The adenoviral early region 1A (E1A) protein has proapoptotic and angiogenic activity, along with its chemosensitizing effect, making it the focus of increased interest in the context of cancer therapy. It was previously shown that E1A-induced chemosensitization to different drugs, including histone deacetylases inhibitors (HDACi), appears to be mediated by Forkhead box O (FoxO) transcription factors. In this study, we explore the relationship between E1A expression and the modulation of FoxO activity with HDACi sodium butyrate (NaBut). We show here that the basal FoxO level is elevated in E1A-expressing cells. Prolonged NaBut treatment leads to the inhibition of the FoxO expression and activity in E1A-expressing cells. However, in E1A-negative cells, NaBut promotes the transactivation ability of FoxO over time. A more detailed investigation revealed that the NaBut-induced decrease of FoxO activity in E1A-expressing cells is due to the NaBut-dependent decrease in E1A expression. Therefore, NaBut-induced inhibition of FoxO in E1A-positive cells can be overcome under unregulated overexpression of E1A. Remarkably, the CBP/p300-binding domain of E1Aad5 is responsible for stabilization of the FoxO protein. Collectively, these data show that the expression of E1A increases the FoxO stability but makes the FoxO level more sensitive to HDACi treatment. The early region 1A (E1A) of adenovirus encodes two major proteins E1A 12S and E1A 13S, translated from two alternatively spliced transcripts, including the 12S and 13S messenger RNAs (mRNAs) encoding 243 (243R) and 289 (289R) amino-acid oncoproteins [E1A is a promising candidate for cancer therapy, due to its proapoptotic , antiangBy changing the expression of different sets of genes, HDACis suppress cell growth and induce cell differentiation, senescence, or apoptosis more effectively in transformed cells than in normal cells ,16,17,18BIM [The molecular mechanisms determining the E1A-mediated cellular sensitivity to apoptosis are not yet fully known. However, E1A-dependent chemosensitization appears to be connected with Forkhead box O (FoxO) transcription factors . A knockBIM . Apart fBIM . This acBIM .FoxOs belong to the Forkhead family of transcription factors sharing the common DNA-binding domain FKH . These tIn this study, we are dealing with FoxO1—the most studied member of the Forkhead family. The functions of FoxO1 and its role in cancerogenesis and tumor progression are quite sophisticated and context-specific. FoxO1 deletions are lethal due to incomplete vascular development in the embryo . ParticiThis study is focused on the stabilizing effects of E1A in relation to FoxO, showing the rise in FoxO level in the presence of E1A and establishing the link between HDACi-induced E1A and FoxO degradation. Taken together, the paper deals with FoxO regulation under the HDACi treatment in relation to E1A expression, and it examines the stabilizing functions of E1A.HindIII-G region of Ad5 viral DNA coding E1A and E1B 19kDa (E1A + E1B). E1A + Ras and E1A + E1B transformed and NIH3T3 normal fibroblasts were cultivated in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics. Cells were treated with 4 mM sodium butyrate (NaBut) for 24–72 h . NIH3T3 mouse fibroblasts, HEK293 human kidney (embryonic) cells, and HCT116 human colorectal carcinoma cells were obtained from the Russian Collection of Cell Cultures . The adenovirus E1A constructs (12S (wild type (wt)), 12S (RG2) and 12S (YH47)) were obtained from Dr. Elizabeth Moran . The hemagglutinin (HA)-tagged full-length p300 was a gift from Elizabeth Wilson . FHRE-Luc was a gift from Michael Greenberg .We used rodent embryonic fibroblasts transformed either with pE1A vector coding the early region of human adenovirus type 5 (E1Aad5) in complementation with pSV-ras-gpt coding the cHa-ras carrying mutations at positions 12 and 61 (E1A + Ras) or with p21/waf1: 5′–GCCGTGATTGCGATGCGCTCA–3′ and 5′–ACAGCGACAAGGCCACGTGGT–3′; mnsod: 5′–GCACTGAAGTTCAATGGTGG–3′ and 5′–ACTGAAGGTAGTAAGCGTGCTC–3′; bim: 5′–GCCAAGCAACCTTCTGATG–3′ and 5′–CACTGAGATAGTGGTTGAAGGC–3′; gapdh (m): 5′–TGTGATGGGTGTGAACCACG–3′ and 5′–CCAGTGAGCTTCCCGTTCAG–3′; gapdh (h): 5′–TCATCAGCAATGCCTCCTGCACC–3′ and 5′–ACAGTTTCCCGGAGGGGCCA–3′). PCRs proceeded for 22–32 cycles: A denaturation step at 950 C for 30 s, an annealing step  for 30 s, and an elongation step at 72 °C for 1 min. PCR products were resolved by electrophoresis in 2% agarose gel. The alpha-tubulin gene was selected as an internal control in the PCR assay.The total cellular RNA was isolated with Trizol . The RT step was performed with 2 µg of RNA and 1 µg of random hexaprimers. The PCR step was performed in the presence of 100 ng of primers to the complementary DNA (cDNA) of mouse or human genes , and probed with appropriate antibodies. As primary antibodies, we used antibodies to Foxo1 #2880 , E1A (M73) sc-25 (Santa Cruz), pan-Ras #OP40 , and alpha-tubulin T5168 (Sigma). Anti-mouse and anti-rabbit antibodies conjugated with horseradish peroxidase (Sigma) were used as the secondary antibodies. Visualization of membrane-bound proteins was performed by enhanced chemiluminescence .Every protein of interest was analyzed at least three times on different sets of samples. The band density was evaluated using ImageJ . Then, density values were scaled to load control and converted to relative units. The figure plots represent the mean values of several experiments; error bars indicate the standard error of the mean (SEM).3 cells per well and transfected with the appropriate constructs  using Lipofectamine-2000 (Invitrogen) as recommended by the manufacturer. For stable plasmid integration, E1A + Ras cells were co-transfected with a vector coding the early region of Ad 5 (E1Aad5), and a selective pBABE-puro construct bearing the puromycin resistance gene. For the luciferase assay, cell extracts were prepared, and luciferase activity was determined according to the protocol supplied with the Luciferase Assay kit . Luciferase activity was determined using a TD-20/20 luminometer . Each experimental point was carried out in triplicate, and each experiment was performed no less than three times. Diagrams represent means ± SEM.For transient and stable transfection, cells were plated in 96-well plates (antibiotic-free DMEM with 10% FBS) at a seeding density of 150 × 10Long-term HDACi treatment was shown to induce FoxO1 and FoxO3 degradation in E1A + Ras-transformed mouse cells . In addiImmunoblotting data reveal that NaBut induced FoxO1 accumulation in E1A-negative cells. On the contrary, in E1A-expressing cells, there was a tendency toward declining of the FoxO1 protein level after prolonged NaBut treatment a,b.The expressions of E1A and FoxO1 correlated positively in E1A-expressing cells treated with sodium butyrate. NaBut-induced decrease of FoxO1 protein level was accompanied by a reduced expression of adenoviral protein E1A. The dynamic of FoxO1 level under NaBut treatment is shown more clearly on the plot b, represE1A gene and its endogenous promoter. The control NIH3T3 and E1A-expressing NIH3T3 cells were prolonged treated with NaBut (up to 72 h) and assayed for FoxO1 expression. Immunoblotting data demonstrate that prolonged action of sodium butyrate increased the initially low level of FoxO1 expression in E1A-negative cells NIH3T3. On the contrary, the initially elevated level of FoxO1 decreased in E1A-expressing cells under prolonged sodium butyrate treatment  including the reatment c.To assess the influence of NaBut on the transactivation ability of FoxO in dependence of E1A expression, the ability of FoxO to activate the target gene transcription was analyzed in E1A-expressing and E1A-negative cells under prolonged NaBut treatment using RT-PCR and a luciferase assay. E1A-negative rodent NIH3T3 and E1A-expressing E1A + Ras and E1A + E1B cells were transfected with a vector, coding a luciferase under control of FoxO-responsive promoter (FHRE-Luc). Cells were left untreated or treated with NaBut for 24–72 h and assayed for luciferase activity. Luciferase assay data demonstrate that NaBut enhanced FoxO transactivity in both cell lines, but temporal dynamics in these cells were different a. NaBut The same results were obtained in human cells. The luciferase activity in E1A-negative HCT116 cells kept increasing even after 48 h of NaBut treatment. FoxO activity increased 10-fold and 20-fold after 24 and 48 h, respectively. On the other hand, in E1A-expressing cells HEK293, FoxO-activity, after a temporary increase, decreased under a prolonged action of NaBut b.p21Waf1, encoding the cyclin kinase inhibitor, and MnSod, the product of which is involved in the ROS inactivation, to validate the FoxO transactivation function changes under NaBut treatment. In general, the dynamics of FoxO target gene expression was consistent with the data obtained with a luciferase assay. RT-PCR analysis of the FoxO target gene expression revealed an increased basal level of mRNA in E1A-expressing cells (E1A + Ras), compared with E1A-negative cells (NIH3T3). Expression of the studied genes did not increase in E1A-expressing cells under prolonged exposure to sodium butyrate. On the other hand, in E1A-negative cells, an intense transcription increase was observed  [E1A promoter, we performed a transient and stable transfection of E1A + Ras cells with the plasmid coding the 12S E1A under the control of a strong promoter of cytomegalovirus (CMV). The regulation and activity of CMV and Ad5 viral promoters differ significantly; thus, they can be used in target cells for different purposes. The high-activity CMV promoter is convenient for efficient transgene expression. Moreover, the CMV promoter was reported to be stimulated by HDACi [We previously showed an HDACi-induced decrease in protein and mRNA level of E1A in HEK293 and E1A + Ras-transformed cells . In E1A  type 5) ,48,49. Tby HDACi . FirstlyThus, our results suggest that, for overcoming NaBut-induced inhibition of the FoxO activity in E1A-expressing cells, increased expression of E1A independent of HDACi is required.To elucidate the mechanisms of E1A influence on the stability of FoxO, we tried to determine which regions of adenoviral E1A protein are important for the induction of FoxO activity. For this, NIH3T3 cells were transfected with different adenovirus E1A constructs. At 36 h after transfection, cells were lysed and assayed for FoxO1 expression. As shown in Next, we investigated whether sodium butyrate modulates the interaction of p300 with FoxO in E1A + Ras cells. To assess the dynamic of FoxO/p300 interaction, E1A + Ras cells were transfected with a vector coding HA-tagged full-length p300 acetyltransferase, which was then treated with NaBut for 24 h. The coimmunoprecipitation assay followed with immunoblotting revealed that endogenous FoxO1 and p300 could associate in sodium butyrate-treated cells only b. In untOur data show that the presence of adenoviral E1A significantly alters the cell response to NaBut treatment, specifically by influencing the dynamics of FoxO expression and activity. First of all, the basal level of FoxO1 expression in E1A-positive cells is generally higher. Moreover, we showed that both rodent (NIH3T3 and REF52) and human cancer cells (HCT116) transfected with a vector encoding the E1A are characterized by an increased basal level of FoxO1 protein. Secondly, our experimental data reveal that the transactivation function of FoxO is generally in line with the FoxO protein level. In E1A-negative cells, the low basal level of FoxO1 greatly increases under NaBut treatment, accompanied by enhanced FoxO transactivation function. As for E1A-expressing cells, there is a downward trend in FoxO1 expression and activity under prolonged NaBut treatment. Several mechanisms leading to FoxO stabilization by E1A protein are suggested. E1A can bind to the proteasomes and inactivate their functions ,52, prevWe established the crucial role of the p300-binding region of the E1A protein in the induction of FoxO a. ComparcHa-ras gene. Ras was shown to suppress FoxO1 expression, activating the Ras/PI3K/Akt pathway, leading to FoxO1 degradation [In E1A-expressing cells, different dynamics of FoxO decrease are observed. In E1A + Ras cells, FoxO undergoes complete degradation, while the degradation of FoxO is less evident in E1A + E1B and HEK293 cells. This discrepancy may be attributed to the Ras oncoprotein. There are no Ras mutations in E1A + E1B and HEK293 cells , whereasradation ,31. As sradation , which wradation .Our present study provides findings that HDACi-induced degradation of FoxO protein can be avoided, provided that the level of E1A protein does not decrease significantly following HDACi treatment. However, in cells expressing E1A under the adenoviral promoter, whose activity is inhibited by HDACi, the amount of E1A decreases following the addition of HDACi, which causes the FoxO expression and activity to decrease. In other words, expression of the early E1A region of adenovirus increases FoxO stability, but it makes the FoxO protein level more sensitive to HDACi treatment.In summary, here, we present evidence showing that FoxO expression and stability under NaBut treatment is firmly associated with expression of E1A and Ras. Oncogenic Ras causes the degradation of FoxO through activation of the Ras/Akt signaling pathway, but expression of E1A in cells carrying activated Ras is able to overcome this inhibition and enhance FoxO expression.FasL, and the proapoptotic Bcl-2 family member Bim. Alternatively, FoxO factors can promote cell-cycle arrest by activating the cell-cycle inhibitors p21Waf1 or p27kip1 to induce G1 arrest, or GADD45 to induce G2 arrest.The interpretation of these findings could be challenging since FoxOs are proven to cause both tumor suppression ,63 and pRas gene mutation, long-term HDACi treatment inhibits FoxO expression and activity. We suggest that the E1A/HDACi co-treatment is ineffective in anticancer therapy of tumors with Ras mutations. On the other hand, there is a hypothesis that FoxO acts as a tumor suppressor in normal cells; however, within tumor cells, it can contribute to their survival and tumor growth [Our results suggest that, in E1A-expressing cells with a r growth ,68. If w


Overall, our data may help to optimize the selection of hematopoietic stem cell donors on the basis of immunogenetic KIR/HLA for ALL patients and identify the best NK cell candidates in immunotherapy for AML.Natural killer (NK) cells are key cytotoxic effectors against malignant cells. Polygenic and polymorphic Killer cell Immunoglobulin-like Receptor (KIR) and HLA genes participate in the structural and functional formation of the NK cell repertoire. In this study, we extensively investigated the anti-leukemic potential of NK cell subsets, taking into account these genetic parameters and cytomegalovirus (CMV) status. Hierarchical clustering analysis of NK cell subsets based on NKG2A, KIR, CD57 and NKG2C markers from 68 blood donors identified donor clusters characterized by a specific phenotypic NK cell repertoire linked to a particular immunogenetic KIR and HLA profile and CMV status. On the functional side, acute lymphoblastic leukemia (ALL) was better recognized by NK cells than acute myeloid leukemia (AML). However, a broad inter-individual disparity of NK cell responses exists against the same leukemic target, highlighting bad and good NK responders. The most effective NK cell subsets against different ALLs expressed NKG2A and represented the most frequent subset in the NK cell repertoire. In contrast, minority CD57 The inh absence  before K absence . NCRs re absence ,9,10,11  absence ,13.We previously documented the beneficial role of KIR and HLA incompatibilities between the donor and the recipient, which significantly limited relapse in the context of haploidentical HSCT . We showbright NK cells express NKG2A and then gradually lose the intensity of expression of CD56, resulting in a pool of CD56dim NK cells [dim NK cell subsets. Human cytomegalovirus (CMV) drastically changes the NK cell repertoire, favoring a massive and lifelong expansion of adaptive-like NK cells that express activating CD94/NKG2C receptors; these cells constitute the most mature subset [+ NK cells preferentially co-express inhibitory KIR, such as KIR2DL2/3 or KIR2DL1, which is involved in functional NK cell education [KIR and HLA genes participate in the structural and functional formation of the NK cell repertoire. The KIR and HLA gene families constitute the most polymorphic and polygenic receptor-ligand pair in humans, and their functional interactions drive tremendous NK cell diversity with a limited number of germline-encoded genes . AccordiNK cells ,19. At te subset ,21,22. Tducation . Of noteducation .In this study, we hypothesized that KIR and HLA immunogenetic markers and CMV status drive the NK cell subset distribution and that NK cell subsets do not share similar degranulation potential against leukemias, depending on the nature thereof. Thus, we carried out an in-depth investigation of the anti-leukemic potential of NK cell subsets against a panel of acute myeloid and lymphoid leukemia cell lines and primary leukemic cells, taking into account KIR and HLA genetic parameters, NK cell development stages and the CMV status of healthy blood donors.® software, taking into account the mean fluorescence intensity (MFI) of each NK receptor ligand  against a panel of myeloid and lymphoid cell lines A. Lymphor ligand B. Two clr ligand B. This dTo undertake our study on a broader cohort of healthy donors, we focused our investigation on H9, an acute lymphoblastic leukemia (ALL) cell line, and KG1, an acute myeloid leukemia (AML) cell line, as two representative cell lines that are differently recognized by NK cells.According to the immunophenotypic analysis, the H9 ALL cell line expressed more ULBP2/5/6, CD48 and B7-H6 than the KG1 AML cell line, which, in contrast, expressed Nectin-2 (CD112) and MICA/B, which were slightly expressed and not expressed on the H9 cell line, respectively . Both H9We further addressed the inter-individual diversity of NK cell responses from a broader cohort of 68 healthy blood donors. We showed that the H9 ALL cell line stimulated NK cell degranulation to a significantly greater extent than the KG1 AML cell line, highlighting bad and good responders against both target cell lines F. In addTo further investigate the intra-individual diversity of NK cell responses, we refined our investigations to take into account immunogenetic KIR/HLA polymorphisms and CMV status. + KIR− CD57− and NKG2A− KIR− CD57− NK cells were the two main subsets in terms of frequency, while NK cell subsets expressing KIR and CD57 markers were represented in low proportions  from our cohort of healthy blood donors did not necessarily exhibit the amplification of NKG2C+ NK cells, we excluded the NKG2C marker in this part of the study to define eight NK cell subsets on the basis of NKG2A, KIR and CD57 expression. Hierarchical clustering analysis (Genesis®) of the degranulation of eight NK cell subsets stimulated with the H9 cell line defined six clusters of individuals (p < 0.01) and C4 and C5 clusters     C. Overal 0.0047) D. The sh 0.0047) E. Intereclusters F. Of not 0.0001) G. This rp = 0.014 between C1C1 and C2C2 individuals, p = 0.004 between C1C2 and C2C2 individuals) (+ KIR− CD57− NK cell frequency significantly correlated with the degranulation of the whole NK cells (p < 0.0001)  . As obseviduals) . NKG2A+  0.0001) . Overallp < 0.0001) (p = 0.0021) in contrast to bad responders  (p = 0.0038) (p = 0.0082)  and KIR+ NKG2A+ CD57−  . C1, C2, C3 and C5 individual clusters presented a strong whole NK cell degranulation in contrast to C4, C6, C7 and C8 individual clusters ( 0.0001) A. Intere and C8) B. We did and C8) C. Howeve 0.0038) C. Moreov 0.0082) C. The mo 0.0082) D. In foc 0.0198) E. These +C2+ CMV− than their bad counterparts (p = 0.024) (p = 0.033)  led us to identify seven clusters of individuals, as illustrated for the most representative AML blasts (WAL-C) in = 0.024) C. These = 0.033) D. The mo= 0.033) E. NKG2A+ML cells E.+ NK cell functionality, we focused only on CMV+ individuals with the amplification of NKG2C+ NK cells . From the 68 healthy blood donors investigated in this study, 32 individuals were CMV seropositive, and only 8 CMV+ individuals presented an amplification of NKG2C+ NK cell subsets  with an amplification of the NKG2C+ KIR2DL2/3+ CD57+/− NK cell subsets  with an amplification of the NKG2C+ KIR2DL2/3− CD57+/− NK cell subsets  was significantly higher than that of NKG2C+ KIR2DL2/3+ CD57+ NK cell subsets , regardless of the HLA-C environment of NK cells (C1+ or C2+) , amplified in C2+ CMV+ individuals, were the most effective subsets against ALL H9 and primary cells, as well as against primary AML targets.The degranulation of NKG2C or C2+) D. Of not+ NK cells to be attractive anti-leukemia candidates for immunotherapy [+ NK cells in these eight CMV+ individuals. We compared that to the degranulation of eight NK cell subsets defined on the basis of NKG2A, KIR and CD57 markers. The degranulation of NKG2C+ NK cells was low against H9, KG1 and primary AML blasts  than Caucasian (20%) and Asian (less than 5%) populations. HLA-C of the C2 group and HLA-Bw4 ligands contribute to the education and maturation of KIR2DL1 and KIR3DL1 NK cells, respectively [+ KIR2DL2/3− CD57− NK cells express KIR2DL1 and KIR3DL1 receptors. In contrast, NKG2A− KIR2DL2/3+ CD57−, NKG2A− KIR2DL2/3+ CD57+ and NKG2A− KIR2DL2/3− CD57+ NK cell subsets were represented in low frequencies in NK cell repertoires. NKG2A− KIR2DL2/3+ CD57− NK cells were favored in A3/A11−/Bw4− genotyped individuals. No significant difference between C1 and C2 ligands was observed in the frequency of this KIR2DL2/3+ NK cell subset. Thus, the absence of A3/A11 and Bw4 ligands suggests that C1 or C2 ligands favored the education and development of the NKG2A− KIR2DL2/3+ CD57− NK cell subset since it has been reported that the KIR2DL3 receptor can strongly recognize the C1 ligand and recognizes the C2 ligand with low affinity [− KIR2DL2/3+ CD57+ and NKG2A− KIR2DL2/3− CD57+ NK cell subsets were particularly favored in A3/A11+ genotyped individuals. As A3/A11 HLA class I molecules, originally identified as ligands for the KIR3DL2 inhibitory receptor [+ NK cells, we can speculate that a proportion of NKG2A− CD57+ NK cells express KIR3DL2. Our data are placed in a broad body of literature on the structuration of the NK cell repertoire [+ NK cell represents the most prevalent NK cell subset in the KIR NK cell repertoire, we focused only on the KIR2DL2/3 inhibitory receptor in this study. However, further investigations are needed to evaluate the role of other inhibitory KIR receptors, such as KIR2DL1, KIR3DL1 and KIR3DL2, in anti-leukemia NK cell responses, particularly against AML.On the basis of clinical data, NK cells appear to play a crucial role in the eradication of acute leukemia ,27,28. Haffinity . NKG2A− receptor , participertoire ,35,36,37pertoire . Indeed,+ KIR− CD57− NK cells as the most effective NK cell subsets against the ALL H9 cell line and ALL blasts, as previously reported [+ NK cells were represented at a high frequency in the NK cell repertoire explains the strong response of these subsets against ALL targets. Moreover, Forslun et al. reported that NKG2A+ KIR− CD57− NK cells exhibit more dynamic migration, which is associated with increased target cell conjugation and a higher probability of killing compared with NKG2A− KIR− CD57− NK cells. The authors suggested that the ability of NKG2A+ NK cells to form more conjugates resulted in their increased motility, leading to more encounters with target cells [− or C2C2 individuals, the latter harboring a higher frequency of NKG2A+ KIR− CD57− NK cells. These parameters are important, as they can help in choosing donors of HSC that harbor more efficient and abundant NKG2A+ KIR− CD57− NK cells to treat acute lymphoblastic leukemia.An in-depth functional analysis identified NKG2Areported . The facet cells . In our + NK cells [+ and CD57+ NK cells represented the most effective NK cells against AML targets, the low frequency of these NK cell subsets explains why AML targets did not