogenic SJNB-JF-IL2/Lptn cell line. Both approaches aimed to enhance the immune response at the tumor site and thus lead to a more effective elimination of cancer cells in relapsed/refractory neuroblastoma [Other approaches have been assessed in a few clinical trials, such as increasing the expression of XCL1 within the tumor in order to recruit CD141blastoma ,118. ProIn recent years, cDC1 have been pointed out as critical elements in the initiation of tumor-specific T cell responses, as well as in sustaining the effector activity of T and NK cells within the tumor microenvironment. However, further studies are essential for deeply understanding the biology of this DC subset in human tumors, particularly its dynamic during cancer progression and how it is conditioned by tumor immunosuppressive mechanisms. + hematopoietic progenitors and from iPSCs, or the direct reprogramming of somatic cells, followed by antigen loading and administration; direct antigen targeting in endogenous cDC1; and strategies aiming to enhance their frequency and functions in tumors. Among these strategies, only cancer cells genetically engineered to express XCL1, and thus to attract cDC1, have been tested or are being currently evaluated in ongoing clinical trials [Despite the difficulties created by their extremely low-abundance, several approaches are being tested in order to exploit cDC1s in anti-tumor immunotherapies: ex vivo differentiation from CD34l trials ,118. It l trials ,125. Conl trials ) (FigureThe use of cDC1 in a therapeutic research context is still in the beginning stage, yet the perspective of exploiting this DC subset to improve anti-tumor immunotherapy is extremely encouraging and validation in future clinical trials is highly anticipated.


Rare hereditary anemias (RHA) represent a group of disorders characterized by either impaired production of erythrocytes or decreased survival . In RHA, the regulation of iron metabolism and erythropoiesis is often disturbed, leading to iron overload or worsening of chronic anemia due to unavailability of iron for erythropoiesis. Whereas iron overload generally is a well-recognized complication in patients requiring regular blood transfusions, it is also a significant problem in a large proportion of patients with RHA that are not transfusion dependent. This indicates that RHA share disease-specific defects in erythroid development that are linked to intrinsic defects in iron metabolism. In this review, we discuss the key regulators involved in the interplay between iron and erythropoiesis and their importance in the spectrum of RHA. Rare hereditary anemias (RHA) are a heterogenous group of diseases caused by genetic defects, resulting in impaired production or increased destruction of red blood cells (RBCs). The incidence of hereditary anemias varies, with hereditary spherocytosis, thalassemia and sickle cell disease being the more common, while Diamond-Blackfan anemia, congenital dyserythropoietic anemia and pyruvate kinase deficiency are among the rarer anemias ,2,3,4. IIron plays an important role in a large variety of cellular processes, yet excess iron can lead to generation of reactive oxygen species which may damage cellular components . The regFam132b, and is also known as myonectin or CTRP15 [Erythropoiesis has a great demand for iron, which is mainly incorporated in red blood cell hemoglobin. Erythroblasts highly express Transferrin receptor (Tfr1), and its expression is further upregulated during increased erythroid drive . During r CTRP15 . It is sr CTRP15 ,26. Uponr CTRP15 . Hence, r CTRP15 . Anotherr CTRP15 ,31. Whilr CTRP15 ,32,33. Hr CTRP15 ,35,36. AHBB) and is characterized by hemolytic anemia and ineffective erythropoiesis [Beta-thalassemia  is a hemoglobinopathy caused by a pathogenic mutation in one or both beta-globin genes  gene, and is characterized by chronic hemolytic anemia [Pyruvate kinase deficiency (PKD) is a congenital glycolytic enzyme defect, caused by mutations in the Liver and Red blood cell Pyruvate kinase  of hemoglobin [In alpha-thalassemia , hemolytic anemia is the result of mutations, mainly deletions, affecting one or more of the four genes encoding for alpha-globin chains , chronic anemia is accompanied by hepatosplenomegaly, jaundice and secondary complications, including pulmonary hypertension and IO [Congenital dyserythropoietic anemia (CDA) is a group of hereditary anemias characterized by a defect in proliferation and differentiation of erythroid precursors (dyserythropoiesis). Patients may present with hyporegenerative anemia, typical morphological abnormalities and hemolysis ,57,58. Bn and IO ,61,62. Tn and IO ,64. In Cn and IO ,63. WherSEC23B gene, encoding for SEC23B protein, which plays a role in the cellular secretory pathway [CDA type II is the most frequently occurring type of CDA. It results from biallelic mutations in the  pathway ,66,67. C pathway ,58,68. I pathway . GDF-15  pathway . In a st pathway . Further pathway . In concKIF23 , KLF1  or X-linked mutations in GATA1  [Other very rare types of CDA are caused by autosomal dominant mutations in  anemia) ,57,58. I anemia) . Interes anemia) ,74,75. T anemia) . Therefo anemia) ,77. ALAS2 , followed by ABCB7 (ATP Binding Cassette Subfamily B Member 7), SLC25A28 (Solute Carrier Family 25 Member 28) and GLRX5 (Glutaredoxin 5). These genes are involved in heme biosynthesis, iron-sulfur cluster biosynthesis and mitochondrial protein synthesis [Sideroblastic anemias (SA) represent a group of inherited and acquired disorders characterized by iron accumulation in the mitochondria of erythroid precursors, leading to an abrogated maturation of erythroblasts. Congenital sideroblastic anemia (CSA) is most frequently caused by X-linked mutations in ynthesis . Whereasynthesis . A recenynthesis . While Iynthesis . The rolynthesis ,83,84. Aynthesis  Moreoverynthesis  Based onPIEZO1 gene  or KCCN4 gene  [PIEZO1-mutated DHS, while ERFE levels were only slightly elevated, suggesting an additional regulatory mechanism of hepcidin suppression and subsequent iron loading in DHS [2+ influx through the mutated PIEZO1 channel directly decreased HAMP gene expression [PIEZO1 DHS. In conclusion, iron loading is common in DHS. Besides ERFE-induced hepcidin suppression, mutations in PIEZO1, and possibly in KCCN4, directly alter iron metabolism, resulting in hepcidin suppression.Dehydrated Hereditary Stomatocytosis (DHS)/Hereditary Xerocytosis (HX) is characterized by increased cation permeability in the red cell membrane, resulting in abnormal hydration, morphological changes and chronic hemolysis . DHS/XS , DHS 2) ,89. Inte, DHS 2) . A receng in DHS ,92. It wpression . While vIn Sickle Cell Disease (SCD), caused by biallelic specific amino acid changes  or compound heterozygous mutations including hemoglobin S (HbS) combined with hemoglobin C (HbSC) or beta-thalassemia , the formation of hemoglobin S (HbS) causes RBCs to sickle under certain conditions, leading to vaso-occlusion, inflammation and hemolysis ,94. InteDiamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome which is characterized by hypoplastic anemia (reticulocytopenia), congenital malformations and cancer predisposition . Since DSPTA1 , SPTB (beta-spectrin), ANK1 (Ankyrin 1), SLC4A1 (Band 3 or solute Carrier Family 4) and EPB42 (Erythrocytic Protein 4.2) [Hereditary spherocytosis (HS) is a relatively common hereditary hemolytic anemia caused by defects in the cellular membrane of RBCs  The majoein 4.2) ,113. IO ein 4.2) ,115. In ein 4.2) . Interesein 4.2) . Splenicein 4.2) . GeneticHere, we have given an overview of important regulators of iron metabolism in the context of RHA  erythropoiesis and hepcidin expression. By determining the rate of erythropoietic drive and degree of ineffective erythropoiesis, the risk of IO could be estimated. At the same time, the knowledge of specific regulators in distinct disease entities, and the direct effect of IO on the quality of erythropoiesis is still largely unknown and primarily based on relatively small case series. In order to improve our understanding of the interplay between erythropoiesis and IO, international consensus guidelines for the screening, investigating and monitoring of IO in combination with uniformal registration of patients with RHA are a prerequisite.


