Recent genomic studies have identified chromatin-spliceosome (CS)-acute myeloid leukemia (AML) as a new subgroup of AML. CS-AML is defined by several mutations that perturb epigenetic regulation, such as those affecting splicing factors, cohesin components, transcription factors, and chromatin modifiers, which are also frequently mutated in other myeloid malignancies, such as myelodysplastic syndrome and secondary AML. Thus, these mutations identify myeloid neoplasms that lie on the boundaries of conventional differential diagnosis. CS-AML shares several clinical characteristics with secondary AML. Therefore, the presence of CS-mutations may help to better classify and manage patients with AML and related disorders. The aim of this review is to discuss the genetic and clinical characteristics of CS-AML and roles of driver mutations defining this unique genomic subgroup of AML.Recent genetic studies on large patient cohorts with acute myeloid leukemia (AML) have cataloged a comprehensive list of driver mutations, resulting in the classification of AML into distinct genomic subgroups. Among these subgroups, chromatin-spliceosome (CS)-AML is characterized by mutations in the spliceosome, cohesin complex, transcription factors, and chromatin modifiers. Class-defining mutations of CS-AML are also frequently identified in myelodysplastic syndrome (MDS) and secondary AML, indicating the molecular similarity among these diseases. CS-AML is associated with myelodysplasia-related changes in hematopoietic cells and poor prognosis, and, thus, can be treated using novel therapeutic strategies and allogeneic stem cell transplantation. Functional studies of CS-mutations in mice have revealed that CS-mutations typically cause MDS-like phenotypes by altering the epigenetic regulation of target genes. Moreover, multiple CS-mutations often synergistically induce more severe phenotypes, such as the development of lethal MDS/AML, suggesting that the accumulation of many CS-mutations plays a crucial role in the progression of MDS/AML. Indeed, the presence of multiple CS-mutations is a stronger indicator of CS-AML than a single mutation. This review summarizes the current understanding of the genetic and clinical features of CS-AML and the functional roles of driver mutations characterizing this unique category of AML. Acute myeloid leukemia (AML) is a group of myeloid malignancies characterized by expanded, undifferentiated myeloid precursor cells and impaired hematopoiesis, however with highly variable clinical manifestations. Despite considerable improvement in the response to initial remission induction chemotherapy, many patients develop refractory disease or experience relapse even after achieving complete remission (CR), which underlines the unmet need for better management and novel therapeutic strategies ,2. AlthoRUNX1, ASXL1, BCOR, STAG2, EZH2, SRSF2, SF3B1, U2AF1, ZRSR2, and partial tandem duplication (PTD) of KMT2A (MLL) gene (MLL-PTD) .,42.5,42] disease . A membe cancers ,49,50. Me manner . Among cn CS-AML ,42. MostSeveral studies have illustrated that cohesin mutations cause enhanced self-renewal and aberrant differentiation of hematopoietic stem and progenitor cells (HSPCs) in both in vitro and in vivo models ,52,53,54STAG2 mutations are almost always accompanied by other driver mutations, such as in RUNX1, SRSF2, and ASXL1 ,9,108,9,and sAML . At leasutations . The co-ed above ,22. Thised above .STAG2, RUNX1, SRSF2, and ASXL1, preferentially co-occur in patients with high-risk MDS who later develop sAML.In summary, CS-AML is a genomic subgroup of AML defined by several gene mutations, which are also frequently identified in MDS and sAML. These mutations may identify myeloid neoplasms lying on the boundaries of conventional categorization of AML. CS-AML exhibits clinical characteristics similar to those of sAML, and the unambiguous presence of CS-mutations may help clinicians to better classify and manage patients with AML, in the absence of detailed clinical history, and apparent morphological or cytogenetic abnormalities. The accumulation of multiple CS-mutations also seems to be important for the development and progression of AML, as multiple CS-mutations, such as in CS-AML is a novel form of AML and is characterized by mutations in several genes, such as those encoding the spliceosome, cohesin complex, transcription factors, and chromatin modifiers, all of which are implicated in various epigenetic pathways. Class-defining mutations in CS-AML are also frequently found in MDS and sAML, indicating a molecular similarity among a subset of CS-AML, MDS, and sAML. Clinically, CS-AML correlates with myelodysplasia-related changes in hematopoietic cells and poor patient prognosis, and has been successfully treated by allogeneic stem cell transplantation, with the potential of using novel therapeutics. Notably, the mutational status in patients with AML is associated with sensitivity to various anticancer drugs, which suggests the need to adjust treatment based on the mutational status .Although the functional role of each CS-mutation in hematopoiesis and leukemogenesis is not completely homogenous, at least some aspects are shared between mutations. Functional studies using mouse models have revealed that CS-mutations typically perturb epigenetic regulation and transcription, and impair hematopoietic functions, causing aberrant hematopoiesis and/or MDS-like phenotypes. Moreover, multiple CS-mutations in mice often synergize to induce more severe phenotypes in the hematopoietic system, such as the development of lethal MDS/AML, suggesting that the accumulation of abnormalities in genes involved in epigenetic pathways may play a critical role in the development and progression of a subset of MDS/AML.Several questions remain to be addressed to better understand CS-AML. For instance, the distinct diagnostic boundaries between CS-AML, MDS, and sAML become obscure when considering the similarity in the molecular signatures of these diseases. This ambiguity hinders the development of optimal diagnosis and therapeutic strategies for patients carrying CS-mutations. Future studies on pan-myeloid tumors should identify ways to integrate the conventional morphological and clinical diagnosis with the mutational profiles to optimize patient diagnosis and disease management. Furthermore, the molecular mechanism by which distinct CS-mutations induce comparable phenotypes and define the same disorder remains nebulous. As CS-mutations may alter epigenetic regulation, integrative analysis of genomic and epigenomic alterations in AML may prove the common molecular pathogenesis of CS-AML. Recent work suggests applying precision medicine that targets CS-mutations, which, although promising and potentially effective, warrants validation in multiple systems before translating to human trails. Given that CS-AML and MDS share common molecular signatures, patients with CS-AML may be treated using the hypomethylating agents azacitidine and decitabine that have been used in MDS treatment. Future studies should comprehensively identify the landscape of genetic and epigenetic features that lead to the pathogenesis of CS-AML, which can be used to develop novel therapeutic strategies for this unique category of AML.


Intracellular Ca2+ levels are tightly regulated; at rest, most Ca2+ is retained in organelles, such as the sarcoplasmic reticulum, or in the extracellular space, whereas depolarization triggers a series of events leading to Ca2+ entry, followed by extrusion and reuptake. The mechanisms that maintain Ca2+ homoeostasis are candidates for modulation at the post-translational level. Here, we review the effects of protein SUMOylation, including Ca2+ channels, their proteome and other proteins associated with Ca2+ signalling, on vital cellular functions, such as neurotransmission within the central nervous system (CNS) and in additional systems, most prominently here, in the cardiac system.Small ubiquitin-like modifier (SUMO) conjugation (or SUMOylation) is a post-translational protein modification implicated in alterations to protein expression, localization and function. Despite a number of nuclear roles for SUMO being well characterized, this process has only started to be explored in relation to membrane proteins, such as ion channels. Calcium ion (Ca The redtraction . A protetraction . SUMO-1 function . The smad so far ). N106 p failure . N106 wa failure .2+ homoeostasis and protein SUMOylation may lead to severe neurological, and also, cardiac pathologies. For example, SUMOylation of proteins involved in Ca2+ signalling can modulate synapse formation and alter neurotransmitter release. Furthermore, SUMOylation of proteins can modulate Ca2+ reuptake in cardiomyocytes and thus affect contractility. As described above and summarized in 2+ homoeostasis, including VGCCs and their proteome, are emerging as SUMO targets; equally, synaptic proteins involved in exocytosis and endocytosis are known to be SUMOylated. Postsynaptic receptor SUMOylation can also impact synaptic function. There is clear potential to exploit this knowledge to improve synaptic function in neurodegenerative and hyperexcitability disorders and to improve cardiac function. Thus, understanding how SUMOylation affects Ca2+ signalling in physiological and pathophysiological conditions is key to novel therapeutic strategies to prevent and/or cure important human diseases.Both alterations in Ca