trigger whole NK cell degranulation. Conversely, NKG2A+ KIR− CD57− NK cells harbor a low responsiveness against AML targets. This observation is in line with the study of Nguyen et al., who showed that increased frequency of immature NKG2A+ NK cells during NK cell repertoire reconstitution after HSCT did not produce a GvL effect in AML patients [+ NK cells with the CD57 phenotype display increased functional potential associated with the increased transcription of genes involved in adhesion and immune synapse [+ and CD57+ NK cell subsets may be more adapted to interact with and eliminate AML target cells. Among different therapeutic approaches developed to enhance NK cell cytotoxicity against cancer [+ and CD57+ NK cell subsets against AML to harness their ability to kill these targets.In the context of haploidentical HSCT, numerous studies have reported that NK cell-mediated alloreactivity is induced by donor KIRNK cells ,41,42. INK cells . In linepatients . In cont synapse . Altogett cancer , the expt cancer . Thus, tt cancer . We can + NK cells in seropositive CMV individuals, consistent with previous reports [+ KIR2DL3+ CD57+/− cells were preferentially expanded in C1+ individuals, whereas NKG2C+ KIR2DL3− CD57+/− NK cells were preferentially expanded in C2+ individuals, demonstrating the important role of the HLA-C environment in the expansion of such NK cell subsets, as previously observed [+ CD57+ NK cells, and they proposed that CD57 might provide a marker of “memory” NK cells in response to CMV infection [+ NK cells with and without CD57 expression, suggesting the presence of phenotypic intra-individual diversity of the NKG2C+ NK cell repertoire in CMV seropositive individuals. Functionally, adaptive NKG2C+ NK cell responses were more diverse than initially expected [+ KIR+ CD57+ NK cells against all leukemia targets, in contrast to other NKG2C+ NK cell subsets. Our study identified NKG2C+ KIR− CD57− NK cells as the most effective NKG2C+ NK cell subset. Of note, taking into account all NK cell subsets investigated on the basis of KIR, CD57 and NKG2A expression, the NKG2C+ NK cell subset was among the least efficient NK cell subset against all leukemia targets. Merino et al. recently showed that chronically stimulated adaptive NK cells were dysfunctional when challenged with tumor cells [Furthermore, we documented the massive amplification of adaptive and more differentiated NKG2C reports ,21,22,23observed ,23,47,48nfection . Our stuexpected . Interesor cells . AlthougIn conclusion, our data may have evident clinical implications, as they can be used to optimize the selection of HSC donors on the basis of immunogenetic KIR and HLA for ALL patients and identify the best NK cell subsets in immunotherapy to cure AML patients.Peripheral blood mononuclear cells (PBMCs) were isolated as previously described . All bloHLA class I allele assignment and KIR gene content typing were performed as previously described  for all −) and 33 seropositive CMV individuals (CMV+).CMV serology was carried out by the technical platform of the Virology Department of Nantes University Hospital (Pr. Berthe-Marie Imbert and Dr. Céline Bressollette) using plasmas from donor biocollections. The CMV serological status of donors was determined by the detection of IgG2 anti-CMV antibodies in plasmas using the LIAISON chemiluminescent immune test  according to the manufacturer’s instructions . Our cohHigh-resolution immunophenotyping of NK cells, cell lines and primary leukemic cells was determined by eight-color multiparameter flow cytometry (MFC) using the mouse anti-human mAbs presented in 6 cells/well) in bottom 96 well plate with brefeldin A  at 10 μg/mL for the last 4 h. An initial cell surface staining was used to target NK cell subsets by MFC using the following mouse-Abs combination: CD57-FITC, NKG2C-PE, NKG2D-PerCP-Cy5.5, NKG2A-PC7, KIR2DL2/3-APC, CD56-APC-Cy7 and CD3-BV510. A second cell surface staining was used to analyze NK cell activating receptor expression by MFC using the following mouse-Abs combination: 2B4-FITC, NKp30-PE, DNAM-1-PerCP-Cy5.5, NKp44-PC7 and NKp46-APC. As some primary leukemic cells expressed CD56, the NKp46 marker was used to target NK cells in the degranulation assay by MFC using the following mouse-Abs combination: NKG2A-FITC, NKG2C-PE, KIR2DL2/3-PE-Cy5.5, CD57-PC7, KIR2DL2/3-APC and NKp46-APC. All flow cytometry data were collected using a FACSCanto II (BD Biosciences) and analyzed with Flowjo™ 10.6 software (BD Biosciences).NK cells from healthy blood donors were pre-incubated with anti-CD107a-BV421 . CD107a is a sensitive marker for the identification of NK cell activity  and can www.genome.tugraz.at.The hierarchical clustering of investigated NK cell subsets was performed following a complete linkage using the Genesis software . The soft-test. Comparisons of multiple groups were performed by one-way ANOVA using the GraphPad Prism v6.0 software . p-values < 0.05 were considered statistically significant.Comparisons of NK cell frequencies between two different series of individuals were performed by Student’s + or/and KIR+ NK cell subsets were more efficient against AML. These results may have evident implications in oncohematology to improve both the selection of hematopoietic stem cell donors for ALL patients and immunotherapies to cure AML patients.In conclusion, this study showed a broad inter-individual disparity of NK cell responses against the same leukemic target, highlighting bad and good NK responders. We observed that the diversity of NK cell responses against leukemic cells was inter- and intra-individual. Indeed, on the basis of NKG2A, KIR2DL2/3, CD57 and NKG2C markers, we identified different clusters of individuals who shared a common NK cell repertoire that was linked to the immunogenetic KIR/HLA profile and CMV status. On the functional side, the most effective NK cell subsets against different ALL targets expressed NKG2A and represented the most frequent subset in the NK cell repertoire. In contrast, minority CD57


Cupriavidus sp. are model organisms for heavy metal(loid) resistance and aromatic compound’s degradation studies and these characteristics make them a perfect candidate for biotechnological purposes. Bacterial strain S14E4C (identified as Cupriavidus campinensis) was isolated from a playground by enrichment method in a 0.25 mM containing medium. The analysis revealed that this bacterium is able to tolerate high concentrations of heavy metal(loid)s: Cd up to 19.5 mM, Pb to 9 mM, Hg to 5.5 mM and As to 2 mM in heavy metal(loid) salt containing nutrient medium. The whole genome data and analysis of the type strain of C. campinensis CCUG:44526T have not been available so far, thus here we present the genome sequencing results of strain S14E4C of the same species. Analysis was carried out to identify possible mechanisms for the heavy metal resistance and to map the genetic data of C. campinensis. The annotation pipelines revealed that the total genome of strain S14E4C is 6,375,175 bp length with a GC content of 66.3% and contains 2 plasmids with 295,460 bp (GC content 59.9%) and 50,483 bp (GC content 63%). In total 4460 coding sequences were assigned to known functions and 1508 to hypothetical proteins. Analysis proved that strain S14E4C is having gene clusters such as czc, mer, cus, chr, ars to encode various heavy metal resistance mechanisms that play an important role to survive in extreme environments.The online version of this article (10.1007/s11033-020-05490-8) contains supplementary material, which is available to authorized users. Extensive use of metals and chemicals in the industrial processes have resulted in accumulation of large quantities of effluents containing toxic heavy metals in the environment, and these effluents pose environmental disposal problems due to their non-degradable and persistent characters . In biolCupriavidus is a genus of the family Burkholderiaceae that is well known for its heavy-metal resistance and diverse metabolic capabilities in different niches, especially from heavy metal and organic-chemical contaminated soils [Cupriavidus  campinensis was isolated first time from Campine, the geographical region of northeast Belgium [Cupriavidus campinensis strain S14E4C was sequenced and analysed in detail, as well as heavy metal resistance genes and genomic potentials were characterised.ed soils . The β-p Belgium . This ba Belgium  and foll Belgium  as well,C. campinensis S14E4C was isolated from the heavy metal contaminated playground (GPS coordinates: 48°05′42.0″N 19°47′32.8″E) of Salgótarján (Hungary), a former industrial city. Sample was obtained by scraping away the upper (grassy) surface of the soil with a sterile knife and the upper 10–15 cm thick soil layer was placed into sterile plastic tubes, then transported to the laboratory at 4 °C temperature condition. The sample was enriched with 0.25 mM of Cd2+ salt containing broth  by constantly shaking (270 rpm) for two weeks at room temperature. Isolation of strains was performed in a random manner from the growing colonies by standard dilution plate technique. Bacterial strain S14E4C was maintained at 28–30 °C on nutrient agar medium (DSM medium 1) supplemented with 0.25 mM cadmium (Cd).Identification of strain S14E4C was done based on 16S rRNA gene sequencing: the genomic DNA of strain S14E4C was extracted using a DNA extraction kit . To confirm the identities of the isolates the 16S rRNA gene was amplified (PCR) from the extracted genomic DNA using the universal primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-GGCTACCTTGTT ACGACTT-3′) [https://github.com/GenomicaMicrob/coverage_calculator).The whole genome shotgun and paired-end sequencing of the strain S14E4C was performed by the Genomics Facility RTSF, Michigan State University (USA), on an Illumina MiSeq platform using the MiSeq standard v2 chemistry. Low quality reads, with excess “N” and low quality score, duplication reads, and adaptor contamination were filtered out from the sequence set. Subsequently, high quality reads were assembled using the SPAdes v3.10.0 assembler in careful mode  and the https://www.ncbi.nlm.nih.gov/genome/annotation_prok/) annotated the genome. Functional genes that were investigated as having possible roles in metabolic pathways were checked by KEGG database [Genome annotation, prediction of genome features and functions were analysed by various tools, such as RAST (Rapid Annotation using Subsystem Technology) , PATRIC database  on PATRIdatabase .C. campinensis S14E4C were determined using nutrient medium (DSM medium 1) supplemented with the following heavy metal(loid) salts 2 or As2O3, respectively). Analysis has started with 0.25 mM of relevant salt concentrations and after one week the adapted cultures transferred to elevated concentrations  media. Additionally, heavy metal tolerance of the strain S14E4C was checked by low phosphate Tris-salt mineral medium with heavy metal salt additives 2 or As2O3,) in various concentrations .MIC values of the 4 heavy metal(loid)s  for www.eucast.org) Version 8.0 (January 2020).Antibiotic resistance of strain S14E4C was tested on nutrient agar medium using disk diffusion method of EUCAST regulation  containing 2 ml bacterial suspension  in sterile 100 ml sealed serum bottle at 24 °C on a magnetic stirrer. Three replicates were tested and the variations were measured after 3 and 7 days by using SPME GC-FID –18.Cupriavidus species, the public and completed 16S rRNA gene sequences of the corresponding Cupriavidus type strains were gathered from the Arb-Silva database [Cupriavidus was created using CIPRES Science Gateway’s MrBayes tool [Polynucleobacter cosmopolitanus CIP 109840T (AJ550672) was used as an outgroup. Phylogenetic tree was visualized by FigTree v1.4.4 [To determine the phylogenetic relationships among database  and aligyes tool  and the e v1.4.4 . In addie v1.4.4  presentse v1.4.4 . In ordee v1.4.4  from thee v1.4.4  and the e v1.4.4  was usede v1.4.4  and prodC. campinensis [, though some of the metabolic pathways and characteristics were revealed, most of them are still unknown due to the lack of whole