The obtained data showed that just the(CA)n, (GA)n, (CAG)n and (GATA)nmicrosatellites were located on sea turtle chromosomes, preferentially inheterochromatic regions of the microchromosomes (mc). Variations in the locationof heterochromatin and microsatellites sites, especially in some pericentromericregions of macrochromosomes, corroborate to proposal of centromere repositioningoccurrence in Cheloniidae species. Furthermore, the results obtained with thelocation of microsatellites corroborate with the temperature sex determinationmechanism proposal and the absence of heteromorphic sex chromosomes in seaturtles. The findings are useful for understanding part of the karyotypicdiversification observed in sea turtles, especially those that explain thediversification of Carettini from Chelonini species.The wide variation in size and content of eukaryotic genomes is mainly attributedto the accumulation of repetitive DNA sequences, like microsatellites, which aretandemly repeated DNA sequences. Sea turtles share a diploid number (2n) of 56,however recent molecular cytogenetic data have shown that karyotype conservatismis not a rule in the group. In this study, the heterochromatin distribution andthe chromosomal location of microsatellites (CA) Dermochelyscoriacea (leatherback sea turtle), and is the sister-taxon to a cladecomprising all other extant sea turtles (i) Chelonini grouping Natatordepressus (flatback sea turtle) and Chelonia mydas(green sea turtle) and; (ii) Carettini groupingLepidochelys olivacea (olive ridley sea turtle),Lepidochelys kempii (Kemp’s ridley sea turtle),Eretmochelys imbricata (hawksbill sea turtle) and,Caretta caretta (loggerhead sea turtle) (Sea turtles (Testudines: Cryptodira) are grouped into Dermochelyidae and Cheloniidaefamilies and only seven living species . DermochAll sea turtle species show different levels of threat of extinction and areconsidered flag species for the conservation of biodiversity  since nui) high 2n (60 - 68 chromosomes) and high amount of mc;(ii) intermediate 2n (50 - 56 chromosomes) and relatively lowamount of mc and; (iii) low 2n (26 - 28 chromosomes) and without mc(Testudines cytogenetic studies demonstrated karyotypes composed of macrochromosomesand a variable number of microchromosomes (mc), a characteristic shared with birdsand squamate reptiles  observed in turtles implies that theirgenomes have been deeply reorganized ,2020b. Heterochromatin is usually composed by an enriched repetitive DNA segment ofsatellite DNAs . SatelliC. mydas, L. olivacea,E. imbricata and, C. caretta, which ruled outthe proposal of conserved structure of the macrochromosomes in Cheloniidae  C. mydas , (ii) C. caretta , (iii) E.imbricata , and(iv) L. olivacea . The collection samples were authorized by the Instituto Chico Mendesde Conservação da Biodiversidade .All experimental procedures were authorized and performed following the EthicalCommittee on Animal Use of the Universidade Estadual de Ponta Grossa. (Protocol:7200/2016).an coast . Fifty sin situ hybridization (FISH) assays, usingmicrosatellites probes.Peripheral blood was used to obtain the chromosomal preparations by temporaryculture of lymphocytes method  and tof15, (GA)15, (CAG)10, (GATA)8,(GAA)10, (CGC)10 and (GACA)8microsatellites probes were directly labelled with Cy5 fluorochrome at the 5’ end during DNA synthesis.FISH was performed according to the protocol proposed by The (CA)Chromosomes were arranged by decreasing size and centromere position, asdescribed by Chelonia mydas, C. caretta, E.imbricata and L. olivacea showed fewheterochromatic blocks in the karyotypes, as follows: (i)C. mydas showed heterochromatic blocks in thepericentromeric regions of macrochromosomes 2-4, 8, 11 and 12, besides in thepericentromeric regions of mc long arms 13-19, 21, 23 and 25 (ii) C. caretta hasheterochromatic blocks in the pericentromeric regions of macrochromosomes 3 and8, as well as in the pericentromeric region of mc 13 and, in the long arm of mc14, 17 and 20 (iii) E.imbricata showed heterochromatic bands located in thepericentromeric regions of the macrochromosomes 1-3 and 12, besides in thepericentromeric regions of mc 13, 14, and 17 and, in the subterminal region ofthe long arm of mc 16, 18 and 19 (iv) inL. olivacea heterochromatin is located in thepericentromeric regions of the macrochromosomes 2-4, 6 and 9-12, as well as inthe pericentromeric regions of mc 13, 15, 17-19 and 21 and, in the long arms ofmc 14, 20, 23 and 25 10 and (GACA)8microsatellites probes were not detected on the chromosomes of these fourspecies by FISH procedure. The microsatellite (CA)n was located as ablock in the short arms of the chromosome pairs 4 and 16 in C.mydas (Caretta caretta has(CA)n block detected in mc 13 and dispersed in mc 17 n signals werevisualized as a block in chromosome pairs 2, 4, 13 and 16, and dispersed alongchromosome pair 9 (Lepidochelys olivaceahas (CA)n markers detected as a block in the short arms of theacrocentric pair 4, besides dispersed signals along the chromosome pairs 4 and14 nsignals were located dispersed along the chromosome pairs 8, 13, 17 and 22nsignals were detected in the mc 13, 16, 17 and 19 olivacea and 5. T9 and 21  and 6.  is made up of systematic intandem repeats favoring the occurrence of ectopic recombination and genic conversion split from Carettini about 34 million years ago(Mya), and in Carettini, Eretmochelys split fromCaretta and Lepidochelys about 23 Mya  in ahomeologue chromosome pair among phylogenetically related species may be the resultof chromosomal rearrangements n, (CAG)n and (GATA)n microsatellites werereported for the first time in sea turtle’s karyotypes and demonstrated that most ofthem possibly make up part of the repetitive units of heterochromatin in C.mydas, C. caretta, L. olivacea and E.imbricata, especially in mc. Furthermore, the chromosomal location ofmicrosatellites sequences in the pericentromeric regions for some chromosomes iscommon in species groups close to turtles, such as birds and crocodilians  locus, except for E. imbricata. Studies haveshown that the frequent association between microsatellites and NOR regions isexplained by large number of microsatellites in rDNA intergenic spacers (The microsatellite (GA) spacers . Furthern signals in the chromosomes of C.caretta demonstrated that this repetition may have contracted (deletionof repetitive units) in the genome n and (GATA)n sites also were lowerin C. caretta when compared to C. mydas,E. imbricata and L. olivacea, corroboratingthat C. caretta has a probable divergent karyotype evolution fromthe other Carettini species.The absence of (CAG)e genome  to a redn sequence can be found in the promoter region of theDoublesex and mab-3-related transcription factor 1(Dmrt1) gene n in threespecies of turtles with TSD, using both sexes, detected markers preferably in mc andwithout evidence of sex-specific chromosomal sites (In situlocation of (GATA)n in C. mydas, C.caretta, E. imbricata and L. olivaceashowed accumulations in mc pairs, in addition to a macrochromosome in E.imbricata. However, the absence of size heteromorphism betweenhomeologous chromosomes carrying (GATA)n sites reinforces the conditionof the absence of heteromorphic sex chromosomes in this group.(GATA)elopment , in whicelopment . AlthougC. caretta.Furthermore, heterochromatic bands and microsatellite sites showed chromosomaldifferences among C. mydas, C. caretta, E.imbricata and L. olivacea karyotypes. These findingsencourage further research on the chromosomal evolution in sea turtle species aimingto understand the diversification of Cheloniidae karyotypes.In conclusion, this study corroborates the proposal of the centromere repositioningto explain chromosomal morphology alterations in sea turtle diversification,addressing important concerns to absence of pericentromeric heterochromatin inmacrochromosomes of Carettini, especially in