The biological function of most mitochondrial proteases has not been well characterized. Moreover, most of the available information on the normal function of these proteases has been derived from studies in model organisms. Recently, the mitochondrial proteases caseinolytic protease P (CLPP) and neurolysin (NLN) have been identified as therapeutic targets in acute myeloid leukemia (AML). Both proteases are overexpressed in approximately 40% of AML patients. Mechanistically, CLPP and NLN maintain the integrity of the mitochondrial respiratory chain: CLPP cleaves defective respiratory chain proteins, while NLN promotes the formation of respiratory chain supercomplexes. In this review, we highlight the functional consequences of inhibiting and activating mitochondrial proteases and discuss their potential as therapeutic targets in AML. The mitochondrial proteases neurolysin (NLN) and caseinolytic protease P (CLPP) have been identified as therapeutic targets in acute myeloid leukemia (AML). Inhibition of NLN by R2 reduces LETM1 complex formation, respiratory chain supercomplex (RCS) assembly, and AML growth. Activation of CLPP by ONC201 or ONC212, as well as inhibition of ClpXP by A2‐32‐01, reduces oxidative metabolism and impairs AML growth. Acute myeloid leukemia (AML) is an aggressive hematological malignancy. Despite recent advances and new therapies for this disease, the prognosis for most patients with AML remains poor. Understanding the biology of this disease is important for developing new therapies. Recently, AML cells and stem cells have been shown to have unique mitochondrial properties, which can be therapeutically targeted. One of these properties is an increased reliance on the mitochondrial matrix proteases, caseinolytic protease P and neurolysin. This perspective discusses the effects of genetically and chemically dysregulating mitochondrial proteases in AML. Moreover, this study considers the potential of targeting mitochondrial proteases as a novel therapeutic strategy.1Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous hematological malignancy that is characterized by the clonal proliferation of immature blast cells.We and others have shown that targeting the unique mitochondrial vulnerabilities in AML and LSCs is a potential therapeutic strategy for this disease.2AML cells and LSCs have unique mitochondrial properties compared to normal hematopoietic stem and progenitor cells (HSPCs). While normal HSPCs rely on anaerobic glycolysis, LSCs are enriched for hallmarks of oxidative metabolism and rely on oxidative phosphorylation (OXPHOS) for their survival.The mitochondrial proteome is tightly regulated by a diverse group of enzymes called mitochondrial proteases. To date, 20 intrinsic mitochondrial proteases have been identified in the different subcompartments of the mitochondria.3CLPP is a mitochondrial serine protease that forms a complex with the AAA ATPase chaperone, CLPX . The ClpXP complex consists of CLPP arranged as a stacked heptameric ring with a proteolytic core capped at both ends by CLPX.CLPP was recently identified in a genetic screen as being important for AML cell viability. Cole et al initiated their study into CLPP by assessing its expression in primary AML samples and normal CD34+ progenitors. CLPP was overexpressed in 45% of AML samples and equally expressed in stem and bulk populations. Moreover, CLPP expression positively correlated with increased expression of genes involved in the mitochondrial unfolded protein response, suggesting that overexpression is associated with mitochondrial stress.To validate the results of the screen and to characterize CLPP's mitochondrial function, CLPP was knocked down using shRNA (short hairpin RNA). Knockdown of CLPP reduced the growth and viability of several AML cell lines with high CLPP expression, but had no effect in HL‐60 cells, which expressed low levels of CLPP. Knockdown of CLPP also targeted AML progenitors, as evidenced by reduced engraftment of TEX cells, a leukemic cell line that has stem‐like properties and hierarchical organization, into mouse marrow.CLPP−/− mice have impaired hearing and are infertile. However, there was no observed effect on hematopoiesis as CLPP−/− mice have normal numbers and function of HSPCs and mature blood cells.Loss of CLPP shows limited toxicity to normal tissues and CLPP deficiency in both humans and mice has not been shown to greatly affect viability. In humans, loss of CLPP causes Perrault syndrome, which is characterized by congenital hearing loss and ovarian dysfunction.β‐lactones were initially identified as potent inhibitors of bacterial CLPP through chemical proteomic screens.Hyperactivation of CLPP has also been shown to be an effective strategy to selectively target AML and LSCs over normal cells. In bacteria, CLPP activation causes uncontrolled degradation of proteins and death.To evaluate the anticancer effects of activating CLPP, AML cell lines were treated with ONC201 and ONC212. Both compounds reduced the growth and viability of AML cells and stem/progenitor cells but had little effect on CLPP knockout cells or cells expressing inactive mutant CLPP. Moreover, sensitivity to ONC201 positively correlated with CLPP expression in primary AML samples. Mechanistically, CLPP activated by ONC201 and ONC212 degraded respiratory chain proteins, reduced basal OCR, and increased mitochondrial ROS. Degradation of complex I subunits by ONC201 suggests that hyperactivation of CLPP changes its substrate selectivity, likely because of an increase in CLPP's axial pore size. Finally, treatment with ONC201 and ONC212 reduced tumor burden and increased survival in a xenograft model of Z138 cells, as well as in a patient‐derived xenograft model.4X‐X‐His sequence motif.NLN is a zinc metallopeptidase with a conserved His‐Glu‐NLN acts on a diverse range of substrates that have been implicated in several biological functions, such as pain, blood pressure regulation, sepsis, and stroke.2CH2)‐Leu‐Pro‐NH2) as a more potent and specific inhibitor of NLN.Several small‐molecule NLN inhibitors have previously been characterized. The dipeptide proline‐isoleucine was the first identified selective inhibitor of NLN. Although proline‐isoleucine is specific for NLN, it has a low affinity for the endopeptidaseNLN was the top scoring protease in the same genetic screen that identified CLPP as a therapeutic target in AML.NLN's mitochondrial function has not been well characterized to date. To gain insight into NLN's function, its mitochondrial interactors were identified using proximity‐dependent biotin labeling coupled with mass spectrometry (BioID‐MS). NLN interacted strongly with proteins involved in respiratory electron transport and respiratory chain complex assembly. Accordingly, genetic knockdown of NLN impaired oxidative metabolism and reduced the formation of large, quaternary structures called respiratory chain supercomplexes (RCS). RCS promote oxidative metabolism and are composed of respiratory chain complexes I, III, and IV.To identify how NLN is mediating the assembly of RCS, we revisited our BioID results. One of NLN's top mitochondrial interactors was the inner mitochondrial membrane protein, LETM1 (leucine zipper‐EF‐hand containing transmembrane protein 1), which has previously been implicated in RCS assembly. LETM1 forms two complexes, termed the minor and the major complex.Chemical inhibition of NLN has also been shown to target AML cells and stem cells. Treatment of AML cells with R2 reduced viability, impaired RCS and LETM1 complex formation, and reduced OCR. In addition, R2 treatment reduced engraftment of patient derived xenograft models. R2 also targeted LSCs, as evidenced by reduced engraftment after secondary transplantation. Of note, R2 treatment did not affect the engraftment of normal cord blood cells, suggesting that chemical inhibition of NLN selectively targeted AML cells and stem cells but spared normal hematopoietic cells and progenitors.5Compared to normal hematopoietic cells, AML cells and stem cells have lower spare reserve capacity and are dependent on OXPHOS. The mitochondrial proteases, CLPP and NLN, maintain the integrity of the mitochondrial respiratory chain and promote oxidative metabolism Figure . TargetiAaron D. Schimmer has received honorariums or consulting fees from Novartis, Jazz, Otsuka, and Takeda Pharmaceuticals and research support from Medivir AB and Takeda. Aaron D. Schimmer owns stock in Abbvie Pharmaceuticals and is named on a patent application for the use of DNT cells for the treatment of leukemia. Sara Mirali declared no potential conflicts of interest.S.M.: manuscript writing and final approval of the manuscript; A.D.S.: manuscript writing and final approval of the manuscript.


Despite extensive research and development of new treatments, acute myeloid leukemia (AML)-backbone therapy has remained essentially unchanged over the last decades and is frequently associated with poor outcomes. Eradicating the leukemic stem cells (LSCs) is the ultimate challenge in the treatment of AML. Emerging evidence suggests that AML remodels the bone marrow (BM) niche into a leukemia-permissive microenvironment while suppressing normal hematopoiesis. The mechanism of stromal-mediated protection of leukemic cells in the BM is complex and involves many adhesion molecules, chemokines, and cytokines. Targeting these factors may represent a valuable approach to complement existing therapies and overcome microenvironment-mediated drug resistance. Some strategies for dislodging LSCs and leukemic blasts from their protective niche have already been tested in patients and are in different phases of the process of clinical development. Other strategies, such as targeting the stromal cells remodeling processes, remain at pre-clinical stages. Development of humanized xenograft mouse models, which overcome the mismatch between human leukemia cells and the mouse BM niche, is required to generate physiologically relevant, patient-specific human niches in mice that can be used to unravel the role of human AML microenvironment and to carry out preclinical studies for the development of new targeted therapies. Acute myeloid leukemia (AML) is a hematologic cancer characterized by the abnormal clonal proliferation of undifferentiated blasts and by their primary infiltration of the hematopoietic organs, such as bone marrow (BM), lymph nodes and spleen. Uncontrolled expansion of leukemic cells suppresses normal hematopoiesis, leading to life-threatening thrombocytopenia, anemia, and immunodeficiency . The AMLIn the last decades, the identification of AML driver mutations such as FLT3 and IDH mutations and the development of specific target therapies have been associated with improved outcomes in this subset of patients ,7,8. DesTherefore, targeting the altered BM microenvironment may represent a more useful approach for overcoming these limitations. Moreover, this strategy could be applied together with chemotherapy to a broad range of patients with various subtypes of disease and driver mutations as interactions between AML and their BM niche are not clone specific. Recent discoveries have identified a series of AML niche-specific features that could be targeted to suppress the self-reinforcing leukemic BM microenvironment and restore normal hematopoiesis. Some approaches for uncoupling leukemic stem cells (LSCs) and leukemic blasts from their protective niche have already been tested in patients and are in different phases of the process of clinical development. Other strategies, like targeting the mesenchymal stromal cells (MSCs) remodeling processes, are very promising but remain mainly at pre-clinical stages.This review summarizes the recent advances in elucidating the AML BM stromal niche characteristics and point out new perspectives for targeting AML-niche interactions to contribute to the improvement of current treatments.The BM microenvironment interacts with normal hematopoietic stem cells (HSCs) and leukemic cells in several ways affecting their cellular functions, including trafficking, adhesion, proliferation, differentiation, and quiescence. AML cells exploit stromal-dependent pro-survival signals and shape the BM microenvironment to create a permissive/self-reinforcing niche favorable for the maintenance and progression of chemotherapy-resistant AML, while suppressing normal hematopoiesis. The mechanism of stromal-mediated protection of leukemic cells and their anchorage to the BM is complex and involves many adhesion