genome information of the species. Whereas, genome sequence of C. campinensis S14E4C revealed the strain has additional capabilities, such as heavy metal resistance, degradation of aromatic compounds, antibiotic resistance, etc. that enhance its potential for use in biotechnological applications. To predict open reading frames and similarity of the species several annotation and alignment programs were used  and compared to be able to detect genes with high accuracy.Since the first isolation of pinensis , though C. campinensis S14E4C adapts and resists to the effect of environmental stresses by functional genes and moreover, genes related to plasmid partitioning and the plasmid initiating (protein RepA) are solid evidence for plasmid existence that perform heavy metal and antibiotic resistance. Through combination of sequencing and comparative genomics our study has provided, for the first time, a comprehensive genomic description of the species Cupriavidus campinensis.It is indicated that Cupriavidus and it is 100% identical to Cupriavidus campinensis WS2 [The 16S rRNA gene sequence of strain S14E4C (NCBI GenBank accession MK660715) was obtained and BLAST search results based on EzTaxon and GenBank databases both indicated that strain S14E4C belongs to the genus nsis WS2 . The WhoThe genome of S14E4C is 6,322,653 bp with a GC content 66.3% after assembly to 52 contigs (contigs shorter than 500 bp were removed) with 78.3 × coverage value. Total of 5968 putative coding sequences (CDSs) were validated by homology and 4460 CDSs that were assigned to one or more function classes  and several plasmids [Cupriavidus species  . The alignments clarified the identical genes in Plasmid1 of the S14E4C, exist in both plasmids of the C. metallidurans CH34, whereas only few positions are similar among plasmid 2 and CH34 plasmids . After the annotation of the plasmid sequences, it is assumed that in the strain S14E4C, the main replicon carried most of the essential housekeeping genes, including those for translation, ribosome production, DNA replication, DNA repair, protein processing, cell component and resistance. Plasmid 1 contains genes mainly encoding mechanisms of heavy metal resistance  and membrane cation transport, additionally genes encoding proteins involved in carbohydrate metabolism and c-type cytochrome biogenesis. Whereas, plasmid 2 carries genes for antibiotic resistance (e.g. tetMOPQST) and operon for ribosomal protein synthesis . The existence of the rRNA operon (rrn) on plasmids and chromids was earlier reported on Bacillus and Paracoccus species [Previous studies depicted that known members of the plasmids –32. In tplasmids . The anaplasmids  with whoC. campinensis S14E4C consists mostly of known genes encoding metabolic modules and various pathways support its growth. The main and common metabolic genes were shown in Fig. cynRXST operon), nitrate and nitrite ammonification , nitrate reductase  and gene clusters responsible for nitrogen metabolism are also present in its genome.The genome of soxABXYZDFCRSWH gene cluster. Basically, sulfur oxidation pathways require only five sox genes whose products form three key periplasmic protein complexes: soxYZ, a sulfur carrier protein, soxXA, a c-type cytochrome complex, and soxB, a sulfate thiol hydrolase [ssuA—alkanesulfonates-binding, ssuB—alkanesulfonate ABC transporter ATP binding, ssuF—organosulfonate utilization, ssuC—alkanesulfonates transport system permease, ssuD—alkanesulfonate monooxygenase, etc. proteins.The biochemistry of the bacterial sulfur metabolism pathways is quite complex and encoded by ydrolase . In caseptsS (putuative periplasmic phosphate binding protein), ptsA (phosphate transport system permease protein), ptsB (phosphate transport ATP binding protein), oprO and oprP (pyrophosphate and phosphate specific outer membrane porins) genes  and mannose metabolism . However, based on the annotation results, compared to previously identified Cupriavidus species the metabolism of ketogluconates (some can serve as the sole source of carbon and energy for various bacteria) is quite different in the Cupriavidus campinensis S14E4C.Spectrum of carbohydrate metabolism is broad, but here only few of them are mentioned. Such as, several operons are responsible for maltose and maltodextrin utilization  and/or Pentose Phosphate pathways (PPP). The pathway is encoded by pglDH gene, whereas, there is no gene for 6-phosphogluconate dehydrogenase (6-PGDH) in any earlier sequenced Cupriavidus species [C. metallidurans, C gilardii, C. necator, etc.).In  species   resistant genes and gene clusters  [Referring to the genome annotation analysis rs Table . CompariCBA, etc.. HoweverzcRDABC) .Table 2G2+, Pb2+, Ni2+, Co2+, such as nccAB, nikABCDEKLMNOQR, cbtACDFGJKLX, ctpD, plasmid-mediated trcD, etc. Unlike the CH34, pbr operon is absent in S14E4C, but previous researches indicate that heavy metal--translocating P-type ATPase genes can perform Pb2+ resistance as well [2+ resistance system at strain S14E4C consisted of merR gene, activates transcription of mer operon (merRTPCADE) in elevated concentrations of mercury, and genes encode the resistance to mercury (Hg) is a well-known property of both Gram-positive and Gram-negative bacteria that generally locate in plasmids [merC, merT, merE and merP genes function as membrane or periplasmic transport of organic and inorganic Hg2+ (Table merA and merB genes (merB) responsible for demethylation of organic mercury compounds by cleavage C-Hg bonds, encoding mercuric reductase and the enzyme organomercurial lyase directly followed by genes encoding transport and transcriptional regulators. The other gene encoding organomercury resistance, merD, a secondary regulatory protein, also binds the same region as merR, involved in transcriptional regulation [The genome also carries system genes and clusters involved in the transport and resistance of Cd as well . Howeverplasmids , 38. Thegulation , 40.Fig.ars gene homologs  were identified in the genome of Cupriavidus campinensis S14E4C for arsenate (AsO43−) resistance and the strategy followed by bacteria depend on the arsenate reductase (ArsC) protein. In the existing operon (arsRBC) the arsC, arsenate reductase is able to transform the arsenate to arsenite and the rest of the process is encoded by arsB, an integral membrane protein, to prohibit arsenic accumulation by expelling out of the cytoplasm [arsR) enables the transcription of the operon by attaching the promoter region [arsH gene) that contributes to the global biotransformation of arsenic [arsH is widely distributed in Proteobacteria (not present in Gram-positive) and similarly to other ars genes locates mostly in chromosome [In addition to the cluster elements, several ytoplasm , 42. Meaenate AsO− resistar region . The str arsenic . Among tromosome , 46.42−) are distributed in the genome of S14E4C as a cluster chrABCEIF, while, available gene clusters, copACDSRZ, cusSARBCF, scsABCD, cutACEF and ycnLKI are encoding proteins that responsible for the uptake, efflux, translocation and periplasmic detoxification of Cu+ and Cu+2 ions. The protein sequences of cusCFBA are similar to the general gene and protein families which pump Cu2+ cations out of cells that had been previously studied. Additionally, except the Cu2+ cations, the cusCBA genes are encoding Ag+ efflux system as well (Table + resistance is putative silver efflux pump (SEP). In the environment Cu2+ is more abundant and less toxic than Cu+ and the presence of protein copA, P-type Cu+ efflux ATPase, controlling the intracellular Cu+ level, whereas, the extracellular periplasmic space is defended by cusCFBA multicomponent efflux transport system [cusCFBA RND efflux has been examined in details [cusF protein displayed as a key periplasmic copper binding protein [Encoded proteins for the efflux system of cations including chromate CrO4− are dist system . The che details  and the  protein .C. campinensis S14E4C primarily occurs by efflux system(s): RND  family of permeases, the CDF (Cation Diffusion Facilitator) family of heavy metal transporters and the P-type ATPase family of ion pumps may operate on a variety of cations [2+, Zn2+, Pb2+, Ni2+, CrO42−, Ag+, Co2+, Cu2+, Cu+, Hg2+, and likely on other heavy metals  detoxification in  cations , includiC. campinensis S14E4C expressed high tolerance/resistance to all four tested heavy metals. The nutrient and Tris-salt mineral medium indicated significant difference in terms of growth inhibition  compared to other Cupriavidus species, though the mercury resistance is noticeable (1.84 mM).In order to verify metal resistance capability of the strain S14E4C MIC assessment was performed and on Table . ResistaC. campinensis S14E4C is resistant to D-cysteine amino acids and Beta-lactam group antibiotics with around 21 resistance genes. D-cysteine is decomposed into pyruvate, H2S, and NH3 by D-cysteine desulfohydrase. Decomposition of D-cysteine by the desulfohydrase (dcyD gene) produces H2S, which the bacterium can use as a sulfur source. In S14E4C strain the desulfhydrase appears in a cluster with two proteins , probably involved in transport processes, and a gene encoding a periplasmic cystine-binding protein (CBP). Additionally, blc, bli, blR, blaI, blaR resistance genes exist in the genome of the S14E4C for resistance of β-lactam group antibiotics. These antibiotics include penicillin, cephalosporin, cephamycin and carbapenem, supply multi-resistance by breaking the antibiotics’ structure. C. campinensis S14E4C carries β-lactam resistance genes that responsible for penicillin binding, repression, hydrolase and regulation [acrB and a substitute for the bridging protein acrA are the homologs of Pseudomonas mexA and mexB [C. campinensis S14E4C contains all of these 4 genes. Other ABR genes were shown on Table The Genome Annotation Service in PATRIC uses k-mer-based ABR genes detection method  that assgulation  to have and mexB , where CThe test with 9 antibiotic agents revealed that the strain S14E4C can resist to the number of antibiotics Table . In 2006C. campinensis S14E4C as a source of carbon and energy. The pathway genes that are found in this strain include, benzoate degradation (benBACDKEF operon) and transport (bt1254 transporters) [salARED, areABCR), additionally, gentisate catabolism and degradation, N-heterocyclic aromatic compounds degradation , etc.Annotation results identified also the presence of genes encoding enzymes involving in the utilization of various aromatic compounds in porters) , salicylThe degradation capacity of our strain was measured connected to phenanthrene and naphthalene and after one week of incubation it was not detectable. It is assumed that the isolation environment (not contaminated by aromatic compounds) of the strain plays direct role in these results, however, the genes for the possible degradation of aromatic compounds could be activated after a long time exposure , 58.C. campinensis S14E4C strain is a member of Cupriavidus genus and its closest relative was C. campinensis LMG 1195T (AF312020)  were concatenated with the 16S rRNA gene and analysed on PATRIC [C. campinensis S14E4C was placed and depicted in Fig. C. metallidurans CH34 was identified [Phylogenetic analysis based on 16S rRNA gene sequence suggested that 1195T AF3020  Syntheny plot of C. campinensis S14E4C vs C. gilardii CR3, C. necator N-1, C. metallidurans CH34 (PNG 433 kb)Supplementary Figure 1. Syntheny plot analysis of the S14E4C sequence vs known Cupriavidus species and their replicons. (B) Chromosome from C. metallidurans CH34 vs C. campinensis S14E4C whole genome (PNG 433 kb)Supplementary Figure 1. Syntheny plot analysis of the S14E4C sequence vs known Cupriavidus species and their replicons. (C) Megaplasmid from C. Metallidurans CH34 vs C. campinensis S14E4C whole genome (PNG 433 kb)Supplementary Figure 1. Syntheny plot analysis of the S14E4C sequence vs known Cupriavidus species and their replicons. (D) Plasmid pMOL28 and pMOL30 from C. metallidurans vs plasmid 1 and plasmid 2 from C. campinensis S14E4C (PNG 433 kb)Below is the link to the electronic supplementary material.