FCGR1A encodes a protein that plays an important role in the immune response. The prognostic impact and immune infiltration of FCGR1A in heterogeneous cancers remain unclear.Differential expression of FCGR1A between tumor and normal tissues and the discrepancies in overall survival (OS) among diverse cancer types were performed by Gene Expression Profiling Interactive Analysis. The correlation between FCGR1A and immune cells or gene marker sets of immune infiltrates was analyzed via Tumor Immune Estimation Resource (TIMER). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and protein-to-protein interaction (PPI) network were used to explore the function and related genes of FCGR1A. The relationships among these genes were further analyzed by TIMER.P < 0.05). High expression of FCGR1A meant a better prognosis besides KIRC. FCGR1A showed significant differences at different stages of KIRC and SKCM (P < 0.05). Furthermore, FCGR1A was notably associated with infiltrating levels of CD4+ T cells, CD8+ T cells, B cells, macrophages, neutrophils, and dendritic cells in the four cancers (P < 0.05). FCGR1A also showed close relevance with different immune gene markers. The copy number variation of FCGR1A significantly influenced the abundance of immune infiltration in KIRC and SKCM. GO, KEGG analysis, and PPI network analysis revealed that FCGR1A is involved in many pathophysiological processes and was most related to FCGR3A. And this gene indicated highly significant positive correlations with FCGR1A in four cancers.FCGR1A is highly expressed in various cancer types. FCGR1A was significantly correlated with the OS of cervical and endocervical cancer (CESC), cholangiocarcinoma (CHOL), kidney renal clear cell carcinoma (KIRC), and skin cutaneous melanoma (SKCM) (FCGR1A may be a potential prognostic biomarker and related to immune infiltration levels in diverse cancers, especially in CESC, CHOL, KIRC, and SKCM. Besides, FCGR1A may be involved in the activation, regulation, or induction of immune cells and diverse physiological and pathological processes. FCGR1A (CD64) is a 72-kDa transmembrane glycoprotein with CD32 and CD16 receptors . It encoCervical cancer is the fourth most common cancer among women worldwide, with a high mortality rate, especially in developing countries . High-riIn this study, we comprehensively analyzed the relationship between the expression of FCGR1A and prognosis of various cancer types using the Gene Expression Profiling Interactive Analysis (GEPIA) database. Besides, Tumor Immune Estimation Resource (TIMER) was utilized to study the correlation between FCGR1A and tumor-infiltrating immune cells. We also elaborated the important role of FCGR1A in cervical and endocervical cancer (CESC), CHOL, KIRC, and SKCM, as well as revealing a potential relationship among FCGR1A, tumor–immune interactions, and neighboring genes.1. GEPIA is an open web that contains 9,736 tumor and 8,587 normal samples from The Cancer Genome Atlas (TCGA) and GTEx projects, which mainly including 33 cancer types  cells, DCs, T-helper 1 (TH1) cells, regulatory T lymphocytes (Tregs), and exhausted T cells. Besides, the relationship between the copy number variation (CNV) of FCGR1A and tumor immune infiltrating level can also be analyzed easily. FCGR1A and relevant marker genes were placed on x- and y-axis, respectively, to depict scatter plots and obtain correlation coefficient as well as P values. The expression level was exhibited with log2 TPM.Tumor immune estimation resource is a public resource dedicated to tumor immune infiltrates across 32 cancer types incorporating 10,897 samples from TCGA2 . This we2 . Differe2 . The gen3. STRING database is an online software that could perform a synthetic analysis of the direct or indirect associations among selected genes. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of FCGR1A were performed using the STRING, and related results were obtained. The protein-to-protein interaction (PPI) networks were also constructed using the protein query function in STRING. We further analyzed the potential relationships between FCGR1A and the most related genes via TIMER database.Related genes with FCGR1A expression were conducted by STRING databaseP values, correlation score, or false discovery rate. The correlation of FCGR1A expression was assessed by Spearman correlation and statistical significance. In addition, the strength of the correlation should follow the following criteria: 0.00–0.19 (very weak or none), 0.20–0.39 (weak), 0.4–0.59 (moderate), 0.6–0.79 (strong), and 0.8–1.0 (very strong). P < 0.05 was regarded as statistically significant.Survival curves were depicted by GEPIA. The results of GEPIA, TIMER, and STRING were displayed with hazard ratio (HR), To determine the differential expression of FCGR1A in various cancer locations, we used RNA-seq data from TCGA database to explore the relationship between prognosis and FCGR1A expression in multiple cancer types via GEPIA. Results of FCGR1A expression between tumor and normal tissues are shown in p = 0.012), CHOL , and SKCM , and also showed better DFS in CHOL . However, low expression of FCGR1A in KIRC was more beneficial to survival . FCGR1A expression levels at different stages of the four aforementioned cancer types were also analyzed. Significant differences were observed in the FCGR1A expression levels between different histological stages in KIRC (P = 4.76e-10) and SKCM (P = 0.00141). However, no significant differences were detected in CESC and CHOL . However, relevant between FCGR1A and diverse immune cells varied substantially in different cancer types. FCGR1A had the highest correlations with DCs among CESC, KIRC, and SKCM, which were 0.682, 0.704, and 0.754, respectively. In CHOL, FCGR1A was more closely related to neutrophils (cor = 0.778). We also analyzed the correlation between FCGR1A and immune marker genes of various immune cells. The results showed that FCGR1A was notably related to most immune marker genes in CESC, CHOL, KIRC, and SKCM after purity adjustment  and SKCM (cor = 0.934). CD86 also showed a close relation with four cancers, especially in SKCM (cor = 0.860). Nevertheless, it indicated very weak or even no correlation with four cancers in B cells, M1 macrophages, and NK cells.To understand the correlation between FCGR1A and different infiltrated immune cells including CD4and SKCM . All immjustment . And FCGjustment . In addi+ T cells, CD4+ T cells, macrophages, neutrophils, and DCs (P < 0.05). Moreover, high amplification was related to the level of macrophage infiltration (P < 0.05). In SKCM, arm-level gain was prominently correlated with six immune cells, whereas arm-level deletion was related only to B cells, CD4+ T cells, macrophages, and DCs (P < 0.05). No association was observed between CNV and immune cell infiltration in the other two cancer types (P > 0.05)  .STRING was utilized to construct the PPI network, and GO and KEGG analyses were performed to identify the enrichment of FCGR1A in GO terms and metabolic pathways . The GO FCGR1A (CD64) is the single high-affinity receptor of immunoglobulin G (IgG) in human. The expression of FCGR1A is closely related to immune inflammation. Although there are few extensive and systematical studies about CD64, its high sensitivity and specificity for the diagnosis of sepsis and systemic infection had been demonstrated, with a sensitivity of greater than 90% and specificity of 90–100% in adults and children . BesidesWe explored the expression levels of FCGR1A in 33 cancer types using TCGA data in GEPIA. The significant differential expression of FCGR1A was observed in many cancer types. In contrast to the normal tissue, FCGR1A expressed highly in BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, KIRC, KIRP, LAML LGG, OV, PAAD, PRAD, READ, SKCM, STAD, TGCT, THCA, UCEC, and UCS, but lowly in LUAD, LUSC, and THYM. Moreover, FCGR1A expression levels were significantly correlated with the OS of CESC, CHOL, KIRC, and SKCM and had notable differences between tumor and normal tissues in these four cancers. Besides, FCGR1A expression in KIRC and SKCM showed conspicuous discrepancies at different stages. Taken together with the above results, FCGR1A was strongly confirmed as a prognostic biomarker in CESC, CHOL, KIRC, and SKCM.H1) and may be involved in the regulatory role of T cells. Taken together, FCGR1A plays a crucial role in the activation and regulation of immune cells in CESC, CHOL, KIRC, and SKCM based on the above evidence.This study also indicated that FCGR1A was associated with multiple levels of immune infiltration in cancers. FCGR1A had the strongest correlation with DCs among six tumor-infiltrating immune cells in CESC, KIRC, and SKCM. It had higher relevancy with neutrophils in CHOL as well. Furthermore, significant positive relevance between FCGR1A expression and six immune cells was discovered in four cancers. CD3E, CD2, CD86, CD163, VSIG4, MS4A4A, HLA-DPB1, HLA-DRA, HLA-DPA1, and TIM-3 were found to be mainly gathering in T cells, monocytes, macrophages, and DCs were significantly correlated with FCGR1A. Similar results were validated in GEPIA. Therefore, the association of FCGR1A with diverse immune cell marker genes suggested its role in regulating tumor immunology in CESC, CHOL, KIRC, and SKCM. M1 macrophage gene markers (such as NOS2 and IRF5) had weak or no correlation with FCGR1A expression, while M2 macrophage markers CD163, VSIG4, and MS4A4A had strong correlations. These consequences indicated the potential regulatory role of FCGR1A in the polarization of TAMs. The increased expression of FCGR1A was positively correlated with the expression of Tregs and T cell exhaustion markers , suggesting that FCGR1A may play a role in the activation of Tregs and induction of T cell exhaustion. The FCGR1A was moderately or