molecules, chemokines, and cytokines.AML cells hijack the BM niche by upregulating receptors on their cell surface, such as very late antigen-4 (VLA-4), CD44, E-selectin ligand-1 (ESL-1), and CD98, resulting in the retention of LSCs in the BM niche via interactions with adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), fibronectin (FN), hyaluronan (HA), osteopontin (OPN), selectins, and integrins. All these molecular interactions are potential targets for therapy since retention of LSCs in the BM niche results in quiescence and survival of LSCs and subsequently resistance to chemotherapy .Very late antigen-4 (VLA-4) is a cell surface adhesion molecule, composed of CD49d (α4) and CD29 (β1) subunits, which belongs to the integrin family . VLA-4 iJacamo et al. showed that VLA-4 is upregulated in AML cells and the interaction with VCAM-1 on stromal cells activates pro-survival and proliferative pathways in both leukemia and stromal cells via the nuclear factor-κB (NF-κB) pathway and favors chemoresistance. Interestingly, blockade of stromal NF-κB signaling can make AML cells more susceptible to chemotherapy . MoreoveNatalizumab is a humanized anti-VLA-4 monoclonal antibody (mAb) used for the treatment of autoimmune diseases, which causes prolonged HSC mobilization . In a xeAnother VLA-4 inhibitor is the synthetic tellurium compound AS-101 tellurate), which induces redox inhibition of adjacent thiols in the exofacial domain of VLA-4 after binding to stromal FN. The inactivation produces cytoskeletal conformational changes that decrease the PI3K/Akt/Bcl-2 signaling  resultinCD44 is a ubiquitously expressed transmembrane glycoprotein that is alternatively spliced, leading to the production of multiple protein isoforms. In the hematopoietic system, CD44 is expressed by hematopoietic precursors and regulates several functions as well as trafficking, lodgment, proliferation, apoptosis, and differentiation by binding to different ligands as HA, OPN, FN, and selectin ,22.CD44 is overexpressed in AML blasts and the presence of certain splice variants is associated with poor prognosis . LSCs arThe use of H90, an anti-CD44 mAb, broke the interaction between CD44 and HA and caused a marked reduction of the leukemic burden in non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice transplanted with primary AML cells. Leukemic cells obtained from primary mice treated with H90 did not engraft into the secondary recipient mice, demonstrating that the antibody directly targeted LSCs . SeveralDifferent mechanisms have been hypothesized to explain the effect of anti-CD44 mAbs on AML cells, such as the induction of cell differentiation and inhibition of cell proliferation . SpecifiOsteopontin (OPN) is another ligand of CD44, which consents anchorage of LSCs in the HSC niche. Thus, besides the CD44-HA interaction, anti-CD44 antibodies may target CD44-OPN interaction as well. OPN expression was increased in BM blasts and in BM serum of AML patients as compared with healthy controls and OPN overexpression was related to a poor prognosis . BlockadE-selectin is a cell adhesion molecule constitutively expressed by BM endothelium where it plays a key role in HSC and HPC homing. In particular, the deletion of E-selectin from endothelial cells increased HSC quiescence and self-renewal, confirming that E-selectin supports HSC functions . MoreoveE-selectin ligand-1 (ESL-1) is expressed not only by HSCs but also by AML cells, where it induces blasts adhesion to the vascular niche and Wnt signaling activation, favoring the survival of AML blasts and promoting cell-adhesion mediated drug resistance . E-selecE-selectin can be inhibited by the small-molecule antagonist Uproleselan (GMI-1271), which favors the reduction of cell survival and the increase of chemosensitivity, showing a contraction in the leukemic burden in xenograft AML models treated with a combination of chemotherapy and this E-selectin antagonist . Thus, cCD98 is a heterodimeric protein constituted by a heavy chain (CD98hc encoded by SLC3A2) that is a type II single-pass transmembrane glycoprotein, disulfide-linked to a multipass light chain that can be any one of six amino acid transporters  . The heaIn AML, CD98 enhances the interaction between blasts and stromal cells and promotes maintenance and proliferation of leukemic cells . For thiAlthough clinical translation of these approaches targeting AML cell adhesion within the BM niche is still in its infancy, emerging early phase clinical data indicate this approach may be beneficial in the adjuvant setting, boosting the effects of conventional treatments.Common chemokine axes, cytokine signaling pathways and pro-inflammatory or immunosuppressive mediators usually regulate homeostasis in the normal BM microenvironment. However, a series of studies suggest that these soluble factors may be hijacked by AML, promoting the maintenance of LSCs and, as a result, contributing to disease progression and recurrence  Figure .CXC motif ligand 12 (CXCL12), also known as stromal-derived factor 1α (SDF-1 α), is a chemokine mainly produced by MSCs in the adult BM . The intConsidering the pathogenic role of CXCL12-CXCR4 axis in AML, much effort has been put toward finding strategies inducing its blockade ,46. OverCC chemokine motif ligand 2 (CCL2), also called monocytes chemoattractant protein-1 (MCP-1), is a powerful chemoattractant for monocytes and macrophages to sites of inflammation. Upon binding to CC chemokine receptor 2 (CCR2), this chemokine mediates the activation of several intracellular pathways associated with survival, adhesion, proliferation, growth, chemotaxis and trans-endothelial migration . Unlike In this scenario, blockage of CCL2-CCR2 interaction might increase chemotherapy efficacy by reducing the BM infiltration of M2-like macrophages, which normally downregulate effector cell types anti-tumor responses . Overall2) display considerably higher levels of CXCL8 compared to those maintained under normal oxygen state (21% O2). Besides, CXCL8 released by AML cells interacts with CXCR1-expressing MSCs, determining their migration into the BM niche, where these stromal cells mediate chemoprotective, anti-apoptotic and pro-survival functions [CXC chemokine ligand 8 (CXCL8), also known as IL-8, is a powerful pro-inflammatory chemokine involved in neutrophil chemotaxis and degranulation. This chemokine exerts its functions binding to two G-protein-coupled receptors, CXCR1 and CXCR2 . High exunctions . Additiounctions .Overall, these studies point out that CXCL8-CXCR1/CXCR2 might be a valid therapeutic target for the treatment of AML. Indeed, CXCR2 blockage may act selectively against LSCs, while sparing normal HSCs. Whereas, CXCR1 blockage may inhibit AML blasts-MSCs crosstalk, determining the loss of support signals for AML cells. By now, several drugs, like small molecule inhibitors, have been tested for the treatment of solid tumors , but in IL-1 family is composed of several factors, among which IL-1α and IL-1β are the most widely characterized. IL-1α is constitutively secreted by different cell types in homeostatic conditions and its production increases following an inflammatory stimulus. On the contrary, IL-1β is released by a restricted selection of cell types, primarily myeloid cells. Both cytokines interact with IL-1 receptor 1 (IL-1R1) and activate a downstream signaling cascade that mediates the transcription of several target genes . Hence, A series of therapeutic agents have been approved by the FDA for the treatment of several chronic and inflammatory diseases, but they have not been tested yet in clinical trials for AML. These drugs can be classified as (1) natural or recombinant IL-1 inhibitors, like IL-1Ra and Anakinra (Kineret), (2) IL-1 decoy receptors, such as Rilanocept  and (3) mAbs, as Canakinumab (Ilaris) . A phaseTNF-α is a pro-inflammatory cytokine and a pleiotropic factor, as it takes part in a wide range of functions, such as inflammatory responses, anti-tumor actions and homeostasis. TNF-α exerts these activities through binding tumor necrosis factor receptors (TNF-R)1 and 2. TNF-R1 is expressed by many different cell types and regulates cytotoxicity, resistance to infections and activation of the NF-κB pathway, whereas TNF-R2 expression is restricted to the hematopoietic lineage and it is activated only in inflammatory conditions. TNF-α role as a HSC growth factor regulator highly depends on the microenvironment which can determine both an inhibitory and a stimulatory effect on HSCs proliferation and maintenance . TNF-α oIL-6 is a multifunctional cytokine released by several cell types, like innate and adaptive immune and stromal cells. This cytokine exerts its functions by binding to the IL-6 receptor (IL-6R/CD126). Primarily, IL-6 is a mediator of acute-phase and immune responses, but it plays a role also in cell proliferation, survival, differentiation and migration. Additionally, IL-6 is implicated in the regulation of normal hematopoiesis, and in malignant conditions it seems to be involved in AML blasts formation . AccordiFor this reason, several strategies have been developed to block IL-6 or its receptor . EssentiInterferon alfa and beta (IFN-α/β) are type I interferons which exert their functions through the interaction with the IFN-α/β receptor. IFN-α and IFN-β are part of a composite group of factors that activate in response to viral antigens and stimulate anti-proliferative and immunomodulatory functions . MultiplInterferon-gamma (IFN-γ) is a type II interferon and a pro-inflammatory cytokine produced by activated T lymphocytes and NK cells. It mediates multiple effects on a wide range of cell types, interacting with its receptors IFNGR1 and IFNGR2. Essentially, IFN-γ is a key mediator between innate and adaptive immune responses. It is involved in the regulation of many other immunomodulatory factors and it plays a role in HSCs regulation, stimulating their proliferation . AccordiTransforming growth factor β (TGF-β) superfamily is a group of 32 factors that includes TGF-β. The most investigated members of this family are the three different isoforms of TGF-β , especially TGF-β1, which is ubiquitously expressed and highly secreted . TGF-β iSeveral attempts to target the TGF-β signaling pathway have been made in solid tumors. Three principal classes of drugs have been developed: (1) small molecule TβRI inhibitors, among which Galunisertib (LY2157299) is the most widely studied. This compound has shown little but consistent therapeutic efficacy in two phase II clinical trials for pancreatic cancer and hepatocellular carcinoma patients. (2) Antibodies directed against TGF-β, like 1D11, an anti-pan-TGF-β antibody and its humanized form, Fresolimumab, that are being tested for the treatment of multiple types of solid tumors, and lastly, (3) receptor-based TGF-β traps; AVID200, which selectively binds and inhibits the TGF-β1 isoform, is of particular interest since it is currently under phase I clinical trials for the treatment of advanced and metastatic solid tumors . To obtaIL-10 is an immunosuppressive multifunctional cytokine produced by several cell types, like Tregs, macrophages, B-cells and keratinocytes. This factor interacts with its two receptors, IL-10 receptor 1 (IL-10R1) and IL-10 receptor 2 (IL-10R2), to exert its pleiotropic effects. Specifically, IL-10 is involved in the inhibition of the effector functions of T lymphocytes, monocytes and macrophages and in the downregulation of pro-inflammatory cytokines to limit and eventually conclude inflammatory responses . Like TGVascular endothelial growth factor (VEGF) is a proangiogenic factor that binds to specific tyrosine kinase receptors on endothelial cells (EC) leading to the induction of angiogenesis. Neoangiogenesis has been shown to play an important role in the pathogenesis of AML and an increased number of ECs have been noted on BM biopsies of patients . AML blaAML may, therefore, represent a disease that is sensitive to a therapy directed against the effects of VEGF . To dateBeyond adhesion molecules and soluble factors, another mechanism has recently been ascribed to AML-niche interaction. Mitochondrial transfer is a newly described way of communication between BM-derived MSCs and AML cells, which consists of the delivery of fully functional mitochondria from the donor stromal cell to the recipient leukemic cell . Given tThe signaling pathways and the molecular processes involved in mitochondria uptake from AML cells are still not well characterized. However, the consensus in the available literature suggests that this process of transfer demands cell-to-cell contact. Indeed, Moschoi et al. observed, in a niche-like co-culture system, that mitochondrial transfer would occur only if MSCs and AML cells were in direct contact in the BM microenvironment. According to their model, mitochondrial uptake was mediated by endocytosis, but they were not able to define the players involved in this system . MarleinAll things considered, mitochondrial transfer might be described as a mechanism employed by AML cells to rescue respiration, acquire a metabolic advantage and survive even after exposure to chemotherapy. Indeed, the majority of chemotherapeutics are based upon processes involving the induction of oxidative stress. Hence, leukemic cells with extra-functional mitochondria might survive after oxidative chemotherapy and eventually regenerate the disease.Targeting mitochondrial transfer might be an appealing approach for the development of improved therapeutic strategies able to eradicate resistant AML cells. Considering that mitochondrial metabolism has emerged as a vulnerable point for LSCs, the inhibition of mitochondrial transfer might contribute to disease clearance . AdditioHypoxia is a key regulator of the niche-mediated quiescence and maintenance of HSCs ,136. LowAML cells are preferentially home to hypoxic niches where their exposure to drugs and immune effector cells is reduced through the compromised blood flow, and these BM areas contain true quiescent and chemoresistant LSCs endowed with a self-renewal capability . HypoxiaHypoxia-activated prodrugs (HAPs) are designed to specifically target cells in a hypoxic milieu. This targeting strategy is conceived by attaching a hypoxia-activated trigger to a chemotherapeutic agent. Under hypoxic conditions, the agent is released and activated while remaining intact in normal conditions and leaving non-hypoxic cells undamaged. TH-302 (Evofosfamide) is a HAP, which releases the DNA alkylating agent bromo-isophosphoramide mustard under hypoxic conditions. Primary human AML cells that were chemoresistant in oxygen-poor conditions became sensitive to Ara-C when treated with TH-302. The prodrug decreased HIF-1α expression, enhanced DNA double-strand breaks, and induced cell cycle arrest and apoptosis . In AML BM stromal remodeling through altered differentiation of MSCs has emerged, besides well-described changes on ECs of BM vasculature , as a keOsteoblastic lineage cells are a compartment of MSC progeny committed to the osteogenesis that comprises many intermediate stages of differentiation, including immature osteoprogenitors and mature bone-lining and bone-forming OBs.In xenograft models, leukemia engrafted preferentially in association with microvascular domains in the BM ,148; espBone turnover is impaired in the leukemic niche. AML has been associated with the accumulation of OB-primed MSCs, which do not seem to be able to mature into OBs, correlating with decreased mineralized bone. Using an immunocompetent murine model of AML, Frisch et al. showed that osteoblastic cells were reduced and inhibited by leukemia, as measured by decreased levels of the bone formation marker, osteocalcin. Mineralized bone was also diminished. The cytokine CCL3 , known to be implicated in bone loss, was increased in AML blasts in mice and patients, suggesting that it may be related to the peculiar phenotype. Regarding the osteoclasts, there is an initial increase followed by a slight decline as the disease progresses to overt leukemia . AnotherMDS and AML patients show a reduction in OB numbers, reflecting a corresponding reduction in bone-formation rate without any changes in osteoclast numbers, and OB recovery correlates with better prognosis . This isTaken together, these observations indicate that AML cells induce and require an osteoprogenitor-rich niche for their expansion, but block MSC differentiation to produce mature OBs . It is tIn vitro experiments identified differentiating OBs as potent protectors of AML cells from various apoptosis-inducing agents, such as SDF-1 and standard chemotherapeutics Daunorubicin and Ara-C ,159,160.Blocking of CCL3 signaling can represent a further strategy to restore OB function, prime the BM for subsequent therapies to ablate leukemic cells and accelerate recovery of normal hematopoiesis. Following treatment with the small molecule Maraviroc which inhibits CCL3 binding to the chemokine receptor CCR5 presents on osteoblastic cells, the increase in MSC population is reversed and leukemic burden was decreased > 2-fold in the BM in a murine model of myelogenous leukemia . ImportaInhibition of BMP/CTGF-mediated signaling may also represent a novel therapeutic concept in AML to reduce leukemia growth. CTGF is a prognostic factor in ALL, and inhibition of its expression by an anti-CTGF antibody (FG-3019) led to increased survival of leukemia-bearing mice . In vitrNeuropathy, which is characterized by decreased sympathetic nerve fibers and ensheathing Schwann cells, is observed in the BM of newly diagnosed AML patients . MoreoveThe sympathetic nervous system is critical for MSC quiescence, OB differentiation and HSC maintenance . In the Although sympathetic neuropathy is observed both in myeloproliferative neoplasm (MPN) and AML, it has been demonstrated using experimental mice that defective β3-adrenergic signals are responsible for the phenotypes observed in MPN, whereas β2-adrenergic signals seem to be mainly involved in AML ,166. ThuExosomes, cell-derived microvesicles of endocytic origin, may be another method that malignant cells use to reprogram the BM niche . In AML It was demonstrated in in vitro co-cultures that both primary AML cells and AML cell lines release exosomes containing coding and non-coding RNAs that can be uptaken by BM stromal cells, altering their secretion of growth factors and changing their proliferation and migration capability . MoreoveRecently, Kumar et al. showed that AML cells use the secretion of exosomes as a means to adapt the niche to support leukemia growth and suppress normal hematopoietic development . They deRab27a has been recently identified as one protein involved in exosome release and it represents a potential therapeutic target . Rab27a Under certain conditions, osteolineage cells have been demonstrated to contribute to the alteration of HSCs, triggering the formation of leukemia-initiating cells. Signals from the microenvironment may indeed select target cells for subsequent transforming events and, therefore, they may represent candidate therapeutic targets in both treatment and prevention strategies . SpecifiSBDS (Shwachman-Bodian-Diamond syndrome) gene mutated in Schwachman-Diamond syndrome, a human congenital BM failure with known leukemia predisposition [In the first model, the conditional deletion of Dicer1 in mouse osteoprogenitors impairs osteogenic differentiation and causes myelodysplasia with sporadic transformation to AML . The losposition .Subsequently, it has been reported that mutations activating β-catenin in OBs in mice induce myelodysplasia, rapidly progressing to AML . These iFinally, activating mutations of the Tyrosine phosphatase SHP-2 (encoded by Ptpn11 gene) in MSCs and osteoprogenitors, already found in Noonan syndrome and associated with an increased risk progression to leukemia, induce juvenile myelomonocytic leukemia-like myeloproliferative neoplasm in mice through the overproduction of chemokine CCL3 . This stWhile these findings in mice offer direct evidence for OB-induced leukemogenesis and although some observations in mouse models have been linked to human diseases, it remains unclear whether alterations to the microenvironment can drive leukemia in humans. Emerging reports of donor cell leukemia in patients receiving allogeneic transplantation  seem to suggest an oncogenic role of the microenvironment that can lead to secondary malignancy also in humans .Adipocytes derive from MSC differentiation are prevalent in the BM stroma and their number augment with age. MSCs from AML patients have a higher propensity to differentiate into adipocytes, and the interactions between adipocytes and AML blasts in the BM niche support their survival and proliferation . We receAML blasts modulate adipocyte metabolism, inducing lipolysis of triglyceride to fatty acid (FA) through induction of hormone-sensitive lipase and growth differentiation factor 15 (GDF15) release ,183. In Increasing attention is being paid on metabolic alterations in AML as potential therapeutic targets and encouraging results have been achieved in preclinical AML models using several inhibitors of FA metabolism. Pharmacological inhibition of FAO by carnitine palmitoyltransferase 1a (CPT1a) inhibitor was reported to decrease the pro-survival effects of adipocytes on AML. Moreover, Lee and colleagues identified a novel FAO inhibitor derived from the avocado fruit, avocatin B, to be a potent inhibitor of AML survival and proliferation . Shafat Nevertheless, FAO inhibition triggers the activation of bypass metabolic pathways to enhance survival of AML in the BM microenvironment. This indicates a possible limited efficacy of these metabolic inhibitors when used as single agents. In AML cells co-cultured with BM adipocytes, FAO inhibition with avocatin B increased glucose uptake and switched to glycolysis . AlternaAdipocytes contribute to the AML protection also by other pro-AML survival factors in