Fl1sa on chromosome 12 (centromere-53.06 Mb) as a significant QTL for fatty liver. Furthermore, isoamyl acetate-hydrolyzing esterase 1 homolog (Iah1) was selected as the most likely candidate gene for Fl1sa. Iah1 gene expression in fatty liver-resistant A/J-12SM mice was significantly higher than in fatty liver-susceptible A/J mice. These data indicated that the Iah1 gene might be associated with fatty liver development. However, the function of murine Iah1 remains unknown. Therefore, in this study, we created Iah1 knockout (KO) mice with two different backgrounds [C57BL/6N (B6) and A/J-12SM (A12)] to investigate the relationship between Iah1 and liver lipid accumulation. Liver triglyceride accumulation in Iah1-KO mice of B6 or A12 background did not differ from their respective Iah1-wild type mice under a high-fat diet. These results indicated that loss of Iah1 did not contribute to fatty liver. On the other hands, adipose tissue dysfunction causes lipid accumulation in ectopic tissues . To investigate the effect of Iah1 deficiency on white adipose tissue, we performed DNA microarray analysis of epididymal fat in Iah1-KO mice of A12 background. This result showed that Iah1 deficiency might decrease adipokines Sfrp4 and Metrnl gene expression in epididymal fat. This study demonstrated that Iah1 deficiency did not cause liver lipid accumulation and that Iah1 was not a suitable candidate gene for Fl1sa.Nonalcoholic fatty liver disease (NAFLD) is a pathological condition caused by excess triglyceride deposition in the liver. The SMXA-5 severe fatty liver mouse model has been established from the SM/J and A/J strains. To explore the genetic factors involved in fatty liver development in SMXA-5 mice, we had previously performed quantitative trait locus (QTL) analysis, using (SM/J×SMXA-5)F2 intercross mice, and identified  Nonalcoholic fatty liver disease (NAFLD) refers to a wide range of liver conditions which occur without excessive alcohol consumption, from simple steatosis to steatohepatitis, fibrosis, and cirrhosis . The incFl1sa (Fatty Liver 1 in SMXA)    mice wer/eucomm)  11]. Th. ThIah1t/eucomm) . The genSM consomic strain (A/J-12SM) was obtained from the Institute for Laboratory Animal Research, Nagoya University School of Medicine. The A/J-12SM consomic mouse strain was established by the introduction of chromosome 12 of the SM/J mouse into the A/J mouse background as previously described [Iah1 gene on the A/J-12SM background was carried out by using the CRISPR/Cas9 method as previously described [5ʹ-CTGTGCGAGCGAGCTGCTAG(CGG)-3ʹ (Iah1_g21316438-21316460) and 5ʹ-CTTCGGGGACTCCATCACGC(AGG)-3ʹ (Iah1_g21316491-21316513). The two crRNAs were designed using the CRISPOR website (http://crispor.tefor.net/) [SM mice to produce offspring with heterozygous mutations in the Iah1 gene. The DNA sequence in the Cas-9 nuclease-target region was confirmed by Sanger sequencing. The primer sequences used were as follows, 5ʹ-GAAGTCAGGCGGTCTACAGTGAG-3ʹ (Forward) and 5ʹ-GGAAGAGAGGAGTGAACTAGTCGG-3ʹ (Reverse). Off-target sites were predicted using CRISPOR [SMIah1 homozygous KO mice (KO_A12), the heterozygous mice were intercrossed. The genomic DNA was amplified by PCR using primers for Iah1 genotyping, namely 5ʹ-TTTCTACCATGTCGCTGTGC-3ʹ (forward) and 5ʹ-AGGCGGACCCTTTAAGCTC-3ʹ (reverse). The size differences between the PCR products obtained from the Iah1-wild type allele and mutation allele were detected by agarose gel electrophoresis. The established KO_A12 line was maintained by (homozygous × homozygous) breeding. A/J-12SM (WT_A12) mice were used as a control for KO_A12 mice.The A/J-Chr12escribed , 14. Theor.net/) . Founder CRISPOR . The topad libitum access to food and water under conventional conditions, in the facilities at the Graduate School of Bioagricultural Sciences, Nagoya University. Mice were weaned at 3 weeks of age and they were housed individual cages at 5 weeks of age.All mice were maintained at 23 ± 2 °C with a 12-h light/dark cycle (light on from 8:00 to 20:00) and Iah1-wild type (WT_B6 or WT_A12) and -KO (KO_B6 or KO_A12) mice were fed with a standard chow, CE-2 , until 6 weeks of age. Subsequently, all mice were fed with a high-fat diet  from 6 to 18 weeks of age (for 12 weeks). The composition of the high-fat diet was previously described [Male This study was carried out in accordance with the Regulations on Animal Experiments of Nagoya University. All procedures and animal care were approved by the Animal Experiment Committee, Graduate School of Bioagricultural Sciences, Nagoya University .Iah1 gene and off-target candidate genes of KO_A12 mice were amplified by PCR and the PCR products were purified using the ExoSAP-IT kit (Affymetrix). The primers used for sequence analysis of off-target effects are shown in The Frozen tissues (approximately 0.2 g each) were homogenized using lysis buffer  containing a protease inhibitor cocktail . After being mixed well, the homogenates were incubated on ice for 1 h. Thereafter, they were centrifuged at 16,000×g for 20 min at 4 °C and the supernatants were collected. The protein content in the supernatants was measured by using the DC Protein Assay Kit  and 10 μg of protein was subjected to SDS-PAGE on a 10% acrylamide gel. Subsequently, the proteins in the gel were transferred on PVDF membranes  by semidry blotting. The membrane was incubated for 30 min with EzBlock Chemi . The membrane was then washed with TBS buffer supplemented with 0.05% Tween 20 (TBS-T) and incubated overnight at 4 °C with the primary antibody, rabbit polyclonal anti-mouse IAH1  and anti-mouse alpha-Tubulin . Afterwards, the membrane was washed with TBS-T and then incubated with the secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG antibody , for 1 h at 20–25°C. Finally, the membrane was washed with TBS-T and the proteins bands were detected with a West Dura Western Blot Detection kit using the ECL method (Thermo Fisher Scientific). All antibodies were diluted with Can Get Signal .Serum triglyceride (TG), total cholesterol (TC), HDL-cholesterol (HDL-C), phospholipid (PL), and non-esterified fatty acid (NEFA) concentrations were measured using Triglyceride-E test Kit, Cholesterol-E test Kit, HDL-Cholesterol-E test Kit, Phospholipid-C test Kit, and NEFA-C test Kit . Serum glucose concentrations were measured using Glucose-CII test kit .et al. [Frozen livers (approximately 0.3 g each) were homogenized with 25 mL of chloroform-methanol (2:1) and statically extracted overnight. The organic extracts (200 μL) were dried and dissolved in isopropanol (200 μL). TG and total TC in isopropanol were measured using the Triglyceride-E test Kit and Cholesterol-E test Kit, respectively. The remaining organic extract was used for the measurement of total liver lipids as previously described by Folch et al. .Iah1. The primers used for the SYBR Green assays are shown in Total RNA from tissue was extracted using the TRI reagent (Molecular Research Center Inc.). The total RNA was treated with the TURBO DNA-free kit (Thermo Fisher Scientific) to remove DNA contamination. Then, cDNA was synthesized using the High Capacity Reverse Transcription kit (Applied Biosystems). We used a StepOne Plus Real-Time PCR System (Applied Biosystems) with the Thunderbird qPCR Mix or the Thunderbird SYBR qPCR Mix  in order to measure gene expression levels. Each mRNA level was normalized to the corresponding β-actin mRNA level. TaqMan probes  were used to determine the mRNA level of Total RNA was extracted from the epididymal fat of WT_A12 and KO_A12 mice fed with a high-fat diet for 12 weeks using the TRI reagent. Then, the obtained RNA was purified using the RNeasy Mini Kit (QIAGEN). Total RNA from three mice per strain was pooled for each array. The transcripts from epididymal fat were measured using a Clariom S Mouse Array (Applied Biosystems). Raw data were normalized with the STT-RMA algorithm using the Applied Biosystems GeneChip Console Software ver.1.3.0. The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) (GSE14750).t-test was used to compare the means between WT (WT_B6 or WT_A12) and KO (KO_B6 or KO_A12) mice. Differences with a p < 0.05 with statistically significant.All results were expressed as mean ± standard error of the mean (SEM). Student’s Iah1 KO mice (KO_B6) by using the Cre/loxP system  by using the CRISPR/Cas9 system to investigate the relationship between Iah1 and fatty liver development. The CRISPR/Cas9 system generated a 53 bp deletion that resulted in frameshift mutations leading to the formation of a premature stop codon (Iah1 mRNA and IAH1 protein were not detected in A/J-12SMIah1-KO mice (KO_A12) (We constructed op codon . This mu(KO_A12) . RegardiIah1 deficiency on an A/J-12SM background  and Cd36 antigen (Cd36) were suppressed in Hepa1-6 cells which overexpressed mouse Iah1 [Srebp1-c, Pparγ, Fasn, and Mtp) in the liver and epididymal fat of KO_A12 mice. Our results showed that the mRNA levels were not significantly different between WT_A12 and KO_A12 mice  which overexpressed mouse A12 mice . TherefoIah1 gene is most abundantly expressed. The Iah1 gene was also shown to be abundantly expressed in the liver and epididymal fat  in KO_A12 mice when compared with WT_A12 mice  and downregulated genes  in the epididymal fat of KO_A12 mice were measured via real-time qPCR (Sfrp4) mRNA levels in epididymal fat were found to be significantly decreased in KO_A12 mice (Metrnl) mRNA levels tended to be slightly decreased in KO_A12 mice when compared to WT_A12 mice (p = 0.05) , there were no significant changes in Sfrp4 mRNA levels in the retroperitoneal adipose tissue  decreased Sfrp4 and Metrnl gene expression. Decreased Sfrp4 mRNA levels may to a decrease in the mRNA levels of downstream genes (Pparγ and C/EBPα) and increase β-catenin mRNA levels. However, significant differences in the expression of these genes were not observed  . The chae tissue . These robserved .Iah1 gene as the most likely candidate gene for Fl1sa, a QTL for fatty liver on chromosome 12 identified in the high-fat diet-induced fatty liver model mouse, SMXA-5 [Iah1 and fatty liver in vivo, we analyzed the phenotype of Iah1-KO mice established on two different backgrounds (C57BL/6N and A/J-12SM).In this study, we focused on , SMXA-5 . TherefoIah1 mRNA and IAH1 protein, and Iah1 gene deficiency did not lead to embryonic death. Moreover, we did not observe a significantly higher TG accumulation in KO_B6 mice when compared with WT_B6 mice, both of which were fed with a high-fat diet for 12 weeks. A previous study has reported that C57BL/6J mice, which is one of the C57BL/6-derived substrains, are more likely to develop severe obesity and type 2 diabetes than A/J mice fed with a high-fat diet [Iah1 deficiency on fatty liver development might be masked in the C57BL/6N background. As such, we created Iah1-KO mice on an A/J-12SM background using the CRISPR/Cas9 system. We previously reported that liver TG accumulation in A/J-12SM mice which had the SM/J allele on Fl1sa was suppressed when compared with A/J mice [Iah1 deficiency on an A/J-12SM background would significantly induce fatty liver development. However, liver TG accumulation in KO_A12 mice did not differ from that in WT_A12 (A/J-12SM) mice  mice . AltogetIah1 deficiency on an A/J-12SM background, and