strongly associated with most immune marker genes in DCs, which can induce immune memory responses in cancer and promote anti-tumor immunity. Thus, the relationship between FCGR1A expression and the infiltration level of DCs may predict the prognosis of the disease and the response to therapies. TIM-3 is a vital surface protein on T cell exhaustion  and is hP < 0.05, ρ > 0)  was the receptor for the Fc region of IgG and bound complexed or aggregated IgG and monomeric IgG. It mainly mediated ADCC and other antibody-dependent responses . SYK was, ρ > 0) . Meanwhi network . AlthougSeveral studies on FCGR1A (CD64) mainly focused on the diagnosis and distinction of infectious diseases, but rarely on cancer. However, an experimental study published in 2013 pointed out that FcRI (CD64) could mediate the protective effect of anti-tumor antibodies on melanoma metastasis and may also have a protective effect on solid tumors . In addiH1, and T cell exhaustion. Thus, FCGR1A may contribute to the activation and regulation of the aforementioned immune cells to varying degrees. FCGR1A may play an important role in immune cell infiltration and serve as a prognostic biomarker for four cancers, especially in SKCM. Also, FCGR1A is involved in many pathophysiological processes and is closely related to FCGR3A, SYK, and HCK in various cancers. It seems that many questions need to be addressed by future research.In summary, high FCGR1A expression leads to different outcomes in diverse cancers. It is associated with better prognosis in CESC, CHOL, and SKCM besides CHOL. Meanwhile, FCGR1A is positively or even strongly correlated with immune cells and various immune cell marker genes in four cancer types, such as CD2, CD3E, CD86, CD163, VSIG4, MS4A4A, HLA-DPB1, HLA-DRA, HLA-DPA1, ITGAX, and TIM-3 in T cell, monocyte, M2 macrophage, DC, Thttp://gepia2.cancer-pku.cn/#general and https://cistrome.shinyapps.io/timer/.Publicly available datasets were analyzed in this study. This data can be found here: J-lX: conceptualization, formal analysis and investigation, and writing-original draft preparation. YG: conceptualization, methodology, writing-review and editing, and supervision. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Camelina sativa, an emerging oilseed crop. In this study, we identified 73 CsPHT members belonging to the five major PHT families. A whole-genome triplication event was the major driving force for CsPHT expansion, with three homoeologs for each Arabidopsis ortholog. In addition, tandem gene duplications on chromosome 11, 18 and 20 further enlarged the CsPHT1 family beyond the ploidy norm. Phylogenetic analysis showed clustering of the CsPHT1 and CsPHT4 family members into four distinct groups, while CsPHT3s and CsPHT5s were clustered into two distinct groups. Promoter analysis revealed widespread cis-elements for low-P response (P1BS) specifically in CsPHT1s, consistent with their function in Pi acquisition and translocation. In silico RNA-seq analysis revealed more ubiquitous expression of several CsPHT1 genes in various tissues, whereas CsPHT2s and CsPHT4s displayed preferential expression in leaves. While several CsPHT3s were expressed in germinating seeds, most CsPHT5s were expressed in floral and seed organs. Suneson, a popular Camelina variety, displayed better tolerance to low-P than another variety, CS-CROO, which could be attributed to the higher expression of several CsPHT1/3/4/5 family genes in shoots and roots. This study represents the first effort in characterizing CsPHT transporters in Camelina, a promising polyploid oilseed crop that is highly tolerant to abiotic stress and low-nutrient status, and may populate marginal soils for biofuel production.Phosphate transporters (PHTs) play pivotal roles in phosphate (Pi) acquisition from the soil and distribution throughout a plant. However, there is no comprehensive genomic analysis of the PHT families in  Phosphorus (P) is an essential macronutrient in all organisms, which provides a backbone to nucleic acids, a polarity to cell membranes, and participates in energy transfer and signal transduction processes ,2. Plant+/Pi symporters located in the plasma membrane of root cells for Pi uptake from the soil and translocation to the shoots [Arabidopsis, among which AtPHT1;1 and AtPHT1;4 are the key players in Pi uptake [In plants, phosphate transporters (PHTs) are classified into five major families, PHT1-5, based on their sequences and subcellular localizations ,4. The Pi uptake ,7. AtPHTi uptake ,9. AtPHTi uptake . Arabidopsis that cotransports H+/Pi into chloroplasts [Arabidopsis, AtPHT3;1–3;3 , that exchange Pi between the mitochondrial matrix and cytosol for ATP synthesis [AtPHT3;1/AtMPT3 in Arabidopsis resulted in a pleiotropic effect, suggesting a vital role of this gene in plant growth and development [+-dependent Pi export from plastids in cells of sink organs [trans-Golgi compartment to cytosol, essential for Golgi functions [The other PHT family transporters are found in different subcellular compartments and play important roles in Pi storage and remobilization. AtPHT2;1 is the only PHT2 member in roplasts ,12. The ynthesis ,14. Overelopment ,16. AtPHk organs . AtPHT4;unctions ,19,20,21unctions ,23,24.Camelina sativa , an emerging oilseed crop for biofuel production. Camelina is an annual plant adapted to temperate growing regions, with centers of origin in southeastern Europe and southwestern Asia [Arabidopsis thaliana, and more distantly to Brassica napus [. Camelina has attracted considerable interest as an oil crop, primarily due to its numerous valuable agronomic traits that support sustainable agriculture. For example, a short life cycle of approximately 85–100 days, low input costs and ease of transformation using a simple floral dip procedure similar to that used for Arabidopsis make Camelina an attractive host for genetic and metabolic engineering [Camelina has high tolerance to adverse environmental conditions , resistance to pests and pathogens, and high oil content with a fatty acid profile suitable for fuel purposes [Camelina has been shown to grow fairly well under P deficiency in field conditions [Camelina accessions to initiate functional studies on the Pi transport system in this crop species.With increasing effort in whole-genome sequencing, genome-wide identification of PHT families has been conducted in a number of plant species, including rice, maize, barley, potato, tomato, soybean, foxtail millet, sorghum, wheat, poplar, apple, and rapeseed ,35,36,37ern Asia ,39,40,41ca napus . Camelinineering ,39,41,42purposes . Althougnditions ,45, nothnditions , we combCsPHT genes in Camelina by blasting the coding sequences of known Arabidopsis AtPHTs against the Camelina genome. These genes belonged to the five PHT families, which include 33 CsPHT1s, 3 CsPHT2s, 9 CsPHT3s, 19 CsPHT4s, and 9 CsPHT5s  between 34.3 and 78.8 kDa, and isoelectric point (pI) value between 5.2 and 9.8. The number of transmembrane domains (TMDs) in CsPHT proteins varied between 4 and 13.We identified 73  CsPHT5s . The proCamelina and 22 Arabidopsis PHTs, their protein sequences were aligned to generate a phylogenetic tree using the neighbor-joining method in MEGA X [Camelina genome appeared to lack homoeologs in this group. AtPHT1;6 along with five CsPHT1s were classified into Group III. The PHT2 family contained one AtPHT2;1 and three CsPHT2 members. Two distinct groups were found for the CsPHT3 family, with AtPHT3;1/3;2 and six CsPHT3 members in Group I, and AtPHT3;3 and three CsPHT3s in Group II. CsPHT4 family proteins formed four separate groups, with seven CsPHT4s corresponding to AtPHT4;1/4;4 homologous pairs in Group I, six CsPHT4s with AtPHT4;2/4;3 in Group II, and three CsPHT4s with AtPHT4;5 and AtPHT4;6 in Group III and IV, respectively. CsPHT5 family consisted of two distinct groups, AtPHT5;1 along with three CsPHT5s in Group I, and AtPHT5;2/5;3 with six CsPHT5s in Group II.To investigate the evolutionary relationship between 73 n MEGA X   for each Arabidopsis ortholog, with the majority of CsPHT families containing three homoeologous genes for each AtPHT gene [Camelina was expected to have 27 CsPHT1 genes that correspond to nine AtPHT genes. However, we identified 33 CsPHT1 genes, although no homoeologous genes were found for AtPHT1;5. The further expansion in the CsPHT1 family largely resulted from tandem duplication events  as queries, we searched the New PLACE database [cis-regulatory elements , or in response to specific conditions, such as defense, phosphate, nitrogen, sugar, dehydration, energy, light, and other abiotic stresses [cis-elements, including P1BS, W-box, PHO-like, helix-loop-helix (HLH) and TATA-box-like elements (CsPHT genes examined. Most of the CsPHT1 family genes (28/31) contained P1BS, suggesting these genes can be transcriptionally induced by PHR1 (PHOSPHATE RESPONSE1) transcriptional factor (TF) in response to low-P for efficient Pi uptake [CsPHT1 genes contained a high frequency of several W-box elements, suggesting their expressions might be regulated by multiple WRKY TFs to modulate Pi uptake in response to the changing P status [CsPHT family genes might be transcriptionally regulated by WRKYs to mediate Pi distribution, as they also harbored a high frequency of those W-box elements. Several PHO-like, TATA-box-like (TATABOX2) and HLH (CATATGGMSAUR) elements were present in the majority of CsPHT family genes, except for CsPHT3s. These cis-elements were implicated to mediate low-P responsive expression, although not experimentally validated [Using 2-kb upstream regions of fully-sequenced database  and founelements . These cstresses ,34. Partelements . Among ti uptake ,58,59. IP status ,61,62. Salidated ,64.CsPHT genes, we analyzed transcriptomic datasets generated for 12 different tissues covering four developmental stages [CsPHT family genes were analyzed  and control (CK) conditions  and the control (CK) conditions using quantitative real-time PCR , thus serving as a strong candidate crop that may utilize non-agriculture lands for fuel purposes [Camelina’s response to low-nutrient status has not been studied at the genome level. In this study, we identified 73 genes encoding CsPHT transporters in the Camelina genome and analyzed their expression patterns in various tissues and in response to low-P, providing a road map to understand the transport and utilization of Pi in this promising oil crop.purposes ,40,42,43Camelina mainly resulted from the whole-genome triplication event that occurred 17 million years ago [AtPHT1;5 was identified, presenting an interesting point for functional analysis in the future. Tandem gene duplication events were the major driving force for the expansion of these CsPHT1 genes on chromosome 11, 18 and 20. These tandem duplicated genes largely correspond to AtPHT1;1/1;2/1;3 orthologs, which are functionally redundant genes that function as key players in Pi uptake [Camelina might contribute to the robust Pi acquisition and low-P tolerance in this polyploidy plant.The vast expansion of CsPHT families in ears ago . In addii uptake . The sig+/Pi symporters since their gene structures, protein sizes and PHO4 domain were highly conserved [Camelina has nine CsPHT3 members with MCF domains, which are also found in AtPHT3 transporters that affect mitochondrial respiration processes [The three CsPHT2 members might share similar functions to their ortholog, AtPHT2;1 as Honserved ,12. Camerocesses ,14,55. HCamelina has 19 CsPHT4 members clustered largely in Group I and II with diverse gene structures, although all display conserved MFS domains. This suggests that CsPHT4 members might have a wide range of functions, as observed in Arabidopsis that transport Pi and other solutes, such as ascorbate, in different subcellular compartments, and are involved in plant defense and salt tolerance [Camelina with conserved SPX-MFS domains that are also found on vacuolar Pi-transporters in Arabidopsis and rice that control vacuolar Pi fluxes [Arabidopsis AtPHT5;1/AtVPT1, and rice OsSPX-MFS1 and OsSPX-MFS3, whereas Pi efflux from vacuoles is mediated by VPEs in rice and Arabidopsis [olerance ,19,20,21i fluxes ,52,53,54bidopsis ,23,52,53bidopsis , which iCsPHT genes in various plant tissues of Camelina DH55 genotype [CsPHT1 genes displayed a broad expression in multiple tissues, implying their roles in Pi transport across various tissues throughout plant growth and development. However, these genes were not expressed in roots under the normal growth condition. This might be because a large number of CsPHT1 transporters are encoded in Camelina, and therefore, a basal expression level in roots might be adequate to efficiently uptake Pi under sufficient-P condition. Nevertheless, these CsPHT1 genes were transcriptionally induced under low-P, probably because these genes harbor P1BS (PHR1 binding site) and several W-box (WRKY binding site) elements on their promoter regions. AtPHR1 and AtWRKY45/75 in Arabidopsis, and OsPHR2 and OsWRKY74 in rice are shown to bind to those cis-elements to promote efficient Pi acquisition and translocation especially under low-P stress [AtPHT1;1, –1;4, –1;8 and –1;9 in Arabidopsis, and OsPHT1;2, –1;3, –1;9 and –1;10 in rice [Understanding when and where a gene is expressed could provide valuable information about its role in plant growth and development. Therefore, we analyzed the expression pattern of genotype . In geneP stress ,67,68,69 in rice ,71,72.CsPHT2 genes displayed tissue-specific expression in leaves, similar to Arabidopsis and rice, suggesting their conserved roles in Pi-transport into chloroplasts [CsPHT3;7–3;9 and their ortholog, AtPHT3;3 showed similar expression patterns in floral organs [CsPHT3;1–3;3 genes exhibited tissue-specific expression in germinating seeds, in contrast to their orthologous gene, AtPHT3;1, that showed ubiquitous expression [CsPHT3 genes in seed germination. The PHT3 family is the least studied among the five PHT families in Arabidopsis due to the lethality of homozygous mutants [Camelina that contains multiple copies, and knocking out one or more homoeologous genes might not pose such a challenge. The roplasts ,12,73,74l organs , indicatpression . This mi mutants . Thus, iCsPHT4 genes were highly expressed in leaves, similar to their orthologous genes in Arabidopsis, suggesting their putative roles in Pi transport into chloroplasts, as observed for AtPHT4;1 [CsPHT4;1–4;3 in senescing leaves confers their additional roles in source to sink Pi transport, as observed for AtPHT4;6 [CsPHT5 genes were expressed in flowers and seeds, and the significance of these vacuolar transporters in maintaining Pi homeostasis during reproductive development has recently been confirmed in Arabidopsis [CsPHT5 family genes, CsPHT5;3 showed a dominant expression from early to vegetative growth, whereas CsPHT5;4 was dominant from flower to seed development. Thus, the functional studies of these two genes as key vacuolar Pi-transporters are worth further investigation. Many AtPHT4;1 ,18,76. HAtPHT4;6 . Intrigubidopsis . Among tCamelina’s response to low-P at a genetic level, we compared the growth and gene expression of two Camelina accessions under low-P and the control conditions. Our results indicate Suneson as the low-P-tolerant genotype, and CS-CROO as the low-P-sensitive genotype. Suneson might have a better Pi utilization compared to CS-CROO based on the gene expression analysis. For example, the high expression of CsPHT1;12 and –1;31 genes in roots of Suneson under low-P suggests a better Pi-uptake capability than CS-CROO, as demonstrated by the roles of these PHT1 genes in Arabidopsis [CsPHT family genes are likely involved in Pi distribution involving Pi remobilization from subcellular compartments and Pi recycling from source to sink in order to maintain cellular Pi homeostasis. For example, the higher expression of CsPHT4;6 in shoots and roots of Suneson implies a better Pi remobilization, as illustrated by the function of its Arabidopsis ortholog, AtPHT4;2 in exporting Pi from plastids [CsPHT4;1 was expressed at a higher level in shoots of Suneson than CS-CROO, and its tissue-specific expression in senescing leaves infers the potential role of this gene in recycling Pi from older to younger leaves under low-P. Similarly, although CsPHT1;1, –1;12 and –1;31 genes are putative Pi-uptake genes, their higher expressions in shoots of Suneson suggest their additional functions in distributing Pi in leaves, which remain unexplored. In Arabidopsis, AtPHT1;5 was shown to be involved in this process [Camelina. Thus, other CsPHT1 members along with CsPHT4s might compensate for the loss. Moreover, the higher expression of CsPHT5;8 in shoots, and CsPHT5;1 in roots of Suneson might maintain vacuolar Pi homeostasis better than CS-CROO under low-P [CsPHT3;7 in shoots of Suneson might be important for respiration processes in mitochondria, as observed in Arabidopsis [To understand bidopsis ,8. The oplastids  In addit process , althouger low-P ,23,24. Abidopsis ,14,55.CsPHT genes for which we could design gene-specific primers. Global transcriptomic analysis of low-P-tolerant and -sensitive genotypes coupled with GWAS analysis of large Camelina varieties under low-P will aid in dissection of the gene-networks involving PHTs, TFs and signaling components that contribute to the higher PUE in Camelina such as Suneson. In addition, this global transcript analysis will provide clues regarding the functional dominance of one homoeolog over the others in response to low-P stress. Further genetic, molecular, biochemical and electrophysiological analyses of the candidate CsPHT transporters will elucidate the biological significance and function of these transporters in Pi acquisition and distribution. These approaches are critical for future breeding efforts to engineer crops with high PUE for sustainable agriculture.Further expression analysis is required as we only covered a subset (15/73) of Arabidopsis thaliana were individually blasted against the Camelina sativa database in the National Center for Biotechnology Information . The coding sequences of AtPHT members were obtained from The Arabidopsis Information Resource (TAIR10) database (https://www.arabidopsis.org/) [−10 and query over 60%. Various splicing variants of a gene and incomplete genes with short length were discarded. The CsPHT chromosomal locations and their genomic DNA were obtained from NCBI. The coding sequences and deduced protein sequences were retrieved from the Kyoto Encyclopedia of Genes and Genomes/KEGG Genome: Camelina sativa (https://www.genome.jp/). The Locus IDs were obtained from “BLAST Database” in The Prairie Gold Novel Industrial Oilseed Crops for Canada Database (http://www.camelinadb.ca/). The properties of putative PHT proteins such as number of amino acids, molecular weights (Mw), and isoelectric point (pI) were calculated using ProtParam (http://web.expasy.org/protparam/). Transmembrane domains (TMDs) were predicted using TMpred , with a score above 500 considered as significant.To identify the candidate CsPHT members, the coding sequences of all known AtPHT members of is.org/) . The choCamelina and Arabidopsis was generated using ClustalW. The Arabidopsis AtPHT protein sequences were obtained from TAIR10 database (https://www.arabidopsis.org/) [CsPHT genes were mapped using the MapDraw program [EnsemblPlants (http://plants.ensembl.org/Camelina_sativa/Info/Index) by close examination of the chromosomal regions of all clustered genes found on the three subgenomes  . The phyis.org/) . The chr program . Tandem bgenomes . HomologCamelina genome annotation GFF file (downloaded from http://www.camelinadb.ca/) [https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) [The gene structures were generated using adb.ca/) . The conpsb.cgi) . The grapsb.cgi) .CsPHT genes were downloaded from NCBI (https://blast.ncbi.nlm.nih.gov/). The multiple promoter sequences of CsPHT genes were scanned in New PLACE (https://www.dna.affrc.go.jp/PLACE/?action=newplace) [cis-regulatory elements (cis elements was plotted using TBtools [Promoter sequences located 2-kb upstream of the transcription start sites (ATG codons) of ewplace)  to identelements . The sub TBtools .CsPHT genes across 12 different tissues covering 4 developmental stages of the Camelina life cycle were extracted from Camelina eFP browser (http://bar.utoronto.ca/efp_camelina/cgi-bin/efpWeb.cgi) [https://software.broadinstitute.org/morpheus/).The RNA-Seq data (Log2 Ratio) of Web.cgi) . The devCamelina accessions, CS-CROO and Suneson, are listed in t test was used to compare biomass between -Pi and CK conditions for each accession.The sources of seeds for the two TM Real-Time System . Each PCR mixture contained 10 μL SYBR Green Master Mix , a pair of 0.5 μM gene-specific primers, and 5 μL of the cDNA sample (∼100 ng) in a final volume of 20 μL. Alpha tubulin  was used as the internal control [CsPHT genes are listed in CsPHTs between the two Camelina accessions after Pi treatments was assessed by Student’s t test (* p < 0.01 and ** p < 0.001).Shoot and root tissues were separated, and quickly frozen in liquid nitrogen. Total RNA was extracted using TRIzol Reagent . After treatment with DNase I , 2 μg of total RNA was subjected to reverse transcription reaction using the M-MLV Reverse Transcriptase  at 42 °C for 1 h. The resulting cDNA was used for PCR amplification with the gene-specific primers. qRT-PCR analysis was performed using the CFX96 control . The speCsPHT genes in Camelina that share more similar gene structures and protein domains within the same CsPHT family. Polyploidization and tandem duplication events drove the vast expansion of these CsPHT families in Camelina. Cis-element P1BS was more specifically conserved in the promoter regions of CsPHT1 genes to mediate Pi uptake under low-P. In silico RNA-seq analysis suggested the importance of CsPHT1 genes throughout plant development, CsPHT2s and CsPHT4s in vegetative growth, CsPHT5s in reproductive development, and CsPHT3s in early growth and reproductive stages. However, several genes showed dominant expression compared to their close homoeologous genes including CsPHT1;27, –5;3 and –5;4. A popular Camelina variety, Suneson showed better tolerance to low-P than CS-CROO, with higher expression of CsPHT1;12, –1;31, –4;1, –4;6 and –5;1 in roots, and CsPHT1;1, –1;12, –1;31, –3;7, –4;1, –4;6, –4;15 and –5;8 in shoots. More in-depth analysis at global transcript level and/or GWAS of a large pool of Camelina accessions under low-P could provide a more holistic view of specific Pi-transporters and regulators that are involved in providing enhanced low-P tolerance to Camelina accessions . This could serve as a better framework for future functional studies of CsPHT transporters in Camelina, and as a model for other crop species.We identified and characterized 73


We detected human metapneumovirus (HMPV) in 72 (7.1%) of 1,021 patients hospitalized with severe acute respiratory infection in Luohe, China, during 2017–2019. We detected HMPV most frequently in young children and less often in adults. HMPV genotype A2c variants 111 nt and 180 nt duplications predominated, demonstrating their continuing geographic spread.  Pneumoviridae, genus Metapneumovirus) is a major cause of acute respiratory tract infections, especially in children and elderly persons , which identifies HMPV, influenza types A and B, human respiratory syncytial virus (RSV), human coronaviruses, human rhinovirus and enterovirus, human adenovirus, human parainfluenza viruses, and human bocavirus.Luohe is a city of 2.8 million people in Henan Province, northern China. In this study, we collected throat swab specimens and clinical data from 1,021 patients with SARI admitted to Luohe Central Hospital during October 2017–June 2019. Patients were 1 month–95 (median 3) years of age; most (76.7%) of the SARI cases involved children <5 years old. All swab specimens were tested by multiplex real-time reverse transcription PCR assay using a nucleic acid diagnostic kit . HMPV was identified in 7.1% (72/1021) of SARI patients, consistent with other studies that identified HMPV in 6%–12% of SARI cases  of SARI patients were positive for RI cases . The epiRI cases . Most (9Forty-three entire coding-region sequences of the G gene were obtained from 72 HMPV-positive samples, as described elsewhere . The nucThe twenty-two 111 nt-dup variants circulating in Luohe during 2018–2019 were closely related to the duplication-variant viruses identified in Japan, Croatia, and Guangdong, China, in 2017, with nucleotide identity of 95.7% to 100%. Six 180 nt-dup variants circulating in Luohe during 2017–2018 were clustered in 1 small cluster with high nucleotide identity at 99.1% and 100% and closely related to the duplication viruses identified in Japan, Croatia, Spain, and Beijing, China, during 2014–2017 , panel AOur findings show that 7.1% of SARI patients in the Luohe study tested positive for HMPV during 2017–2019. The rate of HMPV-positivity decreased gradually with patient age. Among the HMPV patients, 63.9% were co-infected with other respiratory viruses. Four different genotypes were cocirculating, including A2b, A2c, B1, and B2. We identified most of the A2c viruses as duplication variants, including the 111 nt-dup and 180 nt-dup variants, and found that these 2 variants were the predominant viruses circulating in Luohe during 2017–2019. Pneumoviridae. The HRSV duplication variants of both BA9 and ON1 have rapidly spread globally, becoming the predominant viruses in many countries for many years (The 111 nt-dup and 180 nt-dup variants have also been detected in different regions of the world, including in Spain, Japan, Croatia, and Guangzhou, China, in recent years (There were some limitations in this study. The epidemic seasons were not covered for the entire 3 years, and only 1 hospital was involved in this study. Continuous surveillance will be required to determine whether these novel HMPV 111 nt-dup and 180 nt-dup variants will persist as predominant viruses and have a wider geographic distribution in the future.Although no difference in clinical symptoms was observed between the patients infected with HMPV duplication variants and those with non–duplication-variant viruses in this study, the increased transmission frequency of the duplication variants suggests a role for duplication in the G gene in potentially expanding its transmission. Therefore, further study is needed to clarify if and how the duplications result in an evolutionary advantage for HMPV.


TET2, DNMT3A and ASXL1, and global and gene-specific methylation patterns in neoplastic cells. Moreover, we discuss methylation-specific pathways and other epigenetic events that may trigger disease progression in mast cell neoplasms. Finally, we discuss epigenetic targets and the effects of epigenetic drugs, such as demethylating agents and BET-targeting drugs, on growth and viability of neoplastic mast cells. The definitive impact of these targets and the efficacy of epigenetic therapies in advanced SM need to be explored in future preclinical studies and clinical trials.Systemic mastocytosis (SM) is a hematologic neoplasm with abnormal accumulation of mast cells in various organ systems such as the bone marrow, other visceral organs and skin. So far, only little is known about epigenetic changes contributing to the pathogenesis of SM. In the current article, we provide an overview of epigenetic changes that may occur and be relevant to mastocytosis, including mutations in genes involved in epigenetic processes, such as  Systemic mastocytosis (SM) is a neoplastic disease of the bone marrow (BM) characterized by infiltration and abnormal accumulation of mast cells (MC) in the BM, liver, spleen, lymph nodes, skin and other organs. In a subset of patients, release of mediators from MC results in systemic symptoms, and sometimes even in anaphylaxis ,3,4,5,6.Clinical manifestations of mastocytosis are manifold. Several of the symptoms recorded are induced by MC-derived mediators. Other symptoms are caused by MC infiltration in various organs. Main symptoms affecting the patients’ quality of life are symptoms related to skin lesions, anaphylaxis, osteoporosis, gastrointestinal problems, fatigue, anxiety, and depression ,2,3.9/L, Hgb < 10 g/dL, or platelets < 100 × 109/L, (2) hepatomegaly with impairment of liver function, ascites and/or portal hypertension, (3) skeletal involvement with large osteolytic lesions and pathological fractures, (4) palpable splenomegaly with hypersplenism, (5) malabsorption with weight loss [In children and adults, mastocytosis is usually an acquired disease, although rare familial cases have been observed ,8. In a actures,  palpableplenism,  malabsorplenism,  malabsorght loss ,12. KIT proto-oncogene. An aspartic acid to valine substitution (D816V) in exon 17 of 21 exons, which encodes a second catalytic domain, leads to autonomous activation of KIT and thus to enhanced MC differentiation and survival in the absence of SCF [KIT is considered to contribute to the development of SM [KIT, both at codon 816 or in other codons of KIT (for example 560) have also been reported. Familial mastocytosis has also been described to be associated with KIT variants: in these cases, other mutations in KIT, such as K509I, may be detected [Stem cell factor (SCF)-induced KIT activation results in MC differentiation from their progenitor cells as well as MC survival and MC maturation ,14,15. Atution D8V in exone of SCF . This munt of SM ,19. Howedetected .KIT D816V point mutations have been associated with neoplastic phenotype of MC [KIT mutations, e.g., V560G (exon 11), K642E (exon 13) and Y269C (exon 5) have been found in patients with mastocytosis. The KIT mutation V560G is also expressed in the human HMC-1 cell line which has been established from the peripheral blood of a patient with MCL [More than 80% of the observed pe of MC . Intereswith MCL .Epigenetics refers to mechanisms connecting the genetic background with environmental signals in order to provide adaptations to various conditions and threats, like aging, lifestyle factors and also intrinsic pathologic processes . For exaKIT, (3) Methylation status of apoptosis-associated and tumor suppressor genes in HMC-1 cells, (4) Somatic mutations in genes regulating epigenetic mechanisms, (5) TET2, (6) DNMT3A, (7) 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC) level, (8) microRNA, (9) MITF and microRNA.Unfortunately, very few studies have investigated the role of epigenetic modifications in neoplastic cells in SM. In the current study, we present an overview and update of epigenetic changes described in the context of mastocytosis so far: (1) Histone modifications in neoplastic MC, (2) Epigenetic modifications of A study by Martinelli et al. revealed that in advanced SM, specific histone modifications can be detected in neoplastic MC in SM, and the same was found in HMC-1 cells . SeveralKIT inactivation [KIT D816V-mutated BM MC and HMC-1 cells [KIT-mutated human MC lines HMC-1 and ROSA (KIT D816V). Using ChIP qPCR resulted in a decreased active chromatin mark H3K18ac/H3 only in the KIT region but not in control genes upstream and downstream of KIT (PDGFRα and KDR). MC exposed to SAHA showed downregulation of KIT mRNA expression followed by decreased KIT protein expression, and apoptosis. Interestingly, primary MC isolated from SM patients, including one with MCL, were highly sensitive to SAHA-mediated cell death, while normal BM cells were resistant [It has turned out recently that chromatin epigenetic silencing might be one of the key regulators of mutated tivation . However-1 cells . Among tesistant .CIDEB, GADD45A, HRK, TNFRSF25, BIK, BID and TNFRSF21) and six TSG  were found to be methylated in HMC-1 cells but not in normal BM cells, suggesting that these genes are aberrantly hypermethylated in these neoplastic MC.Ghanim et al. explored the methylation status of 24 classical apoptosis-associated genes and 24 classical tumor suppressor genes (TSG) in HMC-1 cells and in control  cells . Their dKIT D816V mutation solely does not explain heterogeneity of SM with different disease phenotypes. Thus, extensive mutational analyses of patients may provide a better explanation of mastocytosis pathogenesis. Interestingly, several somatic mutations have been observed in epigenetically relevant genes.Clinical presentations of the disease vary widely from indolent to advanced forms, and to the exceedingly rare MCL. KIT epigenetic-associated mutations in various types of mastocytosis. Genes related to epigenetic modification that have been found to be mutated in specific SM types are TET2 (TET methylcytosine dioxygenase 2), DNMT3A , ASXL1 , EZH2 (enhancer of zeste homolog 2) and IDH1/2 (isocitrate dehydrogenase 1/2) (There is still scarce research on non-ase 1/2) .KIT genes has been investigated. Bone marrow or peripheral blood were used as a source of target DNA with various sequencing applications [In five studies on patients with different types of SM , a panelications ,33,34,35KIT mutations were less frequent but still comprised a substantial proportion of the mastocytosis patients. In the pilot study, the coding regions of 22 genes were investigated in 19 advanced SM patients. Overall, KIT D816V frequency was 89%, while non-KIT mutation was observed in fourteen patients (74%) who carried at least one additional mutation. The highest prevalence occurred for ASXL1/TET2 , CBL , SETBP1/SF3B1/DNMT3A  and JAK2/CALR/FLT3/IDH-1/RUNX1/TP53  [KIT mutations within 27 investigated gene targets in four SM-AHN groups, MCL and ISM. In the whole group of SM patients, the most frequent mutations have been observed for TET2 (29%), ASXL1 (17%) and CBL (11%) [Non-= 1, 5%) . In a stBL (11%) .KIT D816V and various SM variants: ISM (n = 10), SSM (n = 2), SM-AHN (n = 5) and ASM (n = 15) or MCL (n = 7) with (n = 18) or without (n = 4) SM. Next-generation sequencing was applied to investigate mutational hotspot regions, and complete exon regions of EZH2, ETV6, RUNX1, and TET2 were analyzed [TET2 in 15/39 (39%) SM patients. Ten of 15 (67%) patients carried more than one TET2 mutation. Eight ASXL1 mutations have been identified in 8 patients with 7 frameshift and 1 nonsense mutation. A missense mutation and 2 splice site mutations have been detected in EZH2 [Additional molecular aberrations have been analyzed in a study of 39 patients with analyzed . A totalanalyzed . In addi in EZH2 .KIT D816V alone [KIT mutations led to reduced OS  compared with fewer than 3 mutations (48 months) [TET2, DNMT3A and ASXL1 mutations may affect prognosis, as demonstrated by worse OS in mutated patients. For example, mutation in epigenetic regulators, namely TET2 and/or ASXL1, was associated with reduced OS (11 months) compared with the absence of these mutations (84 months) [KIT-mutated SM and patients with combined TET2/DNMT3A/ASXL1 mutations independent of KIT and sole TET2 mutations (p < 0.001) have been observed [ASXL1, but not TET2, mutation was associated with OS, suggesting different epigenetic changes in heterogeneous SM prognosis [The overall survival (OS) was significantly shorter in patients with additional aberrations as compared to those with 6V alone . Occurre months) . Interes months) . In addiobserved . To add,rognosis .TET2, DNMT3A, and ASXL1, EZH2 and IDH1/IDH2 have been observed more frequently in advanced SM, e.g., ASM, SM-AHN and MCL than in ISM patients (TET2 (29%), ASXL1 (17%), and DNMT3A (6%). The only additional mutations observed in ISM patients are age-related somatic mutations that have also been associated with epigenetic changes, e.g., mutations in TET2 (7%) and DNMT3A (5%) [TET2, DNMT3A, and ASXL1, may be associated with epigenetic features in SM. [Importantly, mutations in patients . MutatioT3A (5%) . Thus, ms in SM. . HoweverKIT, TET2, ASXL1 and CBL in 87%, 27%, 14%, and 11%, respectively. In these patients, mutated ASXL1 had a particularly adverse impact on OS [Patients with SM-AHN presented mutations in ct on OS .TET2, DNMT3A, IDH1/2 mutations have been observed in patients with myeloproliferative neoplasms and other hematological malignancies, such as acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), or myelodysplastic syndromes (MDS) [Mutations in the discussed epigenetic modulators frequently occur in other hematological diseases. Recurrent es (MDS) ,39. Thus2+ TET2 is important in demethylation of 5-mC forming 5-hmC by oxidation that enables dynamic turnover of 5mc and 5hmC. In addition, mice with Tet2 knockdown presented late-onset hematological abnormalities. TET proteins and oxidized methylcytosines are implicated in several pathways of DNA demethylation. They are involved in (1) removal of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) by thymine DNA glycosylase (TDG), and (2) inhibition of DNA methyltransferase (DNMT)1-mediated maintenance of DNA methylation [Ten-Eleven-Translocation proteins (with 3 isoforms) are enzymes dependent on a-ketoglutarate and Fehylation .TET2 and other critical target genes are often detected in primary neoplastic cells in patients with advanced SM, suggesting a possible role of the gene products in abnormal MC accumulation and function in these neoplasms  differentiation. HSC lacking ol cells  and loss in vivo . Therefope cells .KIT D816V mutated oncogene. Neither the loss of 5-hmC correlated with overall MC burden in those patients, nor was it due to inactivating mutations of TET2 or reduced TET2 expression [The only study on global DNA methylation indices, like 5-hmC and 5-mC levels in patients with SM, has revealed that the correlation between SM diagnosis and significantly low 5-hmC levels may represent a systemic effect of SM. Overall reduced 5-hmC levels in all patients with SM have been reported. In SM, reduced level of 5-hmC was associated with the effect of pression Non-coding RNA transcripts (ncRNAs), constituting almost 98% of human genome, have been demonstrated to play a significant role in various pathological processes. It is regarded that microRNAs (miRNAs) possess capacity to act as putative negative regulators of gene expression at transcriptional, posttranscriptional and epigenetic levels of the target genes .miRNAs are recognized to be critical regulators of immune responses in various allergic diseases and are involved in cell differentiation, proliferation, survival and IgE-mediated allergic response . In addiAdditionally, in murine MC, miR-221-222  has occurred to be significantly up-regulated upon cell activation. Overexpression of miR-221-222 resulted in altered cell morphology and cell cycle regulation without altering viability. Overall, this miRNA was involved in cell cycle regulation in MC in response to acute activation stimuli .Currently, there is excessive evidence that exosome might provide cell-to-cell communication. In 2007, Valadi et al. showed that exosomes derived from a mouse and a human MC line , and primary bone marrow-derived mouse MC, contained RNA, both microRNA and mRNA. MC-derived RNAs can be further delivered to another cell, and can be functional in this new location. The authors have thus proposed that this RNA could be called “exosomal shuttle RNA” .TM RNA array analysis. Note that 116 microRNAs were found in exosomes and 134 were identified in donor MC, the most abundant microRNAs in the exosomes, compared to the MC were: hsa-miR-451, hsa-miR-503, miRPlus_27560, miRPlus_2843, miRPlus_27564, hsa-miR-583, miRPlus_1795, miRPlus_17890, hsa-miR-663 and hsa-miR-30b. Predictions according to canonical pathways and networks showed involvement of MC-derived exosomal microRNA in cellular development, cellular movement, hematological system development and function as well as in cell cycle regulation [Characteristics of detailed RNA content have been determined in human MC line, HMC-1 using the microarray assay and miRCURYgulation .However, it remains unknown whether all these miR species play a role in human (neoplastic) MC and it also remains unknown whether such miR molecules contribute to the pathogenesis of SM.MITF is a transcription factor that contains both basic helix-loop-helix and leucine zipper structural features. The encoded protein regulates melanocyte development and is responsible for pigment cell-specific transcription of the melanogenesis enzyme genes [KIT D816V. A study of Lee et al. indicated that MC with activating KIT mutation require MITF expression for MC proliferation [A protein encoded by me genes . The trame genes ,55. MITFferation .Interestingly, it has been observed that miRNA regulation can link these two factors essential for MC function. Lee at al. have revealed that miR-539 and miR-381 are repressed by KIT D816V signaling through conserved miRNA binding sites in the MITF 3’-untranslated region. Normally, expression of these miRNAs is involved in MITF suppression and inhibition of colony-forming capacity of mastocytosis cell lines. Importantly, dysregulation of this pathway may contribute to abnormal MC proliferation and thus to the pathogenesis of aggressive MC diseases, both in humans and in the murine system .Studies on the regulation of miRNA with other epigenetic key players are still at an initial stage. For example, findings from acute myeloid leukemia cells have indicated strong impact of miR-29 family members on three target DNMT izoenzymes: DNMT1, DNMT3A and DNMT3B resulting in global DNA hypomethylation as well as reexpression of tumor suppressor genes .Dnmt3a MC by any microRNA, it has been observed that miR-223 was dysregulated in cells lacking Dnmt3a [In the study on murine MC, although primary screening has not revealed modification of knockout g Dnmt3a . Additiog Dnmt3a , suggestThe classical epigenetic drugs are the hypomethylating agents, such as azacytidine and decitabine. Both drugs are highly active in patients with advanced MDS, CMML and AML ,60. So fFAS mRNA, p21 mRNA, Noxa mRNA, and Bim mRNA in neoplastic MC [A study of Ghanim et al. examined the antineoplastic effects of demethylating agents on human neoplastic MC. They found that the death regulator FAS is hypermethylated in neoplastic MC, but not in normal BM cells, and that azacytidine and decitabine can induce demethylation and thereby can promote expression of astic MC .Another potential epigenetic target in neoplastic MC is the epigenetic reader BRD4 which regulates expression of MYC in normal and neoplastic cells, including AML cells . Wedeh eMore recently, SETD2 histone methyltransferase was identified as a potential target in advanced SM. Indeed, Martinelli et al. have reported that very low or absent SETD2 protein expression was especially found in cases with advanced SM, and proteasomal degradation was found to have a major role in the observed lack this protein. Inhibitors of the proteasome, such as bortezomib, can rescue  expression of SETD2 and H3K36 trimethylation in neoplastic MC, resulting in a marked accumulation of ubiquitinated SETD2. Bortezomib was also found to counteract viability in neoplastic MC and to cooperate with midostaurin in inducing apoptosis in HMC-1 cells and neoplastic MC from patients with advanced SM .The use of epigenetic drugs may pave the way for the development of improved treatment approaches in advanced SM. However, more preclinical studies and clinical trials need to be performed to confirm the impact of epigenetic targets and the efficacy of epigenetic therapies in patients with MC neoplasms.Mastocytosis is a group of heterogeneous diseases with a broad range of molecular patterns and markers and a variable clinical course. Several disease-specific processes may be triggered by the epigenome in these patients. However, only little is known about the role of epigenetic modifications in neoplastic cells in SM.TET2, DNMT3A or ASXL1 may be altered (mutated) in mastocytosis. Such mutations are frequently observed in advanced SM and these mutations may also affect clinical behavior of SM and thus prognosis. In addition, TET2 and DNMT3A may play a role in MC responsiveness to acute and chronic activation. These observations suggest a potential influence of epigenetic processes on the pathogenesis of MC-related disorders.The current paper provides a summary of recent findings regarding epigenetic changes in neoplastic cells in various types of mastocytosis. In general, data published recently suggest that epigenetic changes may indeed trigger the pathogenesis of SM through several different mechanisms, including aberrant methylation of promoters of genes critically involved in DNA/RNA processing, apoptosis, and activation of MC, and modulation of KIT expression by epigenetic regulators. Moreover, genes whose products influence epigenetic processes including