addition to the FA release. BM adipocytes have been shown to secrete high levels of pro-inflammatory cytokines in the context of leukemia, which may have an impact on leukemia cell migration and survival .The first in vitro AML niche models were 2D cultures based on the Dexter system where an established feeder stromal cell layer allowed for the prolonged viability and stemness of hematopoietic cells. van Gosliga and colleagues developed and extensively tested the first long-term co-culture system for the ex vivo maintenance of primary AML cells based on an immortalized mouse MSC line as the feeder layer . To overTo overcome this limitation, numerous 3D models have been developed using a range of biomaterials as scaffolds designed with properties comparable to those of the in vivo BM, including pore size and surface cell-adhesive features, to allow the establishment of cell–niche associations . EffortsThe BM microenvironment is also strictly regulated by the concentration of soluble factors, oxygen levels, and the mechanical stress applied by blood flow. The use of bioreactors can then allow for modeling the niche situation more closely. Rödling et al. developed a bioreactor system for perfusion of 3D scaffolds mimicking the BM in vivo and demonstrated the importance of perfusion during drug treatment as results are different with and without perfusion .The most advanced BM niche model incorporates an in vivo implantation stage in a perfused 3D co-culture ex vivo. One of these hybrid approaches  was developed by the Ingber laboratory by first engineering new bone in vivo, explanting it whole and maintaining it in vitro with culture medium in a microfluidic device . This enPatient-derived xenograft (PDX) mouse models are currently the gold standard for studying the development of human leukemia and testing new therapies. Notably, engraftment and expansion of human AML in vivo remain challenging as a substantial number of samples fail to engraft the most optimized host mice, particularly less aggressive malignancies . It may Due to the mismatch between human leukemia cells and the mouse BM niche, traditional xenograft models are insufficient to study AML microenvironment-targeted therapies, and to overcome this limitation, several humanized xenograft mouse models are developing. Xenotransplantation approaches are rapidly evolving toward injecting human hematopoietic cells into immunodeficient mice implanted with ectopic humanized BM niches. The first study of AML engraftment in humanized microenvironment was reported by Vaiselbuh et al., who developed an ectopic humanized niche by implanting subcutaneously in NOD/SCID mice polyurethane scaffolds coated with human MSCs. The scaffolds formed structures mimicking the human BM niche, including mineralized bone-matrix, OBs, and stromal cells, as well as the appropriate tissue vascularization. Primary AML cells injected directly in pre-implanted scaffold or intravenously in mice, successfully engrafted in the ectopic niche . FurtherFinally, Battula et al. developed a different approach called “human bone implant” that consists of subcutaneous transplants in NSG mice of fragments from freshly collected human BM biopsies using matrigel as a carrier . The impThese models are useful tools for the functional assessment of niche contribution, as well as for the development of novel anti-leukemic modalities aimed at modifying the AML microenvironment. Moreover, these new approaches, which also allow genetic manipulation of human niche components, can likely help in better understanding the role of factors critical for leukemia engraftment/progression. A pilot study demonstrated that the deletion of HIF-1α in human MSCs impaired AML engraftment in BM organoids by decreasing SDF-1α expression .Of note, current 3D models use MSCs isolated from healthy donors, which are molecularly and functionally different from disease-exposed ones. The use of patient-derived niche components may further improve these models and help unravel the role of intercellular communication in disease-relevant systems. We recently reported an AML stromal niche model obtained using MSCs derived from AML patients . QuestioThe final goal of AML research is to find new strategies to effectively eradicate LSCs to prevent therapy relapse and, ultimately, offer a cure to patients.Targeting the altered adhesion and homing of AML cells to the niche is a promising approach to prevent relapse, as to displace LSCs from their protective niche may allow for their full eradication. Development of new approaches to counteracting the soluble factors that promote malignant niche formation also has therapeutic potential. Moreover, finding new systems to directly target the specific features of the malignant BM niche, including aberrant MSC differentiation, also may be helpful at a therapeutic level. Thus, these types of AML niche-targeted strategies may represent a useful approach to complement chemotherapy.A main challenge is represented by the dual role of the microenvironment in regulating normal and malignant hematopoiesis. Therapies targeting the tumor microenvironment must simultaneously eliminate chemo-resistant AML cells and preserve/re-establish normal hematopoiesis.Our understanding of niche contributions to AML has hugely increased over the last decades, however, many questions remain unsolved; in particular, whether the phenotypes and molecular mechanisms identified in mouse models are maintained and therapeutically relevant in the human disease. The use of human leukemia samples to understanding AML niche biology is not allowed as diagnoses are made on BM aspirates that disrupt BM architecture.Moreover, most of the pre-clinical research to study drug effects on AML progression is done in xenograft mice. In the case of therapies targeting the AML microenvironment, this may represent an important limitation in the translation of animal model results to human patients due to the mismatch between human leukemia cells and the mouse BM niche.Functional assessment of niche contribution to AML and pre-clinical testing of new niche-targeted therapies require the establishment of disease-relevant model systems. Researchers’ efforts should be focused to build bone/BM organoids systems that aim to closely recapitulate the human malignant BM niche complexity.


Castanea mollissima Blume (PICB) to produce a new additive. PICB is known to have beneficial effects on monogastric animals. However, the effects of PICB on ruminants need further investigation. This study shows that the addition of PICB to the ruminant diet can significantly reduce methane yields and the ratio of acetic acid to propionic acid, thus improving feed efficiency. The results contribute to the knowledge of tannins from a specific part of the Castanea mollissima Blume plant and provide a theoretical basis for the potential use of involucres. Such an application would limit environmental pollution caused by their disposal and reduce the negative influence of ruminants on the environment.In China, chestnut involucres, an agricultural byproduct, are not appropriately used and are regarded as agricultural waste. To reduce the environmental pollution caused by burning or misuse of chestnut involucres, the authors extracted polyphenols from the involucres of Castanea mollissima Blume (PICB) on in vitro rumen fermentation. Three healthy Angus bulls (350 ± 50 kg), with permanent rumen fistula, were used as the donors of rumen fluids. A basic diet was supplemented with five doses of PICB (0%–0.5% dry matter (DM)), replicated thrice for each dose. Volatile fatty acids (VFAs), ammonia nitrogen concentration (NH3-N), and methane (CH4) yield were measured after 24 h of in vitro fermentation, and gas production was monitored for 96 h. The trial was carried out over three runs. The results showed that the addition of PICB significantly reduced NH3-N (p < 0.05) compared to control. The 0.1%–0.4% PICB significantly decreased acetic acid content (p < 0.05). Addition of 0.2% and 0.3% PICB significantly increased the propionic acid content (p < 0.05) and reduced the acetic acid/propionic acid ratio, CH4 content, and yield (p < 0.05). A highly significant quadratic response was shown, with increasing PICB levels for all the parameters abovementioned (p < 0.01). The increases in PICB concentration resulted in a highly significant linear and quadratic response by 96-h dynamic fermentation parameters (p < 0.01). Our results indicate that 0.2% PICB had the best effect on in-vitro rumen fermentation efficiency and reduced greenhouse gas production.Nutritional strategies can be employed to mitigate greenhouse emissions from ruminants. This article investigates the effects of polyphenols extracted from the involucres of  Anaerobic rumen fermentation produces a large amount of methane  mainly contain HTs such as ellagitannin and gallotannin [Castanea sativa, no literature report has addressed the effects of PICB on rumen fermentation. Aboagye and Beauchemin [4 production, and animal performance vary depending on diet composition, tannin source and type, dose, and molecular weight, part of the plant, stage of growth, and growing conditions. The effects of different tannin sources should be studied case by case. The aim of this study is to investigate the effects of different concentrations of PICB on pH, NH3-N concentration, volatile fatty acids (VFAs) and its components, gas production, and the dry matter degradation rate (IVDMD) during in vitro fermentation.According to the United States Food and Agriculture Organization, the worldwide production of American, European, and Chinese chestnuts and their hybrids is about 2.3 million tons per year . Chestnulotannin . Dong etlotannin  and Xionlotannin  reportedauchemin  indicateThe experimental procedure was evaluated and approved by the Committee for the Care and Use of Experimental Animals, Beijing University of Agriculture . The animals were cared for and handled according to the welfare and ethics standards for experimental animals of Beijing Municipality .Castanea mollissima Blume involucres were sampled from Huairou Chestnut Station. PICB were extracted according to the method described by Shi et al. [g for 15 min. PICB extract was obtained by drying the supernatant with a rotary evaporator. The final PICB had 52.5% total polyphenol content, among which 18.65% was ellagic acid, which is considered the effective component. The nonpolyphenol content of the PICB extract was made up of crude fiber, ash, and water [i et al. . Brieflynd water ,15.All chemicals and reagents used in this trial were of analytical grade and were purchased from commercial sources.The trial was carried out at the experimental farm of the Beijing University of Agriculture. Three healthy Angus bulls (350 ± 50 kg), equipped with permanent rumen fistula, were selected as rumen fluid donors. The cattle were raised in a confined barn, and their total mixed ration (TMR) is shown in 2 to maintain anaerobic conditions. The operation was completed in the shortest possible time.After 14 days of adaptation, samples of rumen fluid were collected from the three donors via the rumen fistula before the morning feed. Rumen fluid was then placed in a thermos flask and brought to the laboratory within 20 min. Rumen fluid was filtered through four layers of gauze into a flask while continuously flushing with COThe trial was carried out three times. The basic fermentation substrate for the in-vitro trials was obtained by drying and grinding the TMR fed to donor cattle to pass through a 1-mm sieve.2 flushing. The artificial rumen culture solution was made by mixing a preprepared mineral buffer solution and rumen fluid (2:1). The substrates were prepared by mixing the basic fermentation substrate with appropriate amounts of PICB to obtain samples with concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5% PICB; each concentration was prepared in triplicate. Substrate (200 mg DM) was weighed into a 100-mL calibrated glass syringe , and 30 mL of artificial rumen culture solution was added. The syringe was kept vertical and shaken gently. By pushing the piston upward, no gas headspace at the liquid inlet end of the syringe was permitted. The silicon rubber tube was clipped, and the initial volume was recorded. Each syringe was placed in an incubator at 39 °C for 24 h, and the gas volume was recorded at different time points; the fermentation parameters and gas composition were analyzed at the end of the trial.The method described by Menke and Steingass  was usedIn-vitro gas production over 96 h was recorded at 16 different time points of fermentation using the method described above. Gas production was calculated relative to the gas volume of a blank control group at every time point. Cumulative gas production was calculated, and a gas production curve was prepared.4 yield was calculated from the resultant data.After 24 h of in vitro incubation, the fermentation syringes were removed and placed in an ice-water bath to terminate the fermentation. Gas production was recorded. Using a disposable syringe, 1 mL of gas was extracted from the fermentation syringe and immediately injected into a TP-2060T gas chromatograph . Chromatographic analysis was performed under the following conditions: thermal conductivity detection, TDX-01 packed column (1 m × 3 mm), inlet temperature 150 °C, column temperature 120 °C, detector temperature 150 °C, helium carrier gas (flow rate 50 mL/min), injection volume 1 mL. The CHImmediately after gas sampling, the pH of the fermentation liquid was measured with a pH meter equipped with a glass electrode .w/v); the mixture was kept at 4 °C for 30 min and then centrifuged . The supernatant (1 mL) was used for the determination of VFA by gas chromatography , as described by Cao and Yang [3-N using the phenol-sodium hypochlorite colorimetric method described by Broderick and Kang [To measure VFA concentration, 5.0 mL of fermentation liquid was mixed with 1 mL of phosphoric acid (25% and Yang . Anotherand Kang . The samFor the in-vitro DM degradation rate (IVDMD) assay, the substrates (600 mg DM) were prepared by mixing the basic fermentation substrate with the appropriate amount of PICB to obtain samples with concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5% (w/w) PICB. Each concentration was prepared in triplicate. The substrates were poured into a filter bag, placed in 100-mL glass culture tubes (in duplicate), and mixed with 70 mL of artificial rumen culture solution. The tubes were then covered quickly and placed in a 39 °C water bath with a shaker. After 24 h of fermentation, the filter bags were removed, rinsed with water, and dried at 105 °C for 12–24 h to constant weight. The in-vitro DM degradation rate (IVDMD) was calculated as follows:3-N concentration, VFA, gas production, gas composition, and DM degradation were analyzed using the ANOVA of SPSS 25.0. The Tukey multi-comparison method was used to compare the effects of different doses of PICB. Linear (L) and quadratic (Q) responses were analyzed to evaluate the dose-dependent trends. The significant level was p < 0.05, and the highly significant level was p < 0.01 [The data for pH, NHp < 0.01 ,23,24.Y is the cumulative gas production (mL) at time t, B is the theoretical maximum gas production (mL), c is the gas production rate (h−1), and t is the fermentation time (h) [The nonlinear method of SPSS 25.0 was used to calculate the dynamic fermentation parameters, and an exponential model (Equation (2)) was adopted to analyze the nonlinear fitting of cumulative gas production at different PICB concentrations:p > 0.05).The pH  was not modified by the addition of PICB, and no significant difference was observed among the different PICB concentrations . A PICB concentration of 0.3% showed the largest decrease, which was significantly different from the other PICB groups except for 0.2%. A significant quadratic response was observed with increasing PICB concentration (p < 0.01).PICB addition significantly affected the NHp > 0.05), the proportions of acetic, propionic, and butyric acids were significantly affected. Compared with the control group, the addition of 0.1%–0.4% PICB significantly reduced the percentage of acetic acid (p < 0.05), with the 0.2% and 0.3% groups significantly lower than the 0.1% and 0.4% groups. In contrast, the addition of 0.1%–0.4% PICB significantly increased the percentage of propionic acid (p < 0.05), with 0.2% and 0.3% groups significantly higher than the 0.1% group. Correspondingly, the addition of 0.2% and 0.3% PICB significantly reduced the acetic/propionic acid (A:P) ratio compared with the control group. The 0.3% PICB inclusion significantly increased butyric acid content during fermentation (p < 0.05). Highly significant quadratic responses (p < 0.01) were observed with the increase of PICB concentration for acetic, propionic, and butyric acids and for the A:P ratio.While the addition of PICB had no significant effect on total VFA concentration during fermentation (p > 0.05) at any dose.PICB had no significant effect on isobutyric, isovaleric, and valeric acids and IVDMD (p < 0.05); 0.2% and 0.3% PICB did not significantly influence gas production even though the values were higher. The CH4 percentage and volumetric yield for 0.2% and 0.3% PICB were significantly lower than those of the control group (p < 0.05), with the 0.2% results lower than the 0.3% results. Highly significant quadratic responses with increasing PICB concentration were observed for CH4 volumetric yield and CH4 percentage (p < 0.01). The addition of PICB did not significantly affect H2, oxygen + nitrogen, and CO2 content during 24 h of in-vitro fermentation (p > 0.05).The results related to gas production and composition after 24 h of in-vitro fermentation are reported in p < 0.01) with increasing PICB concentration. Gas production and maximum gas production showed highly significant quadratic responses with increasing PICB concentration (p < 0.01).As shown in Rumen pH depends mainly on diet characteristics  and can range between pH 5.6 and 7.5 in response to different feed formulations . In the 3-N is a key nitrogen source for fermentation and one of the primary nitrogen sources for microbial growth. High NH3-N concentration in the rumen leads to more nitrogen loss and can even influence reproductive performance [3-N content. First, Al-Dobaib [3-N concentration. HTs can also combine with protein to form HT–protein complexes, but some rumen microorganisms can dissociate the HT–protein complex [3-N production by 3.5% and 4.7%, respectively, which was consistent with the results of Sarnataro and Spanghero [3-N concentration may be attributed to the inhibitory activity of tannin against protozoan symbiotic bacteria [The nitrogen retention rate in the rumen is an important indicator of the efficiency of protein utilization. A higher nitrogen retention rate indicates more efficient utilization of nitrogen compounds and less nitrogen loss in urine and feces. NHformance . Three fl-Dobaib  reportedl-Dobaib ,32. CTs l-Dobaib , thus re complex . Second, complex . Third,  complex . In thispanghero , who reppanghero . The decbacteria .2, H2, CH4, and other minor compounds. VFAs are the main source of energy for ruminants, accounting for 70%–80% of energy absorption; for this reason, the measurement of rumen VFAs is of great significance [4 production, and animal performance vary, depending on the part of the plant, stage of growth, and growing conditions. Therefore, the effects of tannin addition should be studied case by case. In this trial, the addition of PICB significantly affected the relative contents of acetic and propionic acids and significantly reduced the A:P ratio. The butyric acid content for 0.3% PICB was significantly higher than that of the other groups. Jayanegara et al. [Carbohydrates are degraded by rumen microorganisms to form adenosine triphosphate (ATP), VFAs, COificance . Vasta eificance  summarizificance  reporteda et al.  and Bhata et al. , howevera et al.  reported4 production in the rumen [4 production in vitro and demonstrated that the effect was associated with a reduced protozoa population. Liu et al. [4 emissions from sheep by reducing methanogen and protozoa populations. Similar results were reported by Jayanegara et al. [4 production by reducing fiber digestion, while HTs inhibited the activity and growth of methane- and hydrogen-producing microorganisms [4 production more efficiently than CT [4 production. The study compared the function of PICB from Castanea mollissima Blume with chestnut tannins from Castanea sativa in studies reported by Liu et al. [Many authors have reported that the addition of CTs and HTs reduces CHhe rumen ,22. Bhathe rumen  reportedu et al.  reporteda et al. , and Aboa et al. . Vasta ea et al. , after ra et al. . CTs indrganisms . HT redu than CT . Shi [14u et al. , JayanegThe DM degradation rate is a reflection of ruminant diet digestion and absorption efficiency. Jayanegara et al.  and JafaIn-vitro culture gas production reflects diet degradation and microbial activity in the rumen . RegardiB), and gas production rate (c) during the 96-h in-vitro fermentation (see The addition of PICB did not significantly affect 96-h gas production, although there were significant differences and highly significant linear and quadratic responses for gas production, theoretical maximum gas production (tion see . While P3-N concentration, the content of acetic, propionic, and butyric acids, the A:P ratio, and CH4 production after 24 h of in-vitro rumen fermentation. The addition of PICB had no influence on 24-h DM degradation or 96-h total gas production. The addition of 0.3% PICB significantly reduced the NH3-N concentration relative to the control, while the addition of 0.2% PICB significantly increased the relative content of propionic acid and reduced acetic acid content and the A:P ratio. Furthermore, the addition of 0.2% PICB significantly reduced the CH4 yield during in-vitro rumen fermentation. These results indicate that the addition of PICB is a promising dietary strategy for improving ruminant energy utilization and mitigating greenhouse gas emissions.In this study, the addition of PICB to a ruminant diet significantly affected the NH


Vibrio cholerae (MneA), cyanobacterium Synechocystis (SynPAM71), yeast Saccharomyces cerevisiae (Gdt1p), plant Arabidopsis thaliana (PAM71 and CMT1), and human (TMEM165) members. These proteins have all been identified as transporters of cations, more precisely of Mn2+, with an extra reported function in Ca2+ and/or H+ transport for some of them. Apart from glycosylation in humans, the UPF0016 members are required for lactation in humans, photosynthesis in plants and cyanobacteria, Ca2+ signaling in yeast, and Mn2+ homeostasis in the five aforementioned species. The requirement of the UPF0016 members for key physiological processes most likely derives from their transport activity at the Golgi membrane in human and yeast, the chloroplasts membranes in plants, the thylakoid and plasma membranes in cyanobacteria, and the cell membrane in bacteria. In the light of these studies on various UPF0016 members, this family is not considered as uncharacterized anymore and has been renamed the Gdt1 family according to the name of its S. cerevisiae member. This review aims at assembling and confronting the current knowledge in order to identify shared and distinct features in terms of transported molecules, mode of action, structure, etc., as well as to better understand their corresponding physiological roles.The Uncharacterized Protein Family 0016 (UPF0016) gathers poorly studied membrane proteins well conserved through evolution that possess one or two copies of the consensus motif Glu-x-Gly-Asp-(Arg/Lys)-(Ser/Thr). Members are found in many eukaryotes, bacteria and archaea. The interest for this protein family arose in 2012 when its human member TMEM165 was linked to the occurrence of Congenital Disorders of Glycosylation (CDGs) when harbouring specific mutations. Study of the UPF0016 family is undergone through the characterization of the bacterium  Proteins are classified into families in which members share a common evolutionary origin, reflecting their related functions and similarities in terms of sequence and/or structure. Therefore, when a novel protein is identified, its functional properties are often hypothesized based on the functional features of the group it is predicted to belong to. The number of defined protein families increased drastically with the development of genome sequencing. However, some of these families have absolutely no assigned function, for none of their members; these are the so-called Uncharacterized Protein Families (UPFs). This review focuses on one of these UPFs: the UPF0016 family . This protein family was described in 2014 as gathering membrane proteins found in many eukaryotes, bacteria, and archaea –7, wherein silico approaches, the predicted topology of the eukaryotic UPF0016 members consists of two homologous clusters of three transmembrane spans with an opposite orientation in the membrane. These two clusters are separated by a central loop enriched in acidic amino acids  stroma was detected as affected in the two mutant plants compared to the wild-type, thereby reflecting an influence on Ca2+ signaling [2+ in photosynthesis, both in terms of signaling and regulation of involved enzymes, the previously mentioned defects in terms of growth and photosynthesis observed in the two mutant plants could therefore partly be due to disturbed Ca2+ partitioning in the chloroplasts [2+ concentrations and EGTA [2+ content compared to the wild-type [A. thaliana UPF0016 members in Ca2+ homeostasis are available: whereas Schneider et al. reported a higher Ca2+ uptake in the thylakoids of PAM71 mutants [et al. observed a decrease in such Ca2+ uptake [et al. notified differences in terms of chloroplast Ca2+ content between wild-type and CMT1 mutant plants [et al [2+ homeostasis, and to determine whether their impact on photosynthesis is of primary or secondary nature.Finally, the two plant UPF0016 members from the roplasts , 4. Furtand EGTA , and thaild-type . However2+ transport activity of the yeast Gdt1p [2+ influx in bacteria producing one of these UPF0016 members with the Ca2+-sensitive fluorescent probe Fura-2, compared to control cells containing an empty plasmid. Taken together, these data suggest that the Golgi UPF0016 members from human and yeast, and the chloroplast UPF0016 members from plants influence the cellular Ca2+ distribution through their transport ability.Recently, the Cast Gdt1p , the plast Gdt1p , and thest Gdt1p  was furt2+ and Mn2+, there is no current indication of Ca2+ transport by the bacterial SynPAM71 and MneA. This might suggest that, within the UPF0016 protein family, some members could transport the two cations while others would transport only one or the other. Of course, this statement has to be confirmed by further biochemical characterization of these UPF0016 members.At that stage, it is interesting to point out that, while Gdt1p, TMEM165, PAM71, and CMT1 have all been linked to both Ca2+ and/or Mn2+ against H+ and would thereby be involved in pH homeostasis. In this context, Demaegd et al. first noticed a decreased lysosomal pH in both fibroblasts of TMEM165-deficient patients and TMEM165-depleted HeLa cells, in comparison with control individuals or cells [in situ Golgi-localized pH-sensitive probe enabled to highlight acidification of the Golgi apparatus in the absence of TMEM165 [A. thaliana PAM71 mutant plants show a pH-sensitive phenotype and an increased cytoplasmic pH, which is thought to reflect disturbed stromal pH whose proper maintenance is of importance for effective photosynthesis [+-ATPase activity was also observed in PAM71 mutant plants [+ transport. In yeast, the Ca2+ transport activity of Gdt1p depends on the pH gradient across the Golgi membrane created by the Golgi proton V-ATPase. More precisely, Gdt1p seems to promote Ca2+ sequestration within the Golgi lumen when this organelle is correctly acidified by the V-ATPase, but works in the opposite direction, from the Golgi to the cytosol, when such acidification is disrupted by deleting the gene coding for the V-ATPase [2+ influx observed in Fura-2-loaded L. lactis cells producing Gdt1p depends on the external pH in the way that the higher the external pH, the higher the influx rate, being consistent with the model of cation/proton antiporter [TMEM165 during lactating periods may contribute to deacidify the Golgi lumen by transporting the accumulated protons to the cytosol in exchange of Mn2+ and/or Ca2+ [The UPF0016 members are hypothesized to work as secondary transporters. To date, few pieces of evidence indicate that the UPF0016 members would exchange Caor cells . More re TMEM165 . In the ynthesis . Nevertht plants ,5. ThereV-ATPase . Interestiporter . Finally/or Ca2+ .A. thaliana PAM71 in pH homeostasis, there is still a lack of direct H+ transport evidence for the UPF0016 members. Therefore, to date, one cannot exclude that the altered pH homeostasis observed in case of disruption of the UPF0016 members could be a secondary consequence of altered Ca2+ and/or Mn2+ homeostasis.While these data all suggest a role of the human TMEM165, the yeast Gdt1p, and the 2+, Mn2+, and/or H+ as secondary transporters. Figure 2 illustrates the putative mechanisms of action of the studied UPF0016 members according to the biochemical data accumulated to now. Mainly, there is indirect evidence of transport of these three cations for the yeast Gdt1p and the human TMEM165. One of the possible mechanisms of transport with these three actors consists of a Ca2+-Mn2+/H+ antiporter, where Ca2+ and Mn2+ would be transported at the Golgi level in one direction in exchange of protons. However, and in accordance with the limited and relatively indirect evidence of transport of protons, it was suggested that these UPF0016 members would work as Ca2+/Mn2+ antiporters [A. thaliana, data from different studies led to the conclusion that Mn2+ cations and, if transported, Ca2+ cations would also be transported in the same direction, towards the chloroplast lumen [2+ transport. If working as secondary transporters, as suspected from their predicted topology, the nature of the counter-ion still remains to be identified. In other words, combination of the published data suggests putative differential specificity among the UPF0016 members. Further study of the substrate specificity of a wide range of UPF0016 members would answer this question and putatively lead to the establishment of a correlation between the substrate specificity of the UPF0016 members and evolution.When combining the data aforementioned, it appears that bacterial, yeast, plant, and human UPF0016 members all transport Caiporters , 29. In st lumen –7, with st lumen . In cyan2+ and Mn2+ from the cytosol to the Golgi lumen in exchange of protons from the Golgi to the cytosol, if working as Ca2+-Mn2+/H+ antiporters. In case of a Ca2+/Mn2+ antiporter, data suggest that Mn2+ would be imported in the Golgi in exchange of Ca2+ [2+ and, if transported, Ca2+ would be transported from the cytosol to the stroma via CMT1, and then from the stroma to the thylakoid lumen via PAM71 [2+ would be transported from the cytosol to the thylakoid lumen and/or the extracellular medium, depending on the considered subcellular localization of SynPAM71 [2+ from the cytosol to the extracellular medium [Another missing piece of the puzzle is the direction of transport of these cations. Nevertheless, based on data interpretation, the human and yeast orthologs TMEM165 and Gdt1p could transport Ca of Ca2+ , 29. In ia PAM71 –7. In cySynPAM71 , 14. Finr medium , 16, wit2+-regulated riboswitch yybP-ykoY, thereby indicating a transcriptional Mn2+-dependent regulation [Anabaena and four halobacteria) are under the control of the two-component system ManS (histidine kinase that senses the extracellular concentration of Mn2+)/ManR (DNA-binding response regulator that regulates expression of targeted genes) [2+ concentration [V. cholerae mneA is induced after addition of Mn2+ to the extracellular medium [CMT1 expression in plant is reduced in the presence of Mn2+ excess [2+ excess [2+ excess triggers (i) increased abundance of the bacterial UPF0016 members to detoxify the cytosol through their export activity, and (ii) decreased abundance of the eukaryotic UPF0016 members to prevent toxic Mn2+ excess in the lumen of the organelle this cation is sent to.In the light of their cation transport activity, it would not be surprising that the UPF0016 members would be regulated at the transcriptional and/or translational levels according to the concentrations of the transported substrates. In bacteria, 328 out of 333 screened strains that possess a UPF0016 member have the corresponding gene under the control of the Mngulation . Additiod genes) –33 that ntration , 35. Morr medium , while C+ excess . Althoug2+-induced calcineurin pathway [2+-dependent regulation of the UPF0016 members. This aspect should therefore be addressed in the near future.The activity of the yeast Gdt1p is reported to be negatively regulated by the Ca pathway . No more126Arg>His, 126Arg>Cys, 304Gly>Arg, and 108Glu>Gly) and one mutation that activates a cryptic splice site (c.792+182 G>A)), identified in six patients, are reported to cause CDGs [Specific mutations in the gene coding for the human Golgi-localized UPF0016 member TMEM165 were identified as causing a sub-type of CDGs , 36. Sinuse CDGs  (revieweuse CDGs ).2+ [At the molecular level, patients suffering from TMEM165-CDGs, as well as TMEM165-depleted HeLa and HEK cells, show affected protein glycosylation profile, mainly in terms of relative hypo-sialylated and hypo-galactosylated N-glycans , 17. Int2+ . Accordi2+ . Antisen2+ .S. cerevisiae Golgi-localized UPF0016 member, in glycosylation efficiency has also been investigated. Surprisingly, no glycosylation deficiency could be detected in the strain deleted for GDT1. Protein glycosylation is however affected when growing the gdt1Δ strain in a Ca2+-rich medium (500 mM) [2+ cations [gdt1Δ mutant cultured in presence of high Ca2+ concentrations presents strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination [The role of Gdt1p, the (500 mM) , 19. Sim cations , 19. Strmination .TMEM165 expression in developing embryos causes altered initiation, processing, and extension of N-glycans, together with altered cartilage and bone development [Finally, it was shown in zebrafish that inhibition of elopment . Taken t2+ but also Ca2+ and/or H+ for some of them. Disruption of the ionic intracellular pools in case of malfunction of the UPF0016 members might in turn influence essential physiological processes in their respective organism, as further detailed in this section. Of course, while focused on the UPF0016 members, it is important to mention that these transporters most likely act in concert with other transporters from other protein families that are also essential for proper cation homeostasis.The previous sections clearly illustrate that the UPF0016 members influence the cellular cation distribution, most likely through their common ability to transport Mn2+ as cofactor for proper activity. This has for instance been directly demonstrated for the enzymes α-1,3-N-acetylgalactosaminyltransferase, α-1,3-galactosyltransferase, β-1,3-glucuronosyltransferase, and β-1,4-galactosyltransferase among others [2+ cations as cofactor for proper activity [2+ disturbances, which would in turn impair the enzymatic activity of this specific glycosyltransferase. Similarly, some S. cerevisiae glycosyltransferases also require Mn2+ as cofactor: Och1p, Mnn1p, Mnn2p, Mnn5p, and Mnn9p [gdt1Δ strain grown in the presence of Ca2+ actually correspond to altered activity of the Mn2+-dependent mannosyltransferases Mnn1p, Mnn2p, and Mnn5p [2+ content within the Golgi. Nevertheless, as TMEM165 and Gdt1p both seem to transport also Ca2+ at the Golgi membrane, one cannot rule out the possibility that the glycosylation defects are also partly caused by a disturbed Ca2+ balance within the Golgi lumen (and/or cytosol). Indeed, Ca2+ cations are essential for proper vesicular trafficking through their role in membrane fusion, and for activity and stability of enzymes involved in glycosylation [2+ by Gdt1p most likely modulates cellular Ca2+-induced signaling in yeast  [2+ and/or Mn2+ cations in the Golgi lumen by the UPF0016 members might constitute a detoxification pathway to prevent toxic accumulation in the cytosol in case of cation excess. Indeed, once inside the Golgi lumen, these ions can be directed out of the cell through the secretory pathway.The link between TMEM165 and glycosylation can first be examined in the light of its transport activity. Many glycosyltransferases involved in glycosylation actually require interaction with Mng others –45. Inteactivity . Therefond Mnn9p –49. The nd Mnn5p , suggestsylation –52. Aparof GDT1) . Finally2+ and Mn2+, TMEM165 and Gdt1p are also suggested to modulate the pH homeostasis of the Golgi. According to this latter statement, Gdt1p would be a putative actor in eliminating the proton load generated by glycosylation, in exchange of transport of divalent cations from the cytosol to the Golgi [TMEM165 during lactation might reflect its role in providing the Golgi lumen of lactating mammary cells with Ca2+ as nutrient and Mn2+ as cofactor of enzymes, but also in removing the protons generated as by-product of lactose production out of the Golgi lumen [Apart from providing the secretory pathway with Cahe Golgi . In the gi lumen , 53. Sin2+ and Ca2+ cations were delivered into the chloroplast lumen remained elusive. The current model for Mn2+, and putatively for Ca2+ delivery in the chloroplast of A. thaliana is that these cations would first be transported from the cytosol into the chloroplast stroma through the inner envelope CMT1, and further transferred to the thylakoid lumen by PAM71 [2+ content within the chloroplasts. Indeed, Mn2+ is a structural component of the PSII. In photosynthetic organisms, the PSII mediates splitting of water into oxygen, protons, and electrons. While O2 is released, the PSII thereby provides electrons for further photosynthetic reactions and generates a proton gradient used by the ATP synthase to generate ATP. Affected function of the PSII therefore leads to less reducing power and ATP synthesis [2+-requiring enzymes in the plastid stroma. As illustration, Mn2+ cations are known to activate the rubisco in the chloroplasts [2+ homeostasis from their localization at the chloroplast, which could in turn also impair photosynthesis. Indeed, Ca2+ is a regulator of chloroplast enzymes, and proper modulation of the Ca2+ concentration within the chloroplast is essential for signaling during photosynthesis [A. thaliana additionally possesses two ER-localized (PML4 and PML5) and one Golgi-localized (PML3) UPF0016 members [Before the characterization of the plant chloroplast UPF0016 members CMT1 and PAM71, the mechanism by which Mnynthesis , which iynthesis ). In addroplasts . Besidesynthesis . On the 2+ supply to the Mn4CaO5 oxygen-evolution center, but also in sequestering Mn2+ within the thylakoid lumen to prevent toxic accumulation in the cytosol. At the plasma membrane, this protein would balance the otherwise harmful effects of Mn2+ accumulation in the cytoplasm, hence preventing Mn2+ toxicity. In this context, the photosynthetic deficiency observed in the mutant line would derive from reduced Mn2+ bioavailability for the PSII, and/or from toxic cytosolic Mn2+ accumulation [Since the subcellular localization of the cyanobacterial SynPAM71 is still under debate . Therefore, the structure-function relationship of the UPF0016 members was analyzed mainly in terms of importance of the residues that constitute the two motifs to evaluate their putative role in ion coordination, but also of the residues that are found mutated in the TMEM165-CDG patients to gain insight into the mechanism of pathogenicity. Table 1 provides an overview of the mutations that have an effect on protein function, stability, or subcellular localization.Alignment of the protein sequences of the UPF0016 members reveals that the most conserved regions are the two copies of the featured motif Glu-ϕ-Gly-Asp-(Arg/Lys)-(Ser/Thr)  and polar uncharged (58Thr and 209Ser) residues of the motifs in the gdt1Δpmr1Δ strain, that is sensitive to high Ca2+ concentrations and shows impaired Ca2+ response to a salt stress, failed to suppress these defects, whereas non-mutated Gdt1p and the other mutated versions from the motif did [GDT1 coding for proteins with mutation of the acidic residues of the conserved motifs  in the gdt1Δ strain fails to suppress the glycosylation defects observed in the presence of high Ca2+ concentration [2+ transport activity of Gdt1p , positively charged (Lys or Arg), and hydroxyl-containing residues (Thr or Ser) within the UPF0016 motif predicted to be incorporated in hydrophobic transmembrane spans, these two copies were perceived as good candidates for the formation of the pore for cation transport. In yeast, production of Gdt1p with mutations in Ala of the acidic (otif did . In the ntration . Altogetified by ). In humified by . Interestrations . These d126Arg>His, 126Arg>Cys, and 304Gly>Arg affect the Golgi subcellular localization of TMEM165. Interestingly, the mutations corresponding to 126Arg>His and 126Arg>Cys were also introduced in GDT1 and expressed in the gdt1Δ yeast strain . Since their expression restores the ability of gdt1Δ to grow in Ca2+-rich medium, these proteins seem to be functional, thereby suggesting that the pathogenicity of these mutations is linked to altered localization rather than deficient protein activity [gdt1Δ strain, its mutated versions were produced in this host. Using this yeast model of the disease, combined with transport assays carried out in TMEM165-producing L. lactis cells loaded with the fluorescent probe Fura-2, it appears that the mutation 108Glu>Gly leads to decreased protein activity while such reduced activity is not clear for the other mutants [126Arg>His, 126Arg>Cys, and 304Gly>Arg), or altered transport activity (108Glu>Gly) [The effect of the naturally-occurring mutations among TMEM165-CDG patients were also examined in terms of protein localization, expression, and activity. In this context, the mutation c.792+182 G>A (that activates a cryptic splice site) was the only one identified as conferring protein instability . In contactivity . More re mutants . AltogetGlu>Gly) , 60.Molecular characterization of the UPF0016 members tremendously increased since their identification in 2012. The implication of the UPF0016 members in regulating cation homeostasis, thereby influencing various essential processes, is now well established in diverse organisms. For this reason, the UPF0016 family has been recently renamed as the Gdt1 family, according to the name of the yeast ortholog whose study significantly contributed to the characterization of this protein family (UniProt). Nevertheless, several aspects related to the UPF0016 family still remain to be investigated. Development of direct transport assays for the UPF0016 members in their endogenous host or proteoliposomes appears as one of the key approaches to confirm the identity of the transported substrates and their direction of transport. High-resolution structural analysis of the UPF0016 members also appears as essential for a better understanding of their mode of action.Besides, recent results suggest that the spectrum of action of the Gdt1 family could be broader than what is currently known. Indeed, TMEM165 was found overexpressed in hepatocellular carcinoma and involved in cancer invasive activity, through a mechanism that still needs to be uncovered . Another