the data showed that Iah1 deficiency in vivo might decrease adipokines (Sfrp4 and Metrnl) gene expression in epididymal fat  signaling pathway. Sfrp4 inhibits Wnt from binding to the Frizzled receptor and leads to β-catenin degradation. Wnt signaling maintains preadipocytes in an undifferentiated state by suppressing the expression of adipogenic transcription factors (C/EBPα and Pparγ) [et al. reported that Sfrp4 mRNA levels were high in the visceral adipose tissue of obese or type 2 diabetes patients and that Sfrp4 promotes de novo lipogenesis in mice hepatocytes [Sfrp4 levels in the epididymal fat of KO_A12 mice activates the Wnt signaling pathway and leads to inhibition of lipid synthesis in the liver. However, in the epididymal fat of KO_A12 mice, the expression of Sfrp4 downstream genes  remained unchanged  promotes differentiation of adipocytes by increasing the expression of lipid metabolism genes [Metrnl adipocyte-specific KO mice under a high-fat diet showed insulin resistance [Metrnl in mice by intravenous injections of adenoviral vectors stimulates thermogenesis in beige fat and improves glucose tolerance [Sfrp4 and Metrnl protein levels in adipose tissue and serum.Reduction of lipid storage-capacity and dysregulation of secretion adipokines in adipose tissue are involved in the development of fatty liver , 20. Theymal fat . Sfrp4 id Pparγ) . Horbeltatocytes . These rnchanged . Metrnl sm genes . Metrnl sistance . Moreoveolerance . HoweverIah1 is not a gene responsible for Fl1sa, we take into consideration another candidate gene, namely, Lpin1 (encoding the Lipin1 protein) which is located at 16.7 Mb on mouse chromosome 12. Lipin1 was identified as a mutated gene in the fatty liver dystrophy (fld) mouse [lipin1 gene was shown to impair adipose tissue development, which led to fatty liver development. Phan and Reue reported that modulation of lipin1 expression levels leads to dramatic alterations in adiposity [Fl1sa, including Lipin1, to elucidate the mechanism of fatty liver development in SMXA-5 mice.As d) mouse . The mutdiposity . TherefoIah1 gene (most likely candidate gene for Fl1sa) and fatty liver in vivo, we constructed Iah1-KO mice on two different genetic backgrounds (C57BL/6N and A/J-12SM). Data from both types of Iah1-KO mice (KO_B6 and KO_A12) demonstrated that the absence of the Iah1 gene did not affect lipid accumulation in the liver. We conclude that the Iah1 gene is not a gene responsible for Fl1sa.In this study, to clarify the relationship between the S1 Figet al. [http://www.mousephenotype.org/about-ikmc/eucomm). The tm1a mice (C57BL/6NTac genetic background) have a heterozygous knockout-first allele with the loxP sites and Frt sites. The CAG-Cre transgenic mice (C57BL/6NCrSlc genetic background) show constitutive expression of the Cre recombinase gene under the control of the CAG promoter. The tm1b mice have the genome construct of Iah1 which has exon 3 and 4 deleted. (B) Breeding scheme of KO_B6 mice.(A) The scheme of KO_B6 mice generation by using the Cre-loxP system. Figure modified from Skarnes et al.  and EUCO(PDF)Click here for additional data file.S2 FigIah1 mRNA levels in WT_B6 and KO_B6 mice. Data were expressed as mean ± SEM. . The mRNA levels were measured by real-time qPCR. (B) Western blot analysis of the IAH1 protein (28 kDa) with α-tubulin as a loading control in WT_B6 and KO_B6 mice. The size marker  was loaded into lane M. Tissues were collected from WT_B6 and KO_B6 mice fed with a high-fat diet for 12 weeks. BAT, brown adipose tissue; eWAT, epididymal white adipose tissue.(A) Real-time qPCR analysis of (PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.S2 Raw images(PDF)Click here for additional data file.


IFITM3 results in a truncated IFITM3 protein lacking its first 21 amino acids, which is much higher in the Han Chinese population and associated with severe illness in adults infected with pandemic influenza H1N1/09 virus. To investigate if IFITM3 or IFITM3 rs12252-C could affect the antibody response after influenza vaccination, we detected the haemagglutination inhibition (HI) of 171 healthy young adult volunteers  and in an IFITM3-deletion mouse model (-/-Ifitm3) after trivalent inactivated vaccine (TIV) immunization. Seroconversion rates for H1N1, H3N2 and B viruses in IFITM3 rs12252-C/C genotype carriers was lower compared with C/T and T/T donors. Significantly lower levels of specific antibodies to H1N1, H3N2 and B viruses and total IgG were observed in -/-Ifitm3 mice. Correspondingly, the numbers of splenic germinal centre (GC) B cells, plasma cells, TIV-specific IgG+ antibody secreting cells and T follicular helper cells in -/-Ifitm3 mice were lower compared with wild type mice. However, the number of memory B cells was higher in -/-Ifitm3 mice at day 7 after booster. The HI level of -/-Ifitm3 mice remained lower than WT mice after third vaccination. Moreover, the transcriptional network regulating GC B cell and plasma cell differentiation was abnormal in -/-Ifitm3 mice. Our results indicate that IFITM3 deletion attenuated the antibody response. The mechanism of influenza-IFITM3 interactions affecting the antibody response requires further investigation.Interferon-induced transmembrane protein 3 (IFITM3) as an antiviral factor can inhibit replication of several viruses including influenza virus. A single-nucleotide polymorphism rs12252-C of  Dissections and orbital blood collections were performed under anaesthesia that was induced and maintained with isoflurane. All efforts were made to minimize suffering to the mice.IFITM3 rs12252 were operated following the method described previously ,3029,30] B cells . Prolifeantibody ,33. GC Bdm1 gene . Blimp-1dm1 gene ,36. Bachdm1 gene .Figure -/-Ifitm3 mice showed a substantial reduction in the expression of Blimp-1 protein in spleens at day 7, whereas the expressions of BCL6 and AID were similar when WT mice were compared with -/-Ifitm3 mice  antigens . TFH celxpansion . Prolife-/- mice . BCL6 in-/- mice . So, BCL-/- mice ,42. In ton assay . Our finIFITM3 SNP rs12252-C/C carriers show lower antibody levels after TIV immunization. The frequency of the IFITM3 SNP rs12252-C is much higher in the Han Chinese population than in Caucasians [IFITM3 SNP rs12252-C/C. IFITM3 SNP rs12252-C could be used to identify patients at high risk of severe illness, which would be of immense benefit for prioritizing treatment.Our results show that IFITM3 deletion diminishes the antibody response and ucasians . A compr


The situation faced by breast cancer patients, especially those with triple-negative breast cancer, is still grave. More effective therapeutic targets are needed to optimize the clinical management of breast cancer. Although collagen type VIII alpha 1 chain (COL8A1) has been shown to be downregulated in BRIP1-knockdown breast cancer cells, its clinical role in breast cancer remains unknown.Gene microarrays and mRNA sequencing data were downloaded and integrated into larger matrices based on various platforms. Therefore, this is a multi-centered study, which contains 5048 breast cancer patients and 1161 controls. COL8A1 mRNA expression in breast cancer was compared between molecular subtypes. In-house immunohistochemistry staining was used to evaluate the protein expression of COL8A1 in breast cancer. A diagnostic test was performed to assess its clinical value. Furthermore, based on differentially expressed genes (DEGs) and co-expressed genes (CEGs) positively related to COL8A1, functional enrichment analyses were performed to explore the biological function and potential molecular mechanisms of COL8A1 underlying breast cancer.COL8A1 expression was higher in breast cancer patients than in control samples . Elevated expression was detected in various molecular subtypes of breast cancer. An area under a summary receiver operating characteristic curve of 0.80 (95% CI 0.76–0.83) with sensitivity of 0.77 (95% CI 0.69–0.83) and specificity of 0.70 (95% CI 0.61–0.78) showed moderate capacity of COL8A1 in distinguishing breast cancer patients from control samples. Worse overall survival was found in the higher than in the lower COL8A1 expression groups. Intersected DEGs and CEGs positively related to COL8A1 were significantly clustered in the proteoglycans in cancer and ECM-receptor interaction pathways.Elevated COL8A1 may promote the migration of breast cancer by mediating the ECM-receptor interaction and synergistically interplaying with DEGs and its positively related CEGs independently of molecular subtypes. Several genes clustered in the proteoglycans in cancer pathway are potential targets for developing effective agents for triple-negative breast cancer. Breast cancer poses a grave threat to female health. According to the latest American cancer statistics, breast cancer is estimated to be the most common cancer and the second most common cause of cancer-associated deaths in women . Owing tBRCA1) and P53 mutations, EGFR upregulation, and cytokeratin 19 downregulation have been associated with TNBC [Molecular events occurring in breast cancer help us better understand the onset and progression of breast cancer. It is generally agreed that chromosome 1q amplification, chromosome 16q deletion, and PIK3CA mutations are the most common pathways leading to luminal breast cancer , 18. Morith TNBC . It has ith TNBC . Recentlith TNBC . On the ith TNBC –25. Baseith TNBC . Althougith TNBC –29, the Collagen type VIII alpha 1 chain (COL8A1), also named C3orf7, is located at chromosome 3 and encodes alpha 1 chain in collagen type VIII, which is an essential component of extracellular matrix (ECM) . PreviouConsidering this knowledge gap, our study aimed to explore the role of COL8A1 in breast cancer. We were focused on investigating the expression of COL8A1 messenger RNA (mRNA) in various molecular subtypes of breast cancer by analyzing gene microarray and RNA sequencing data sets. The COL8A1 protein expression level was validated by immunohistochemistry staining. We also aimed to determine prognostic value of COL8A1 in breast cancer to pave the way for future clinical applications. Moreover, we explored the molecular mechanisms of COL8A1 underlying breast cancer to improve our knowledge of breast cancer carcinogenesis and progression.Homo sapiens; (ii) the studied specimens should be tissue dissected from patients or healthy individuals rather than cell lines. In the case of duplicated studies or samples, the most recent version was retained. The exclusion criteria were as follows: (i) expression profiles not including COL8A1; (ii) breast cancer patients receiving hormone therapy or chemotherapy; (iii) stromal rather than epithelial tumors; and (iv) metastatic rather than primary tumors. The included data sets were carefully checked and a log2 transformation was performed if any matrices had not been normalized. Additionally, the data sets were integrated into larger matrices according to various platforms, and batch effects between studies were removed using the limma-voom package in R v3.6.1. Subsequently, COL8A1 expression values were extracted and grouped according to specimen types. Standardized mean difference (SMD) were calculated to compare the expression of COL8A1 mRNA between breast cancer patients and control samples using STATA v12.0. Heterogeneity between the included studies was assessed with the I2 statistic. Statistical significance was set to an I2 value greater than 50% with a P-value less than 0.05. A random effects model was used in the case of significant heterogeneity. Sensitivity analysis was performed to probe the potential source of heterogeneity, and a publication bias test was used to evaluate the stability of the SMD results. Subgroup analysis was performed to compare the COL8A1 expression levels between molecular subtypes .We integrated gene microarrays and mRNA sequencing data downloaded from Gene Expression Omnibus, The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression, the Sequence Read Archive, ArrayExpress, and Oncomine. The search formula, based on MESH terms, was as follows: ((Breast OR mammary) AND ). Studies were screened according to the following criteria: (i) the studied species should be A diagnostic test was performed to assess the clinical significance of COL8A1 in breast cancer. Based on the expression value of COL8A1, a receiver operating characteristic (ROC) curve was plotted to compute the area under the curve (AUC) using IBM SPSS Statistics v19.0. AUC values of less than 0.7, between 0.7 and 0.9, and greater than 0.9 represented weak, moderate, and strong discriminatory capability of COL8A1, respectively, between breast cancer patients and control samples. The true positives, false positives, true negatives, and false negatives rates were calculated, and the cutoff values were identified. A summary receiver operating characteristic (sROC) curve was drawn using STATA v12.0 to assess the general discriminatory capability of COL8A1 between breast cancer patients and control samples. The significance of the area under the sROC curve was consistent with that of the ROC curve. The diagnostic odds ratio (DOR), sensitivity, specificity, positive diagnostic likelihood ratio (DLR P), and negative diagnostic likelihood ratio (DLR N) were also calculated to precisely determine the accuracy and validity of COL8A1 in distinguishing breast cancer patients from control samples.P-value of < 0.05 was considered statistically significant. Kaplan–Meier curves were used to compare high and low COL8A1 expression groups in terms of survival. The log-rank test was used to determine whether the prognostic difference was statistically significant.To explore the relation between COL8A1 mRNA expression and prognosis of breast cancer patients, information on clinicopathological parameters was collected. The independent samples t-test or one-way analysis of variance was used to identify statistically significant differences in COL8A1 expression between two or more groups. A A total of 115 non-specific invasive breast carcinoma and 65 normal breast tissue specimens were obtained from the First Affiliated Hospital of Guangxi Medical University, P.R.CHINA. All patients had previously signed informed consent forms, and our research was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University. The breast cancer and normal breast tissue specimens were fixed with formalin. The two steps immunohistochemistry method was used to determine the protein expression of COL8A1. The primary antibody was polyclonal Antibody to COL8A1 (concentrated 1:150 dilution) purchased from Wuhan Pujian CO.LTD. Supervision TM Mouse/Rabbit-HRP Broad Spectrum Detection System (Product No. D-3004-15) was purchased from Shanghai Long Island. The experimental procedure conformed to the manufacturer’s instructions. The patients’ clinicopathological information was analyzed to determine the relationship between COL8A1 protein expression and prognosis.http://cbioportal.org) proved to be a powerful tool and facilitated our online search and cancer genomics data analysis. Using cBioPortal, we gained insight into the genetic alterations of COL8A1 in breast cancer patients and obtained information on the association between COL8A1 alterations and breast cancer patient survival. We selected the Breast Invasive Carcinoma  cohort, which contains 1,108 patients, and used a method of mRNA expression z-scores relative to diploid samples (RNASeqV2 RSEM). We also considered the mutation types of COL8A1 in breast cancer patients using the Catalogue of Somatic Mutations in Cancer (COSMIC), which has been recognized as the most detailed resource for somatic mutations in cancer.The cBioPortal for Cancer Genomics |relation coefficient | > 0.3 and (ii) P-value < 0.05. Upregulated DEGs and CEGs positively related to COL8A1 were intersected. Similarly, downregulated DEGs and CEGs negatively related to COL8A1 were intersected.To gain insight into the role of COL8A1 in breast cancer, we identified DEGs and COL8A1 CEGs using the limma-voom package. The DEG criteria were as follows: (i)|log2FoldChange | > 1 and (ii) adjusted https://string-db.org) to investigate protein interactions. Hub genes and functional modules were identified using Cytoscape v3.6.1. Based on 1,020 breast cancer patients, the mutation landscapes of genes clustered in essential pathways were visualized using the TCGAmutations package in R v3.6.1.Overlapping genes were used to perform function enrichment to shed light on the potential mechanisms of COL8A1 underlying breast cancer. The R clusterProfiler package was used to conduct Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Disease Ontology (DO), and Reactome pathway analyses. Protein-to-protein interaction (PPI) network was constructed using STRING , a random effects model was used. An SMD value of 0.79  showed that COL8A1 expression was significantly higher in breast cancer than in non-breast cancer tissue . Moreover, elevated COL8A1 expression correlated with estrogen-negative (ER-) breast cancer (P = 0.018).Clinical data of breast cancer samples used to perform immunohistochemistry was summarized in Additional file P > 0.05). Furthermore, according to COSMIC, substitution missense mutations were the most frequent types  were associated with the breast cancer pathway (ID: hsa05224), even though is not in the top 30 KEGG pathways. Moreover, DO analysis indicated that these genes are closely associated with myeloma and bone marrow cancer  were associated with the breast cancer pathway (ID: hsa05224), even though this pathway is not in the top 30 KEGG signaling pathways. Eight of these genes  have been extensively studied in breast cancer [More importantly, our study provides important clues about the role of COL8A1 in breast cancer for the first time. The intersected CEGs positively related to COL8A1 and upregulated DEGs were significantly aggregated in several cancer pathways . Interestingly, we noticed that 14 genes related to COL8A1  and non-BRCA tissues.Additional file 3: Figure S3. Heterogeneity detection based on the enrolled data sets. a Sensitive analysis. The included studies were not sources of heterogeneity. b Funnel plot. No significant publication bias existed.Additional file 4: Figure S4. Comparison of COL8A1 expression levels between: a Luminal A subtype breast cancer and control normal or adjacent breast cancer tissues; b Luminal B subtype breast cancer and control normal or adjacent breast cancer tissues.Additional file 5: Figure S5. Comparison of COL8A1 expression levels between HER-2 + subtype breast cancer and control tissues.Additional file 6: Figure S6. Comparison of COL8A1 expression levels between Three Negative Breast cancer (TNBC) and non-TNBC tissues.Additional file 7: Figure S7. Calculation of standard mean deviation (SMD) based on COL8A1 expression in Three Negative Breast cancer (TNBC) and non-TNBC tissues.Additional file 8: Figure S8. Receiver operating characteristic (ROC) curves based on COL8A1 expression value in breast cancer (BRCA) patients. An AUC value > 0.70 signified COL8A1 possessed moderate capability in distinguishing BRCA from non-BRCA patients. AUC ,area under the curveAdditional file 9: Figure S9. DLR positive and negative in breast cancer diagnostic trial based on COL8A1 expression level. DLR, Diagnostic likelihood ratio.Additional file 10: Figure S10. Association between COL8A1 expression and clinicopathological parameters in breast cancer patients. a Elevated COL8A1 expression correlated with races of breast cancer patients. COL8A1 expression was higher in white compared to Black or African American. b Elevated COL8A1 expression correlated with subtypes of breast cancer. COL8A1 expression was lower in Three Negative Breast Cancer compared to Luminal A or Luminal B subtypes of breast cancer. c Elevated COL8A1 expression correlated with ER status. d Elevated COL8A1 expression correlated with PR status. e Elevated COL8A1 expression correlated with HER-2 status.Additional file 11: Table S1. The relevance between COL8A1 expression and clinicopathological parameters of breast cancer patients. Independent-samples t-test or one way analysis of variance (ANOVA) was used to compare the COL8A1 expression level between two groups or more groups, respectively. *A P-value < 0.05 indicates statistically significant.Additional file 12: Table S2. Clinical data of breast cancer samples used to perform immunohistochemistry.Additional file 13: Figure S11. Genetic alteration types of COL8A1 in breast cancer. a Amplification, mRNA high, and mRNA low were predominant alteration types of COL8A1 in breast cancer. b, c. indicated no significant difference between overall or disease-free survival conditions of COL8A1 altered and unaltered groups in breast cancer.Additional file 14: Table S3. Mutation types of COL8A1 in breast cancer patients based on the Catalogue Of Somatic Mutations In Cancer (COSMIC).Additional file 15: Figure S12. The identification of DEGs and COL8A1 related CEGs in breast cancer. TCGA dataset was selected to partially show A. DEGs and B. COL8A1 related CEGs. DEGs, differentially expressed genes; CEGs, co-expressed genes.Additional file 16: Table S4. Functional enrichment based on 632 overlapping genes of upregulated DEGs and CEGs positively related to COL8A1. Only the top four terms or pathways were exhibited. KEGG, Kyoto Encyclopedia of Genes and Genomes; DO, Disease Ontology; GO, Gene Ontology; BP, biological process; CC, cellular component; MF, molecular function; DEGs, differentially expressed genes; CEGs, co-expressed genes. Table S5. Functional enrichment based on 322 overlapping genes of downregulated DEGs and CEGs negatively related to COL8A1. Only the top two terms or pathways were exhibited. KEGG, Kyoto Encyclopedia of Genes and Genomes, GO, Gene Ontology; BP, biological process; CC, cellular component; MF, molecular function.Additional file 17: Figure S13. Intersected DEGs and CEGs positively related to COL8A1 clustered in the proteoglycans in cancer pathways Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway: Proteoglycans in cancer (ID: hsa05205).Additional file 18: Figure S14. Intersected DEGs and CEGs positively related to COL8A1 clustered in the ECM-receptor interaction pathways Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway: ECM-receptor interaction (ID: hsa04512).Additional file 19: Figure S15. Co-expressed network of COL8A1 in breast cancer. Genes clustered in the top two KEGG pathways or the top two functional modules were displayed. KEGG, Kyoto Encyclopedia of Genes and Genomes.


HMG-CoA reductase degradation protein (Hrd1), an E3 ubiquitin ligase, is involved in protein trafficking. H2S may play a role in controlling fatty acid uptake in diabetic cardiomyopathy (DCM) in a manner correlated with modulation of Hrd1 S-sulfhydration; however, this role remains to be elucidated. The aim of the present study was to examine whether H2S can attenuate lipid accumulation and to explain the possible mechanisms involved in the regulation of the H2S-Hrd1/VAMP3 pathway. Db/db mice and neonatal rat cardiomyocytes treated with high glucose, palmitate and oleate were used as animal and cellular models of type 2 diabetes, respectively. The expression of cystathionine-γ-lyase (CSE), Hrd1, CD36 and VAMP3 was detected by Western blot analysis. In addition, Hrd1 was mutated at Cys115, and Hrd1 S-sulfhydration was examined using an S-sulfhydration assay. VAMP3 ubiquitylation was investigated by immunoprecipitation. Lipid droplet formation was tested by TEM, BODIPY 493/503 staining and oil red O staining. The expression of CSE and Hrd1 was decreased in db/db mice compared to control mice, whereas CD36 and VAMP3 expression was increased. NaHS administration reduced droplet formation, and exogenous H2S restored Hrd1 expression, modified S-sulfhydration, and decreased VAMP3 expression in the plasma membrane. Using LC-MS/MS analysis, we identified 85 proteins with decreased ubiquitylation, including 3 vesicle-associated membrane proteins, in the cardiac tissues of model db/db mice compared with NaHS-treated db/db mice. Overexpression of Hrd1 mutated at Cys115 diminished VAMP3 ubiquitylation, whereas it increased CD36 and VAMP3 expression and droplet formation. siRNA-mediated Hrd1 deletion increased the expression of CD36 in the cell membrane. These findings suggested that H2S regulates VAMP3 ubiquitylation via Hrd1 S-sulfhydration at Cys115 to prevent CD36 translocation in diabetes.Hydrogen sulfide (H Type 2 diabetes is a strong risk factor for the development of cardiovascular disease and atherosclerosis, which is related to dyslipidemia. Recent evidence suggests that lipid accumulation within the myocardium may be involved in the progression of heart failure . In healAdditional evidence has demonstrated that intracellular membrane trafficking and exocytosis are controlled by several superfamily proteins, including N-ethylmaleimide sensitive factor (NSF); its cofactor, soluble NSF attachment protein (αSNAP); and SNAP receptors (SNAREs) . SNAREs Ubiquitylation is thought to be an important signal regulating intracellular trafficking . The end2S), the third identified gasotransmitter after nitric oxide and carbon monoxide, is involved in a wide range of physiological and pathological processes. Our previous study suggested that H2S inhibits the ubiquitylation of superoxide dismutase (SOD) to promote the initiation of autophagy in diabetic cardiomyopathy [et al. demonstrated that H2S regulates Krüppel-like factor 5 (KLF5) transcriptional activity via specific protein S-sulfhydration to prevent myocardial hypertrophy [2S in modifying Hrd1 or on the role of H2S in modulating LCFA uptake in diabetic cardiomyopathy. Our findings raise the possibility that H2S participates in VAMP3 degradation through Hrd1 S-sulfhydration in the cardiac tissues of db/db mice.Hydrogen sulfide   and age-matched lean littermates (C57BL/6) (n=25) were used in this study. The mice were bred in a conventional animal facility with a 12-hour light/dark cycle. The mice were divided into four groups. Half of the db/db mice and their lean age-matched C57BL/6 littermates were treated with NaHS  by intraperitoneal injection for 6, 12 or 20 weeks . Simultaneously, the remaining half were injected with equal amounts of saline. The animal experiments were carried out following the recommendations of the Guide for the Care and Use of Laboratory Animals published by the China National Institutes of Health. The feeding regimen was approved by the Animal Care Committees of Harbin Medical University, China.Before tissue collection, the mice were fasted overnight. Blood was collected from the orbits of the mice. The tissues were rapidly removed and frozen in liquid nitrogen. Cardiac sections were embedded into Tissue-Tek OCT compound (Sakura) for histology. The ventricles were rapidly dissected and processed as follows: (1) a small (3×1×1 mm) section from the ventricle was placed into fixation buffer for TEM analysis, (2) a 20-30 mg section was rapidly frozen for TAG analysis, (3) a 20-30 mg section was used to make frozen sections, and (4) the remainder was used for Western blot analysis.2S in cardiac tissues and neonatal rat cardiomyocytes (NRCMs) was tested with 7-azido-4-methylcoumarin . This probe has been testified to selectively respond to H2S [The fluorescence response of Hd to H2S . The samLipid droplets were stained with BODIPY 493/503 (Life Technologies) diluted in 10% (v/v) dimethyl sulfoxide in PBS to a concentration of 1 mg/mL . Frozen After induction of lipid droplets, cells were stained with oil red O using an adipogenesis assay kit from the Nanjing Jiancheng Bioengineering Institute . The cells were fixed with 4% paraformaldehyde for 10 min and then incubated with oil red O solution for 20 min at room temperature. The stained lipids were observed using a fluorescence microscope .2PO4, 8.0 NaCl, 0.35 NaHCO3, and 0.06 Na2HPO4·7H2O, pH 7.2) at 4 °C. Then, the tissue was cut into pieces and incubated with 0.25% trypsin at 37 °C for 8 min. An equal volume of cold Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum was added to terminate the digestion. The digestion step was repeated six times. The supernatant cells were filtered and isolated by centrifugation . The cells were resuspended in DMEM containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin and incubated at 37 °C in humidified air containing 5% CO2. After 1 hour of culture at 37 °C, the unattached cells were transferred to fresh medium. The medium was replaced with fresh culture medium periodically.Cultures of NRCMs were prepared according to previously described methods . Hearts 2S donor, 100 μM)-treated group; (4) an HG+Pal+Ole+propargylglycine -treated group; (5) an HG+Pal+Ole+dithiothreitol -treated group; and (6) an HG+Pal+Ole+PYR41 -treated group. The reagents were added to the culture medium for 24 or 48 hours.NRCMs were divided into six groups: (1) a control group ; (2) a high glucose (HG)+palmitate +oleate (Ole)-treated group ; (3) an HG+Pal+Ole+NaHS . The coverslips of cells were blocked with 5% BSA for 1 hour at 37 °C. The cells were then incubated with anti-CD36 antibodies  and anti-VAMP3 antibodies  at 4 °C overnight before being incubated for 2 hours with anti-rabbit IgG or anti-mouse IgG. Images were captured and analyzed with a Zeiss LSM 510 inverted confocal microscope.7 cells or 50 µg of cardiac tissue using a Nuclear-Cytosol-Mem Extraction Kit (Applygen) following the manufacturer’s instructions. The isolated protein fractions were analyzed by Western blotting.Cell plasma membrane proteins were isolated from 1×10g for 3 min. Agarose and antibodies (anti-Hrd1 or anti-VAMP3) were added, and the samples were incubated with gentle rotation overnight. The precipitates were collected after centrifugation at 10,000 × g for 5 min and three washes with buffer. Samples meeting the purity standards were subjected to Western blotting with anti-VAMP3 or anti-ubiquitin antibodies.Protein samples were incubated with agarose  at 4 °C for 20 min and centrifuged at 10,000 × g for 30 min at 4 °C. The cell lysates were incubated with blocking buffer (sonication buffer with 2.5% SDS and 20 mM MMTS) for 20 min at 50 °C with frequent vortexing. The proteins were precipitated with acetone at -20 °C for 20 min, and then the samples were resuspended in blocking buffer (with 1% SDS) following the addition of 4 mM biotin-HPDP. After incubation for 3 hours at 25 °C, the biotinylated proteins were mixed with streptavidin-agarose beads. Then, the cells were washed with HENS buffer. The biotinylated proteins were eluted by SDS-PAGE and subjected to Western blotting using an anti-Hrd1 antibody.Briefly, cells were sonicated in buffer containing 250 mM HEPES buffer (pH 7.7), 1 mM EDTA, 0.1 mM neocuproine and 100 μM deferoxamine and then centrifuged at 13,000 × Intracellular production of polysulfide was examined with a new type of fluorescent probe, SSP4 . Briefly, isolated cells were incubated with 50 μmol/L SSP4 in serum-free DMEM containing 0.003% Cremophor EL at 37 °C for 15 min in the dark. After washing, SSP4 was detected using a fluorescence microscope .®, life technology) using Lipofectamine 3000 reagent (Invitrogen). The adenoviruses were added to NRCMs directly, and after 4 hours of transfection, the medium was replaced with fresh medium. The NRCMs were treated with different reagents after 24 hours, and the related proteins were detected by Western blotting.Adenoviruses expressing GFP and Hrd1 were purchased from Cyagen Biosciences Inc . Full-length mouse Hrd1 with a single mutation of cysteine 115 to alanine and GFP cDNA were inserted into a pM vector (Cyagen Biosciences) between the Kozak and T2A sites. Subconfluent (70%) monolayers of cells were transfected with the pHrd1-cys-115-GFP plasmid in Opti-MEM reduced-serum medium (Gibco2 incubator at 37 °C.H9c2 cells (Organism Rattus norvegicus Disease Comments Breed/subspecies: BDIX. Proper Citation ATCC Cat# CRL-1446) (80% confluent) were treated with Hrd1 short interfering RNAs (siRNAs) (Sangon Biotech Shanghai) for 48 hours to inhibit Hrd1 expression according to the manufacturer’s instructions. Transfection was performed with Lipofectamine 3000 (Invitrogen). Briefly, transfection reagent and Hrd1 siRNAs were mixed and incubated for 20 min to form complexes, which were then added to plates containing cells and medium. For further analysis, the cells were incubated in a COThe results were analyzed with the Prism software package (GraphPad Software). The results are shown as the mean ± standard deviation (SD). Differences among three or more groups were analyzed using one-way ANOVA and Bonferroni’s correction. Differences between individual groups were analyzed using Student’s t-test.12) and was significantly increase in the HG+Pal+Ole (with or without PPG)-treated groups compared with the NaHS-treated and control groups. We also examined beta-oxidation and glucose uptake levels, and our results showed that fatty acid beta-oxidation was increased and that glucose uptake was slightly decreased in NRCMs in response to HG, Pal and Ole treatment for 24 or 48 hours. These results indicated that under diabetic conditions, the heart is forced to increase the uptake of fatty acids instead of glucose.  and fraction shortening (FS) were decreased at 12 weeks and 20 weeks in db/db mice compared to control mice, whereas they improved after treatment with NaHS. All the results indicated that cardiac pump function was reduced in db/db mice. -induced diabetic cardiomyopathy. In this study, we tested the expression of cystathionine-γ-lyase (CSE), a principal enzyme for H2S production, in the hearts of db/db mice , a reducing agent that reverses sulfhydration, was used to treat cardiomyocytes for 10 min. The results showed that exogenous H2S enhanced SSP4 fluorescence intensity, suggesting that polysulfide could accumulate after H2S application (It has been reported that S-sulfhydration, the addition of one sulfhydryl or persulfide group, is a novel posttranslational modification by Hlication .2S on Hrd1 by S-sulfhydration, we detected the S-sulfhydration of the Hrd1 protein. The biotin switch assay revealed that exogenous H2S could regulate Hrd1 S-sulfhydration (To further investigate the effect of Hydration . Furtherydration .2S could not reduce the expression levels of VAMP3 or CD36 in the group overexpressing mutant Hrd1 and treated with Pal and Ole (2S modified Hrd1 at the Cys115 site.To investigate how Hrd1 interacts with VAMP3 and to elucidate which specific cysteine residues may be responsible for this interaction, we conducted a computational modeling study of Hrd1. The optimized structure of Hrd1 is depicted in  and Ole . We also and Ole . Stainin and Ole . The aboTo further investigate whether Hrd1 regulates the formation of lipid droplets, we successfully knocked down Hrd1 expression using siRNA in H9c2 cells . Deletio2S has been suggested to be beneficial for a range of cardiovascular diseases, such as cardiac ischemia and atherosclerosis. Our data revealed that H2S levels were reduced in the hearts of db/db mice. A large number of lipid droplets were produced in the db/db mice; however, the number of droplets was reduced by treatment with NaHS, and we thus postulated that H2S regulated endocytosis by S-sulfhydration of target proteins.H2S levels and CSE expression were clearly decreased in db/db mice compared to control mice. The number and size of the droplets increased in a time-dependent manner; however, administration of exogenous H2S could reduce the numbers and sizes of the droplets in the hearts of db/db mice. These results demonstrated that impairment of the H2S/CSE system is involved in dysregulated free fatty acid transportation. We also report a novel finding that H2S regulated the interaction between Hrd1 and VAMP3 through Hrd1 S-sulfhydration. Collectively, these data demonstrate that H2S plays an important role in modulating VAMP3 ubiquitylation to regulate free fatty acid transporters in the context of diabetes.In the present study, we found that cardiac H2S, and coimmunoprecipitation revealed that Hrd1 interacted with VAMP3 in cardiomyocytes.The transport of LCFA is primarily a protein-mediated event involving several transport proteins, including CD36 and FABP . Both of2S action in which H2S adds sulfur atoms to cysteine residues of target proteins to induce conformational changes, leading to modification of target protein activity [2S. We also found that VAMP3 expression in the cell membrane was clearly reduced in the db/db mice treated with exogenous H2S. To further investigate whether exogenous H2S affected Hrd1 activity, we overexpressed Hrd1 in which Cys115 was mutated to Ala and demonstrated that treatment with exogenous H2S decreased VAMP3 ubiquitylation levels and CD36 translocation to the cell membrane. This finding suggested that sulfhydration of Hrd1 at Cys115 might be one of the detailed mechanisms by which H2S reduces CD36 translocation to the cell membrane, reduces LCFA uptake and inhibits lipid droplet formation (Sulfhydration has been proposed as a mode of Hactivity . Accordiormation .2S increases Hrd1 sulfhydration and participates in regulating VAMP3 ubiquitylation to prevent LCFA uptake. Our findings raise the possibility that H2S supplementation might be a clinically useful therapeutic strategy to attenuate diabetic cardiomyopathy.In conclusion, this study led to the novel finding that Hwww.aginganddisease.org/EN/10.14336/AD.2019.0530.The Supplemenantry data can be found online at: