In particular, B7-H4 expression appeared to be associated with lymph node metastasis (P = 0.008) and TNM stage (P = 0.012). Taken together, our study demonstrates a strong promoting role of B7-H4 in lung tumor growth, progression and metastasis, and supports its potential as a therapeutic target for the treatment of the disease.Aberrant expression of B7-H4 occurs across a broad spectrum of human cancers. The aim of this study was to investigate the key role of B7-H4 during tumorigenesis and metastasis of human lung cancer. Our data showed that the shRNA-mediated disruption of B7-H4 markedly inhibited tumor cell proliferation, invasion and migration, increased cell apoptosis and arrested cell cycle at G0/G1. These changes were accompanied by a marked increase in Bax and caspase-3/caspase-8, but a decrease in Bcl-2, cyclinD1 and activation of AKT. In addition, our shRNA-mediated disruption of B7-H4 led to a marked decrease in tumor growth in the immune-compromised mice. Importantly, B7-H4 was expressed in 53.33% of lung carcinomas from our patient cohort ( Lung cancer is one of the leading causes of cancer-related death in men and the second cause (after breast cancer) in women worldwide . AlarminB7-H4, which is also known as VTCN1, B7x or B7S1, is an important member of the B7 family. It has been shown to control cytokine secretion, cytotoxicity development and activation of T cells . B7-H4 ivitro and vivo analyses demonstrate that B7-H4 is a key driver and a promising therapeutic target for human lung cancer.Here, we applied shRNA-mediated gene silencing technology to investigate the functional and signaling roles of B7-H4 in lung tumor growth and progression, as well as underlying molecular mechanisms. Results from our To evaluate the clinical importance of B7-H4 in lung cancer, we conducted IHC analyses of paraffin–embedded tumors and adjacent non-tumor tissues of lung cancer patients. As shown in Figure p = 0.008, p = 0.012). However, there was lack of association between B7-H4 expression and patients’ age, gender, smoking status, pathological type, differentiation and three year survival rate after surgery. Thus, our data imply a strong role of B7-H4 in lung cancer progress and lymph node-associated metastasis.Next, we assessed the link between B7-H4 expression and key clinicopathological parameters for our lung cancer patient cohort. As shown in Table p < 0.05). Meanwhile, the B7-H4 protein expression level in A549-B was lower than A549-NC group , B7-H4 mRNA expression level was evaluated by real-time PCR. As shown in Figure p Figure . Therefop < 0.001).The potential effect of RNAi-mediated B7-H4 down-regulation on A549 cell growth was evaluated by CCK-8 assay. In Figure p < 0.001). These data demonstrate a promoting role of B7-H4 in the motility and promoted invasion of A549 cells.To address the role of B7-H4-shRNA in the aggressive behaviors of A549 cells, we evaluated the influence of B7-H4-shRNA on migration and invasion with transwell assays. As shown in Figure p < 0.05); while the percentage of S+G2/M phase in A549-shRNA cells were decreased relative to A549-NC cells . The results showed that the percentage of G0/G1 was increased and the proportion of S+G2/M phase was decreased upon B7-H4 ablation. The data indicated that inhibition of B7-H4 resulted in G0/G1 arrest. The G0/G1 arrest in cell cycle could inhibit cell growth and proliferation of cancer. At same time, we detected cell apoptosis in above cells by using Annexin V–PE and 7-ADD double staining. In Figure p < 0.05); there was no difference in percentage of late apoptosis between the two groups . It indicated that inhibition of B7-H4 expression could promote apoptosis of tumor. These results suggested that B7-H4 contributed to tumor progression by apoptosis inhibition.Moreover, the influence of B7-H4 gene silencing on tumor cell cycle was assayed. In Figure To investigate which apoptosis and cell cycle pathway components were involved in A549 cells response to B7-H4 RNAi, we detected the expression of apoptosis and cell cycle associated molecules by real time PCR and Western blot. We observed the increase of the pro-apoptotic Bax, decrease of anti-apoptotic Bcl-2 and cell cycle associated cyclinD1, and activation of caspase-3 and caspase-8 relative to controls. Furthermore, knockdown of B7-H4 decreased AKT phosphorylation, but not changed total AKT protein expression level in A549-shRNA cells Figure .in vivo, the xenograft tumor model of BALB/c nude mice was established by injecting stable A549-shRNA cells and A549-NC cells into the groin of nude. The sizes of tumor volume were shown in Figure p < 0.001). Our data indicated that B7-H4 gene silencing results in inhibition of tumor growth.To confirm the function of B7-H4 in lung cancer To date, B7-H4 gene has been intensively studied in many tumors , 14–18. in vitro and vivo studies have demonstrated a strong role of B7-H4 in tumor growth and metastatic progression of lung cancer by use of the RNA interference approach. Notably, our data showed that B7-H4 gene silencing markedly inhibited cell proliferation, invasion and migration, increased cell apoptosis and arrested cell cycle at G0/G1. Apoptosis is critical step for tumor development. The induction of apoptosis is a significant therapeutic strategy for tumor [Our or tumor . Bcl-2 bor tumor –23. Incror tumor . This alor tumor . It is wor tumor . B7-H4 min vivo. Two groups of xenograft growth provide direct evidences that B7-H4 gene RNAi resulted in the obvious variation in tumor mass. It indicated that B7-H4 gene silencing suppress growth of xenografts in vivo and has a potent therapeutic effect against tumor cells.It is worth noting that the orthotopic xenograft tumor model of nude mice was also established to confirm the effect of B7-H4 on the growth of tumors The aberrant activation of AKT signaling pathway has a critical influence in many biological processes of human malignancies, including NSCLC . KnockdoIn summary, our work from vivo and vitro demonstrated that B7-H4 might be an important cancer promoter and a novel therapeutic target for human lung cancer. B7-H4 inhibition might offer an exciting opportunity to inhibit the progression and metastasis of human lung cancers.Formalin-fixed, paraffin-embedded tumor tissues were obtained from 90 cases of lung carcinomas and 50 cases of adjacent non-tumor specimens (within at least 5cm of tumor margin) were also obtained in Affiliated Hospital of Jiangnan University , China. The protocol in this study was approved by the ethics committees of the Affiliated Hospital of Jiangnan University.The paraffin-embedded human lung tumor tissues were cut into 4μm-thick serial sections, Immunohistochemical (IHC) staining was performed on human tumor specimens and non-tumor specimens by using Envision methods, these slides were labeled by using primary antibodies specific for B7-H4 , a negative control was carried out by replacing the primary antibody with PBS, then sections were incubated with horseradish peroxide-labeled goat anti-Rabbit second antibody (Merck & Millipore). Semi-quantitative measurements of staining intensity  and the proportion of stained cells  were determined as previously described . All sliA549 cells, derived from human lung adenocarcinoma, were purchased from cell bank of Chinese Academy of Science, Shanghai, in China, and maintained in Roswell Park Memorial Institute  medium containing 10% fetal bovine serum (Gbico), 1% penicillin and streptomyc (Life Technologies) at 37°C, in a humidified incubator with 5% CO2.Four DNA template oligonucleotides corresponding to B7-H4 gene were designed and synthesized. Four pairs of B7-H4 specific shRNA were also synthesized according to four DNA template oligonucleotides sequence. The synthesized single DNA chain was ligated with empty vector GV115 . The connected product was transferred to Escherichia coli and cultured on the Amp+LB plate for an over night at 37°C. The positive colonies were selected and amplified using PCR primers, then inoculated into Amp+LB liquid medium in shaking bottle at 37°C for an over night. The plasmid was extracted by using Axygen plasmid extraction kit  and sequenced (invitrogen). Recombinant lentiviral vector was then transfected into 293T cells. Supernatants containing lentiviruses were harvested 48h later after transfection. A549 cells were infected with viral supernatants for 12h at an appropriate concentration of viral supernatants. After 3 days post-transfection, 90% of the cells were transfected by observing the expression of GFP. There were respective two experimental groups for A549 cells: cells transfected with lentivirus mediated shRNA-targeted B7-H4 were named A549-shRNA-A, A549-shRNA-B, A549-shRNA-C, A549-shRNA-D cells, cells transfected with lentivirus mediated shRNA  were named A549-NC cells.TM Reverse Transcriptase kit (Life Technologies), then, RT-PCR was performed using Power SYBR® Green PCR Master Mix (Life Technologies). The mRNA expression of various genes was assessed using ABI 7700 Sequence Detection System (Applied Biosystems). Relative gene expression was quantified.A549-shRNA cells and A549-NC cells were seeded and harvested after culture 96h. Total RNA was extracted from each group of cells using the Trizol (Invitrogen), cDNA synthesis was obtained using the ThermoScript5/well, were respectively seeded into 96-well plates, and incubated in the water-saturated carbon dioxide incubator. The absorbance at 450 nm was measured by using a Model AMR-100 microplate reader.The cell viability of A549-shRNA and A549-NC cells were measured by Cell Counting Kit-8Assay . Two groups of cells, at 1 × 10Apoptosis assays were performed by using an Annexin V–PE apoptosis detection kit (BD PharMingen). A549-shRNA and A549-NC cells in 6-well plate were digested and collected at 96h, cells were suspended in PBS and stained with Annexin V–PE and 7-ADD , and detected in a FACS Calibur cytometer . Annexin V-PE and 7-ADD double-negative cells were considered to be non-apoptotic cells. PI staining kit (Vazyme) was used to detect cycle of cells by using flow cytometry. DNA content of cell was detected according to the relative proportions of cells in the G1/G0, S and G2/M phases of the cell cycle.5 cells respectively) suspended with serum-free RPMI medium were seeded to upper chamber with 8 μm pores membrane, and 10% FBS contained medium were added into lower chamber, then cells were incubated for 12h at 37°C. Cells removed from upper chamber to lower chamber. In invasion assay, A549-shRNA and A549-NC cells ( 2.0 × 104 cells respectively) suspended with serum-free RPMI medium were seeded to upper chamber, 10% FBS contained medium were placed in the lower chamber. Then cells were incubated for 24 h at 37°C. Cells were removed from upper chamber surface of 8 μm pores polycarbonate filter coated with 1mg/ml Matrigel  to lower chamber. The cells on the lower surface of the filter were fixed in methanol and stained with 0.1% crystal violet. The stained cells were washed three times with PBS, and counted in five random high-power fields.In migration assay, A549-shRNA and A549-NC cells (1 × 107 cells) were injected into groin of 8-week-old male BALB/c nude mice. The size of tumors was observed at intervals of 3 days. The orthotopic xenograft tumors were measured using a caliber and harvested at 42 days after the injection.Briefly, A549-shRNA cells and A549-NC cells (both 2.0 × 10A549-shRNA cells and A549-NC cells were lysed in 50ul lysis buffer (Beyotime) on ice for 30 min respectively. Cells lysates were centrifuged and denatured with SDS polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer for 10 min at 95°C. Proteins were electrophoresed on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in TBST and then incubated with specific antibodies  for 2 h at room temperature. After thorough washes, membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibody .x ± SD), the differences between the groups was analyzed by two-tailed Student's t-test. The software of SPSS version17.0  was used for statistical analysis. Statistical significance was considered at p < 0.05 and markedly significance was considered at p < 0.01.The association between B7-H4 expression and clinic pathologic features was analyzed by chi-square test. The difference between the groups showed mean ± standard deviation (


Dlk1-Dio3 locus. Functional analysis revealed that ground-state miRNAs embedded in the Dlk1-Dio3 locus  promoted pluripotency via inhibition of multi-lineage differentiation and stimulation of self-renewal. Overall, our results demonstrate that ground-state pluripotency is associated with a unique miRNA signature, which supports ground-state self-renewal by suppressing differentiation.Ground-state pluripotency is a cell state in which pluripotency is established and maintained through efficient repression of endogenous differentiation pathways. Self-renewal and pluripotency of embryonic stem cells (ESCs) are influenced by ESC-associated microRNAs (miRNAs). Here, we provide a comprehensive assessment of the “miRNome” of ESCs cultured under conditions favoring ground-state pluripotency. We found that ground-state ESCs express a distinct set of miRNAs compared with ESCs grown in serum. Interestingly, most “ground-state miRNAs” are encoded by an imprinted region on chromosome 12 within the   •Ground-state pluripotency is associated with a unique miRNA signature•Dlk1-Dio3 locus on 12qF1 encodes the majority of ground-state miRNAs•Dlk1-Dio3 locus is hypomethylated in ground-state ESCs•Ground-state miRNAs promote ESC self-renewal and inhibit differentiation Dlk1-Dio3 locus, contribute to the maintenance of ESCs through stimulating self-renewal and inhibiting differentiation.Ground-state pluripotency is a cell state in which pluripotency is maintained through inhibition of differentiation. In this paper, Baharvand and colleagues report that ground-state pluripotency is associated with a unique microRNA signature. They find that ground-state microRNAs, which are mostly encoded by the  ESCs wein vitro . Furtherin vitro . Based oin vitro . Notablyin vitro .Recently, we devised alternative culture conditions, dubbed R2i, which allow ground-state cultivation and efficient generation of ESCs from pre-implantation embryos . R2i conMicroRNAs (miRNAs) are ∼22-nt long non-coding RNAs that post-transcriptionally regulate a large number of genes in mammalian cells, thereby modulating virtually all biological pathways including cell-fate decisions and reprogramming . In ESCsIn the present study, we analyzed the global expression patterns of miRNAs in ESCs cultured in ground-state conditions of 2i and R2i compared with serum using small RNA sequencing. We provide a comprehensive report on the “miRNome” of ground-state pluripotency compared with serum cells, which enabled us to identify miRNAs specific to each cell state. Furthermore, we found that selected ground-state miRNAs contribute to the maintenance of ground-state pluripotency by promoting self-renewal and repressing differentiation.To obtain a comprehensive expression profile of miRNAs in ground-state ESCs, we used the RB18 and RB20 ESC lines maintained under feeder-free conditions in serum, 2i, or R2i cultures. RB18 and RB20 ESC lines were initially derived from C57BL/6 mice using the R2i + LIF protocol . IsolateAnalysis of the small RNA profiles of ESC samples revealed a total of 79,520,099 raw reads. After removal of adaptors and reads with <15–35> bases, the processed reads were aligned to mouse genome, which yielded a total of 35,287,315 mappable reads B. We fouA Pearson correlation coefficient heatmap D revealeBioinformatics analysis identified a set of 20 miRNAs, which were abundantly expressed under all conditions in ESCs B. MemberIn contrast, members of the miR-302–367 cluster were mostly upregulated in 2i/R2i cells compared with serum ESCs B. This fWe next asked whether miRNAs associated with differentiation were differentially expressed F. We fouTo validate the expression patterns of miRNAs obtained by small RNA sequencing, we performed TaqMan miRNA qRT-PCR assays of RNA isolated from serum, 2i, and R2i cells, and observed highly similar results compared with small RNA sequencing . HoweverOur small RNA-sequencing approach detected 1,233 miRNAs in each ESC state. Scatterplots indicated that miRNAs were significantly differentially expressed under different conditions Ai–iii. SA global assessment of miRNAs exhibiting >2-fold differences in expression revealed that 179 miRNAs were differentially expressed between serum and 2i cells  and S2. Next, we sought to pinpoint miRNAs associated with serum or ground-state pluripotency. We defined ground-state-associated miRNAs as miRNAs that are significantly upregulated in both 2i and R2i. Our data identified 91 miRNAs associated with serum and 101 miRNAs associated with the ground state . Top serNext, we sought to profile the global chromosomal distribution of miRNAs detected in the different ESC samples. miRNAs were mapped to different chromosomes and the relative expression of mature miRNAs per chromosome was determined. miRNAs were encoded on and expressed in ESCs by all chromosomes other than the Y chromosome A and S4Ath intron of the imprinted Sfmbt2 gene located on chromosome 2 were expressed much higher in serum compared with 2i/R2i (Dlk1-Dio3 locus on chromosome 12 (12qF1) were expressed much higher in 2i/R2i than in serum  based on the following: (1) their high abundance compared with other ground-state-specific miRNAs RNAs see . After cRNAs see C, we treRNAs see A. qRT-PCRNAs see B. In addRNAs see C. MoreovRNAs see D. AdditiRNAs see E. FurtheRNAs see F and absRNAs see G. NotablRNAs see D, furtheSince ground-state-specific miRNAs promoted ESC viability and 2i and R2i cells exhibit a much higher cloning efficiency than serum cells , we want1 phase, which is shortened in ESCs compared with differentiated cells and the extension of G1 phase upon differentiation  most differentially expressed miRNA genes are located in imprinted regions of the genome and (2) serum cells own higher DNA methylation levels than 2i , we reas2i cells . The aveectively A, indicao3 locus B. We hypP4ha1) (Srsf11) and poly(C)-binding protein 2 (Pcbp2) (Map4)  B, miR-41 (Pcbp2) C, and mi) (Map4) D. P4ha1 neration . The extneration . Srsf11 splicing . Pcbp2 eytoplasm  and is iytoplasm . Map4 isrization . Map4 phrization . AlthougDlk1-Dio3 locus. Interestingly, activity of the Dlk1-Dio3 locus seems crucial to maintain pluripotency of iPSCs, and its reduced expression is associated with incomplete iPSC reprogramming and poor contribution to mouse chimeras   . Since wDlk1-Dio3 locus contribute to the maintenance of ground-state pluripotency by enhancing viability, clonogenicity, AP activity, and ESC cycling as well as inhibiting differentiation. Our analysis revealed that the putative targets of miR-541-5p, miR-410-3p, and miR-381-3p are involved in cytoskeletal organization, ECM dynamics, control of RNA processing/decay, and cell cycling. These processes have previously been found to be important for pluripotency in general and ground-state pluripotency in particular  and passaged every other day. Serum ESCs were cultured in the presence of 15% ES-qualified fetal bovine serum (HyClone), and 2i/R2i cells were cultured in serum-free N2B27 medium.Additional experimental procedures are detailed in H.B. and S. Moradi conceived and designed the study. S. Moradi, H.B., and T.B. designed experiments and analyzed and interpreted the data. S. Moradi performed most of the experiments and wrote the manuscript. H.B., T.B., and S.A. provided financial and administrative support, discussed the results, and approved the manuscript. S. Mollamohammadi and A.S. contributed to cell-culture and cell-cycle analysis. A.S.-Z. performed bioinformatics analysis. A.A. contributed to qRT-PCR and DNA methylation analysis. S.A., G.H.S., and S.G. contributed to data analysis and interpretation. All authors reviewed and confirmed the manuscript before submission.


However, the mechanism underlying the coordination of metabolism and the G2/M-phase transition in mammalian cells remains unclear. Here, we show that the activation of AMP-activated protein kinase (AMPK), a highly conserved cellular energy sensor, significantly delays mitosis entry. The cell-cycle G2/M-phase transition is controlled by mitotic cyclin-dependent kinase complex (CDC2-cyclin B), which is inactivated by WEE1 family protein kinases and activated by the opposing phosphatase CDC25C. AMPK directly phosphorylates CDC25C on serine 216, a well-conserved inhibitory phosphorylation event, which has been shown to mediate DNA damage–induced G2-phase arrest. The acute induction of CDC25C or suppression of WEE1 partially restores mitosis entry in the context of AMPK activation. These findings suggest that AMPK-dependent phosphorylation of CDC25C orchestrates a metabolic checkpoint for the cell-cycle G2/M-phase transition.From unicellular to multicellular organisms, cell-cycle progression is tightly coupled to biosynthetic and bioenergetic demands. Accumulating evidence has demonstrated the G The ansition –36. Thes2/M-phase transition is driven by a series of tightly regulated and coordinated signaling events that eventually lead to the activation of CDC2-cyclin B  or A 769662 (A7) . By contrast, the percentage of cells in mitosis indicated by phosphorylation of histone H3 (pH3) is reduced following AICAR and A7 treatment (A). Notably, the disappearance of BrdU incorporation in AICAR group is probably due to the substrate competition between BrdU and AICAR, both of which are nucleotide analogs. Next, we repeated the experiment in the presence of nocodazole, a reversible inhibitor of microtubule polymerization, which blocks mitosis exit and therefore highlights the changes of mitotic entry following treatments. Both AICAR and A7 reduced the percentage of cells in mitosis compared with the control group (B).Previous studies have demonstrated an AMPK-dependent cell-cycle checkpoint at the GG1 phase , 33. How662 (A7) . AICAR i662 (A7) –66. We fporation A. By conreatment A. Notablol group B.1 phase or G2 phase. We also included cells starved by serum removal as a control to indicate the baseline metabolic activity. Compared with cells at G1 phase or serum-starved cells, cells at G2 phase significantly up-regulated glycolysis, indicated by the detritiation of [5-3H]glucose; glucose consumption via the pentose phosphate pathway (PPP), indicated by 14CO2 release from [1-14C]glucose; and glutamine consumption through oxidative catabolism (glutaminolysis), indicated by 14CO2 release from [U-14C]glutamine (A). In contrast, both mitochondria-dependent pyruvate oxidation through the tricarboxylic acid (TCA) cycle, indicated by 14CO2 release from [2-14C]pyruvate, and fatty acid β-oxidation, indicated by the detritiation of palmitic acid, were comparable among all three groups (B). These data suggest that cells at G2 phase actively engage glucose and glutamine catabolic programs to meet their bioenergetic and biosynthetic demands.We next applied radiochemical-based approaches to determine the activity of major catabolic pathways that could fuel the biosynthetic programs in cells released into Glutamine A. In cone groups B. These 2 phase would cause a delay of mitosis entry. This would determine whether the delay of mitosis entry is a secondary effect from the G1/S-phase transition in the presence of AMPK activators. For this, we first synchronized cells at the G1/S boundary by double thymidine blockage and then released the cells into S phase and treated them with AMPK activators and nocodazole once they reached G2 phase (A). In addition to AICAR and A7, we also included metformin and phenformin, two respiration chain complex I inhibitors, to indirectly activate AMPK by suppressing ATP production (B and C). Moreover, none of the acute treatment with these compounds caused significant apoptosis, as measured by PI uptake and cell-surface annexin V staining . Collectively, our results suggest that activation of AMPK in cells at G2 phase delays mitosis entry.Next, we sought to determine whether the acute activation of AMPK at GG2 phase A. In addoduction . Our resoduction , B and C2 phase with damaged DNA are prevented from entering into mitosis, and the control mechanisms behind this are known as the G2 checkpoint  and histone H2AX (H2AX), two characteristic biomarkers of the DNA damage response (A). Previous studies have demonstrated that AMPK phosphorylates p53 and causes the accumulation of p21, a transcriptional target of p53 that mediates p53-dependent regulation on cell cycle . Our findings are also consistent with earlier reports showing no detectable p53 protein in HeLa cells . Importantly, neither rapamycin nor torin treatment delayed mitosis entry (C). Collectively, these findings suggested that mTORC1 is not involved in mediating AMPK-dependent regulation of the G2/M transition.It has also been reported that AMPK regulates cell-cycle progression through directly inhibiting mTORC1 complex, which is the central player in sensing nutrients and coordinately regulating cell-cycle progression , 33–36. 33–tivities B. Importis entry C. Collec2 phase. CDC25 activates CDC2-cyclin B and drives cells into mitosis by dephosphorylating the inhibitory phosphorylation sites on CDC2-cyclin B (A). Given that the mitotic regulators mentioned above are all regulated by phosphorylation and AMPK is a signaling protein kinase, we asked whether AMPK is capable of directly phosphorylating and modulating any of the above mitotic regulators. Previous studies have revealed the optimal consensus phosphorylation motif for AMPK (B). The phosphorylation of serine 216 plays a key role in the temporal and spatial regulation of CDC25C and the G2/M transition in response to DNA damage and during normal cell-cycle progression . Second, we generated recombinant CDC25C fused to glutathione S-transferase (GST-CDC25C) and a non-related GST-tagged protein and phosphorylated these proteins with recombinant AMPK or extracellular signal-regulated kinase 1 (ERK1) or PBS control. ERK readily phosphorylated CDC25C at threonine 48, as we reported previously (C). Third, we treated cells with AICAR or transfected cells with either a control construct or a construct expressing a constitutively active mutant of the AMPK catalytic subunit (AMPK-CA) . Finally, we applied a chemical genetic approach to validate Ser-216 in CDC25C as a genuine AMPK-mediated phosphorylation site in cells . Then we followed up the above experiment by co-transfecting cells with a construct expressing AS-AMPK as well as a construct expressing either Myc-tagged WT or S216A CDC25C. The recognition by thioP-specific antibody was significantly compromised by S216A mutation , suggesting that Ser-216 is a direct phosphorylation site and very likely the only phosphorylation site of AMPK on CDC25C.The activity of CDKs oscillates throughout the cell cycle and determines the transition between different cell-cycle phases , 77. Mitcyclin B A. Given for AMPK , 80. We for AMPK ) to searfor AMPK B. The phgression , 61, 62.gression , 80. Wheeviously . Howevereviously C. Third,AMPK-CA)  and thenin cells –85. Thisn cells –, 86. Thu2/M-phase transition through the phosphorylation and inhibition of CDC25C. CDC25C activates CDC2-cyclin B and promotes the G2/M-phase transition by antagonizing WEE1 (i.e. by dephosphorylating WEE1-dependent phosphorylation sites on CDC2-cyclin B) (2/M-phase transition (A). To test this, we first took a pharmacological approach, using MK1775, to block WEE1 activity in the presence of AMPK activators. MK1775 is a potent WEE1 inhibitor and allows us to acutely block WEE1 activity in cells synchronized at G2 phase without interfering with the cell-cycle synchronization process (2-phase progression (B). Supporting the idea that the inhibition of WEE1 may antagonize AMPK-mediated suppression on CDC25C, neither A7 nor AICAR treatment delayed mitosis entry in the presence of MK1775; however, A7 and AICAR treatment consistently reduced the percentage of cells in mitosis in the absence of MK1775 . Next, we applied a genetic approach to knock down WEE1 by transfecting a WEE1-specific siRNA. To avoid perturbing the normal cell-cycle progression following a long-term WEE1 knockdown, we transfected cells with WEE1 siRNA when synchronized cells were released into S phase and collected samples 12 h later to examine the protein level of WEE1 and cell-cycle status (B). The protein level of mammalian WEE1 oscillates during the cell cycle and thus indicates a short half-life of WEE1 protein. This allows us to deplete WEE1 in a short time frame by siRNA (C) . Last, we sought to genetically modulate the phosphorylation of Ser-216 on CDC25C and examine its effect on G2/M-phase transition in the context of AMPK activation. To minimize the perturbation of constitutive overexpression of CDC25C on normal cell-cycle progression, we established a cell line stably expressing a doxycycline-inducible CDC25C (S216A) mutant, which enables the acute induction of CDC25C (S216A) following doxycycline treatment when synchronized cells were released into S phase . We confirmed the acute induction of CDC25C (S216A) by immunoblotting and showed that such treatment partially relieved AMPK-mediated suppression of the G2/M-phase transition . Of note, neither the WEE1 siRNA nor the induction of CDC25C (S216A) in the context of AMPK activation caused additional stresses and induced cell death during the time frame of experiments . Taken together, our results suggest that AMPK-mediated phosphorylation of Ser-216 on CDC25C represents a mechanism of cell-cycle checkpoint.Our data on AMPK-dependent phosphorylation of Ser-216 on CDC25C suggested that AMPK suppresses the Gyclin B) , 87, 88.ansition A. To tes process . Becausegression B. Suppore status B. The prby siRNA C 90–92)–922/M-ph2/M-phase transition may represent a metabolic checkpoint that ensures the coordination of sequential cell-cycle transitions with metabolic status. As such, abrogation of the checkpoint may reduce the ability of cells to survive. To test this idea, we treated cells with AMPK activator, WEE1 inhibitor, or a combination of these two and monitored the cell growth curve. Whereas single-agent treatment demonstrated a moderate ability to suppress cell growth, either A7 or AICAR in combination with MK1175 led to a synergistic effect on suppressing cell growth (A). Moreover, the growth suppression correlated with cell death induction, as measured by cell surface staining of annexin V and cellular uptake of PI, following the treatment with these reagents (B). Our studies suggest that abrogation of AMPK-dependent G2 checkpoint response induces cell death and may represent an attractive cancer therapeutic strategy.AMPK is a central sensor of cellular energy status and therefore plays a key role in maintaining metabolic and bioenergetic homeostasis , 93. We l growth A. Moreovreagents B. Our st2/M transition. We first applied glucose-free conditional medium and hexokinase inhibitor, 2-deoxyglucose (2DG), to induce an acute metabolic stress and assess the activation of AMPK. Given that the average duration of G2 phase is between 2 and 5 h, we reasoned that the acute condition (1 h) is more relevant to assess the impact of metabolic stress on cell-cycle G2/M transition. Under this condition, we found that U2OS cells but not HeLa cells respond to acute metabolic stress by enhancing AMPK activity, as indicated by the increase of AMPK autophosphorylation and phosphorylation of ACC (A). Previous studies have shown that HeLa cells are deficient in liver kinase B1 (LKB1), an AMPK-activating kinase (B). LKB1 deficiency probably renders HeLa cells resistant to metabolic stress-induced activation of AMPK because AMP/ADP promotes the conformational change of AMPK that will facilitate the phosphorylation by LKB1 and consequentially enhance AMPK activity . Both the procedure of transient transfection and the time frame (48 h) required to achieve an efficient knockdown of AMPKα1 interfered with the standard protocol for double thymidine synchronization. We therefore chose to treat cells with the indicated siRNA for 48 h and then applied nocodazole, to hold cells in mitosis, with or without metabolic stress. As Fig. 8D shows, metabolic stress imposed by either glucose starvation or glycolysis inhibitor 2DG significantly suppressed the percentage of cells entering into mitosis following nocodazole treatment. Importantly, AMPKα1 knockdown partially relieved metabolic stress-mediated suppression on the G2/M-phase transition . Collectively, our results suggested that acute metabolic stress (nutrient starvation or metabolic inhibitor) activates AMPK and suppresses the G2/M-phase transition.We next sought to assess the impact of metabolic stress on AMPK and cell-cycle Gn of ACC A. Previog kinase , and we g kinase B. LKB1 dactivity , 96. As OS cells C. Both t2-phase checkpoint mechanism in mammalian cells in response to both DNA damage and metabolic stress  supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin (Corning). Cell line identity was authenticated by short tandem repeat analysis. Glucose-free DMEM was supplemented with 10% (v/v) heat-inactivated dialyzed fetal bovine serum, which was made dialyzing against 100 volumes of distilled water (five changes in 3 days) using Slide-ALyzerTM G2 dialysis cassettes with cut-through MW size 2K (Thermo Fisher Scientific) at 4 °C. AICAR, A769662, rapamycin, torin, metformin, phenformin, 2-deoxy-d-glucose, and nocodazole were purchased from Caymen Chemical . MK1775 was purchased from MedKoo Biosciences .HeLa  and U2OS  were grown at 37 ºC/5% COm) were transfected into cells using Lipofectamine RNAiMAX reagent (Invitrogen). After 12 h of transfection (transfection when cells were released from the second thymidine block) or after 48 h of transfection (transfection in non-synchronized cells), immunoblots were carried out to examine the knockdown of targeted proteins.The siRNA oligonucleotides corresponding to human WEE1 and AMPKα1 were purchased from Fisher. siRNA oligonucleotides (20 n2-microglobulin to determine relative expression levels. Primer sequences (Table S2) were obtained from the Primer Bank  and was reverse-transcribed using random hexamer and Moloney murine leukemia virus reverse transcriptase (Invitrogen). SYBR Green-based quantitative RT-PCR for specific genes was performed using the Applied Biosystems real-time PCR system. Samples for each experimental condition were run in triplicate and were normalized to βmer Bank .m Tris-HCl, pH 7.4, 150 mm NaCl, 0.5% SDS, 5 mm sodium pyrophosphate, protease, and phosphatase inhibitor tablet). Cell lysates were centrifuged at 13,000 × g for 15 min, and the supernatant was recovered. The protein concentrations were determined using the PierceTM BCA protein assay kit (Thermo Fisher Scientific). After 5 min boiling in 4× NuPAGE® LDS sample buffer with 10× reducing solution (Thermo Fisher Scientific), the proteins were separated by NuPAGE 4–12% protein gels (Thermo Fisher Scientific), transferred to polyvinylidene difluoride membranes by using the iBlot gel transfer device (Thermo Fisher Scientific), and probed with the appropriate primary antibodies (Table S1). Membrane-bound primary antibodies were detected using secondary antibodies conjugated with horseradish peroxidase. Immunoblots were developed on films using the enhanced chemiluminescence technique. Density of each band was analyzed by ImageJ software, and all values were normalized with actin. All image data are representative of at least two independent experiments.Cells were harvested, lysed, and sonicated at 4 °C in a lysis buffer , cells were fixed in ice-cold ethanol, treated with HCl to denature the DNA, and stained with PI and anti-BrdU antibody. Mitotic cells were examined by intracellular staining of phospho-histone H3 using a hypotonic buffer protocol described previously . Cell-cym ATP), AMPK (1 unit), and 1 μg of the indicated substrates for 30 min at 30 °C. The reaction mixtures were boiled in SDS sample buffer and subjected to SDS-PAGE analysis and a Western blot assay.The bacterial expression vectors for GST-tagged CDC25 fragments were generated by PCR amplification of the encoding human CDC25C cDNA, followed by subcloning of the cDNA into pGEX-4T3 vector. Activated recombinant ERK (p42 MAPK) was prepared by incubating MBP-MAPK with constitutively activated MBP-MKK1 . GST- anTable S3. The activity of various metabolic pathways was determined by the rate of detritiation or 14CO2 released from radioactive tracers, as described previously  with a PCR tube containing 50 μl of 0.2 m KOH glued on the sidewall. After adding 0.5 μCi of radioactive tracer, the vials were capped using a screw cap with a rubber septum . The assay was stopped 2 h later by injection of 100 μl of 5 n HCl, and the vials were kept at room temperature overnight to trap the 14CO2. The 50 μl of KOH in the PCR tube was then transferred to scintillation vials containing 10 ml of scintillation solution for counting. A cell-free sample containing 0.5 μCi of radioactive tracer was included as a background control.Glutaminolysis, TCA cycle (oxidation of pyruvate), and glucose oxidation flux through the PPP were determined by the rate of lutamine , the ratpyruvate , and theications . Whereas5 cells/35-mm dish and were transfected with 1.25 μg of WT- or AS-AMPKα2 and 1.25 μg of WT- or S216A-Myc-CDC25C using the Lipofectamine (Thermo Fisher Scientific). Forty-eight hours after transfection, cells were washed twice with serum-free DMEM and incubated for 2 h before stimulation with 300 μm A769662 for 20 min. Following stimulation, 200 μl of phosphorylation buffer (20 mm HEPES (pH 7.3), 100 mm KOAc, 5 mm NaOAc, 2 mm MgOAc2, 1 mm EGTA, 10 mm MgCl2, 0.5 mm DTT, 5 mm creatine phosphate, 57 μg/ml creatine kinase, 30 μg/ml digitonin, 5 mm GTP (Abcam), 0.1 mm ATP, 0.1 mmN6-(phenethyl) ATPγS (Abcam), 0.45 mm AMP (Abcam), 1× phosphatase inhibitor mixture I and II (Sigma), and 1× complete protease inhibitors (Sigma)) was added. After a 20-min incubation at room temperature, 200 μl of 2× radioimmune precipitation assay buffer  with 2.5 mmp-nitrobenzyl mesylate (Abcam) and 5% DMSO was added, and cells were incubated for an additional 1 h at room temperature. The cell lysates were then subjected to immunoprecipitation using agarose beads coupled to Myc tag antibody.HeLa cells were seeded at 4 × 10Plasmid pECE-AMPKα2 WT, M93G, and pEBG-AMPKα1 (1–312) were obtained from Addgene  , 86. TheThe Amphopack 293 cells were transfected with the pRetro-TRE3G vector using Lipofectamine (Thermo Fisher Scientific) to produce retrovirus. HeLa cells that stably expressed reverse tetracycline-controlled transactivator  were infected with the retrovirus and treated with 2 μg/ml puromycin (Sigma) and maintained in puromycin-containing medium.Cell proliferations were measured by the IncuCyte cell proliferation assay. Cells were harvested by trypsinization, counted on a Countess automated cell counter (Invitrogen), and plated at 4000 cells/well on 96-well tissue culture plates in four replicates. Photomicrographs were taken every 3 h using an IncuCyte live cell imager , and confluence of the cultures was measured using IncuCyte software (Essen Biosciences) over 48 h in culture. For the cell death assay, cells were stained with annexin V–APC and PI and evaluated for apoptosis by flow cytometry according to the manufacturer's protocol .p values were calculated with Student's t test. p values < 0.05 were considered significant, with p values < 0.05 and p values < 0.01 indicated as with single and double asterisks, respectively.Y. S. data curation; Y. S. formal analysis; Y. S. validation; Y. S. and J. W. S. investigation; Y. S., J. W. S., and X. C. methodology; Y. S. and R. W. writing-original draft; Y. S., J. W. S., X. C., and R. W. writing-review and editing; R. W. conceptualization; R. W. supervision.


Diagnosis of viral myocarditis is difficult by clinical criteria but facilitated by detection of inflammation and viral genomes in endomyocardial biopsies. Parvovirus B19 (B19V) targets endothelial cells where viral nucleic acid is exclusively detected in the heart. Microparticles (MPs) are released after cell damage or activation of specific cells. We aimed to investigate whether circulating endothelial MPs (EMPs) in human and experimental models of myocarditis are associated with B19V myocarditis.MPs were investigated in patients with myocarditis (n = 54), divided into two groups: B19V+ (n = 23) and B19V- (n = 31) and compared with healthy controls . MPs were also investigated in B19V transgenic mice (B19V-NS1+) and mice infected with coxsackievirus B3 (CVB3). MPs were analyzed with fluorescent activated cell sorting (FACS).In human samples, EMP subpopulation patterns were significantly different in B19V+ compared to B19V- and HCTR (p<0.001), with an increase of apoptotic but not activated EMPs. Other MPs such as platelet- (PMPs) leukocyte-(LMPs) and monocyte-derived MPs (MMPs) showed less specific patterns. Significantly different levels of EMPs were observed in transgenic B19V-NS1+ mice compared with CVB3-infected mice (p<0.001).EMP subpopulations are different in B19V+ myocarditis in humans and transgenic B19V mice reflecting vascular damage. EMP profiles might permit differentiation between endothelial-cell-mediated diseases like myocardial B19V infection and other causes of myocarditis. Myocarditis is a non-ischemic inflammatory heart disease, which is potentially leading to severe heart failure and death ,2. CliniBlood samples were obtained from patients with clinical evidence for myocarditis n = 54), divided into two groups after endomyocardial biopsy (EMB), B19V+  and B19V-  and then compared with healthy controls . All patients underwent left ventricular EMB and histological, immunohistological and molecular workup as previously described [4, divideB19V DNA was isolated from deparaffinized myocardial tissues of patients with fatal B19V-associated myocarditis as described previously to generate transgenic mice (accession no. AY768535 and AF162273) . C57BL/6CVB3 used in this study was derived from the infectious cDNA copy of the cardiotropic Nancy strain, and virus stocks were prepared as previously described . C57BL/6Microparticles were isolated as described previously . DetailsThe details of the technique are described elsewhere  and summData are expressed as mean ± standard error of the mean (SEM). Continuous variables were tested for normal distribution with the Kolmogorov-Smirnov test and compared using a two-way ANOVA test, followed by a two-sided Bonferroni post-hoc testing. Tests for equal variance normality were performed using the Levene Median test. A p-value of <0.05 was considered statistically significant. Assumptions of normality and equal variance were automatically tested using the statistic program. Normal distribution of the parameters  was tested here using a Kolmogorov-Smirnov test. Both parameters showed normal distribution and are reported as mean +/- SD. Statistical analyses were performed using SigmaStat version 3.5. All data analyses and event classifications were performed by investigators blinded to the microparticle-status of patients and controls.The clinical and demographic data of the studied individuals are given in Subpopulations of EMPs (CD144+) were significantly different in B19V+ compared to B19V- and HCTR p<0.001, . The incPMPs (CD62P+CD42b+AnnexinV+) increased in B19V+ compared to B19V- patients and healthy controls  were increased in both, B19V+ and B19V- in contrast to healthy controls  were studied for comparison. A small increase of EMPs was detectable in CVB3-infected mice at the early stage of infection  with a rapid decline during acute infection at 8 days p.i. later at 28 days p.i . CompariPMPs measured in C57BL/6 control mice compared to transgenic B19V-NS1-mice without doxycyclin showed comparable levels . PMP of B19V-NS1 after induction and of CVB3 mice after infection did not rise significantly (not shown). MMPs and LMPs measured in C57BL/6 control mice compared to transgenic B19V-NS1-mice without doxycyclin showed about the same levels .The ratio of CD62E/CD31 EMPs reflects endothelial activation versus apoptosis. The changed patterns of EMPs in humans  or mice EMPs patterns were changed in humans with B19V myocarditis and in transgenic B19V-mice compared to healthy controls and control mice, respectively. In CVB3-infected mice, there were no or minor changes in EMPs, but increased levels of LMPs.B19V DNA is the most frequent viral genome observed in endomyocardial biopsies (EMBs) with left ventricular dysfunction ,11. PrevIn order to add plausibility, we investigated transgenic B19V mice. Also, in this B19V associated model, an increase of EMPs was detected. It was accompanied by high levels of platelet-derived PMPs and LMPs, which could reflect the response to vascular damage reflected by significantly elevated EMPs. It is known that PMPs generated from apoptotic human platelets induce human monocyte chemotaxis and polarization into resident M2 monocytes, implying that these MPs possess immunomodulating properties . MPs couIn comparison to the murine model of B19V-induced heart disease, CVB3-infected mice showed higher levels of LMPs. CVB3 is known to infect primarily cardiomyocytes, and due to extensive virus replication, a rapid cytolysis of these cells occurs. Before reaching their cardiac target cells, the cardiomyocytes, CVB3 transmigrates through the endothelium for a short period of time during viremia . The risOne limitation of the present study is the absence of coxsackievirus B infection in humans in this study. Unfortunately, human samples with CVB-myocarditis are rare. Therefore, the murine model of CVB3 myocarditis was used as it reflects human enteroviral myocarditis with regard to myocardial damage and virus-induced immune response . EndomyoOur data provide first evidence that differential endothelial microparticle changes are detected in different virus-associated heart diseases. Differences in the subtypes of MPs can be attributed to specific myocardial virus infections targeting different cell types. B19V persists in the endothelium  and, thuIt is concluded that microparticle profiles vary between different myocardial diseases and could facilitate early differential diagnosis between endothelial-cell-mediated disease due to B19V and other causes of myocarditis and pave the way to early diagnoses and potentially to early initiation of treatment.S1 Appendix(DOCX)Click here for additional data file.S1 FigHuman platelet-derived microparticles (PMPs) in patients with myocarditis divided into B19+ and B19V- patients and compared with age-matched healthy controls (HCTR). The B19V- group consisted of either no virus detection or HHV6+ and EBV+ samples. A: PMPs were significantly increased in B19V + patient samples compared to B19V- and HCTR. B19V- had increased EMP levels as well, but not significant versus HCTR. B: CD42b-AV+ PMPs represent apoptotic PMPs. Apoptotic PMPs were significantly higher detectable than activated PMPs in B19V+ and B19V-. C: CD62P+ PMPs represent activated PMPs.(TIFF)Click here for additional data file.S2 FigMonocyte-derived microparticles  and leukocyte-derived microparticles  in patients with myocarditis divided into B19V+ and B19V- patients and then compared with age-matched healthy controls (HCTR). The B19V- group consisted of either no virus detection or HHV6+ and EBV+ samples. A: MMPs were increased in both, B19V+ and B19V - in contrast to healthy controls (p<0.001 and p<0.003) but no significance between themselves. B: LMPs were significantly increased in B19V+ compared to B19V- (p<0.011) and healthy controls (p<0.004).(TIFF)Click here for additional data file.


Supplemental Digital Content is available in the text + T-cell counts in some antiretroviral therapy suppressed participants (immunodiscordance) has been related to perturbed CD4+ T-cell homeostasis and impacts clinical evolution.The failure to increase CD4+ T-cell counts (cutoff) or CD4+ T-cell increases from nadir value (ΔCD4) using supervised random forest classification of 74 immunological and clinical variables from 196 antiretroviral therapy suppressed individuals. Unsupervised clustering was performed using relevant variables identified in the supervised approach from 191 individuals.We evaluated different definitions of immunodiscordance based on CD4+ cell count 400 cells/μl performed better than any other definition in segregating immunoconcordant and immunodiscordant individuals (85% accuracy), using markers of activation, nadir and death of CD4+ T cells. Unsupervised clustering of relevant variables using this definition revealed large heterogeneity between immunodiscordant individuals and segregated participants into three distinct subgroups with distinct production, programmed cell-death protein-1 (PD-1) expression, activation and death of T cells. Surprisingly, a nonnegligible number of immunodiscordant participants (22%) showed high frequency of recent thymic emigrants and low CD4+ T-cell activation and death, very similar to immunoconcordant participants. Notably, human leukocyte antigen - antigen D related (HLA-DR) PD-1 and CD45RA expression in CD4+ T cells allowed reproducing subgroup segregation (81.4% accuracy). Despite sharp immunological differences, similar and persistently low CD4+ values were maintained in these participants over time.Cutoff definition of CD4+ T-cell count 400 cells/μl classified better immunodiscordant and immunoconcordant individuals than any ΔCD4 classification. Immunodiscordance may present several, even opposite, immunological patterns that are identified by a simple immunological follow-up. Subgroup classification may help clinicians to delineate diverse approaches that may be needed to boost CD4+ T-cell recovery.A cutoff value of CD4 Thesel counts . The subllnesses –11. Sevel counts –15.+ T-cell count under 200 cells/μl after suppressive ART is considered an immunodiscordant response, although higher cutoff values of 350 and 500 cells/μl are also commonly used in the literature [+ T-cell increases from ART initiation.Despite the clinical relevance of immunodiscordance, the lack of a consensus definition hampers the identification of immunodiscordant individuals, their clinical follow-up and ultimately the assessment of prevalence of immunodiscordance among HIV-treated individuals ,17. Undeterature ,17, as i+ T-cell homeostasis, in particular with poor thymic output, high cell turnover and sensitivity to ex-vivo cell death [+ T-cell recovery that in turn would help both to quantify the clinical impact of immunodiscordance and to explore new clinical interventions to reverse it. We approached this issue by using supervised random forest classification, a powerful machine learning algorithm characterized by high classification accuracy and the capacity to rank variables according to their importance for classification [+ T-cell count of 400 cells/μl is the best immunological cutoff for classification. Importantly, after variable selection, unsupervised hierarchical clustering showed that immunodiscordant participants could be subgrouped according to three different immunological patterns that will probably require specific clinical interventions to reverse immunodiscordance.In the absence of standard definitions, multiple analyses have explored the immunological basis for immunodiscordance, that are strongly related to CD4ll death ,17. We cfication . Our datn = 230) performed to analyze the immune status of participants with paradoxical immune response [+ and viral load (VL) were collected from medical records.We analyzed immunological data from 196 participants on suppressive ART (HIV RNA levels <50 copies/ml) for at least 2 years. All participants having the full immunological dataset were selected from a larger previous cross-sectional, descriptive and comparative study -PerCP-Cy5.5, CD3-APC-Cy7, CD4+-APC and CD8+-PE-Cy7; and HLA-DR-FITC, CD38-PerCP-Cy5.5, CD45RO-APC, CD3-APC-Cy7 and CD8+-PE-Cy7. CD4+ and CD8+ T-cell death data from fresh peripheral blood mononuclear cells and data from soluble CD14 in plasma samples had been also previously reported [n = 50)  or based on the increase in CD4+ cell counts from recorded CD4+ nadir values to CD4+ T-cell counts at the time of visit . Participants below or above the indicated values were considered as discordant (unfavorable immunologic response) or concordant (favorable immunologic response), respectively.Participants were grouped as immunodiscordant or immunoconcordant based on either the CD4Supervised random forest classification was performed using all sociodemographical, clinical and immunological variables collected as predictors using R Statistical Software (Free Software Foundation)  using thUsing the variables with the highest contribution to sample discrimination (according to Gini's index), unsupervised hierarchical clustering in conjunction with heatmaps were performed to find patterns in our data in an unbiased fashion. Each variable was centered with respect to the mean and scaled by the SD. The distance between samples and variables was measured by means of Euclidean distance, and hierarchical clustering was performed using the Ward method. Principal component analysis (PCA) was performed to visualize the behavior of the cluster groups regarding different cellular processes.U with permutation  or signed-rank test . Discrete variables were described as percentages and compared using the Fisher's exact test. Multiple comparisons were adjusted for false discovery rate. Statistical analyses were also performed using R Software version 3.0.2 [Continuous variables were expressed as the median (interquartile range) and compared using Kruskal–Wallis (for multiple comparisons), Mann–Whitney on 3.0.2  with two+ T-cell recovery defined by CD4+ T-cell counts at the sampling time. However, the full set of immunological parameters for random forest analysis was available for 196 participants . The main characteristics of the analyzed cohort are shown in Table + T-cell count strata showed similar sex representation, ART composition, hepatitis C virus (HCV) or hepatitis B virus (HBV) coinfection, time from diagnosis and time on ART; however, participants with poorer CD4+ T-cell recovery tended to be older . In addition, differences in CD4+ T-cell counts and CD4/CD8 ratio, individuals with poorer CD4+ T-cell recovery showed lower nadir CD4+ T-cell count values and lower CD8+ T-cell counts .Participants included in this analysis have been previously characterized ,20. The +FOXP3+ cells), proliferation (Ki67 expression), differentiation and immunosenescence [+ T-cell stratification of this subset of individuals is also shown in Supplementary Fig. 1.A wider range of immunological data, including Treg (CD25nescence  were ava+ T-cell counts by establishing different cutoffs of immunodiscordance (CD4+ T-cell counts from 200 to 600 cells/μl) or based on the increase of CD4+ T-cell counts from nadir values . For cutoff values, the best classification was achieved using a value of 400 cells/μl that provided a specificity of 83% and a sensitivity of 88%, resulting in an OOB error of 15%. For ΔCD4 values, the best classifier was 350 cells/μl; although this classification showed a 84% of specificity, the sensitivity was significantly lower (76%), resulting in a OOB error of 20%, failing to achieve better classification than the cutoff values  than for ΔCD4 value 350 cells/μl (AUC 84%).To define the main immunological features that distinguish immunodiscordant and immunoconcordant participants, we analyzed the performance of different definitions of immunodiscordance using a random forest approach in which 74 immunological and clinical parameters were analyzed (Supplementary Table 1). Definitions were based on the CD4+ T-cell counts analyzed. Restrictive definition of discordance (cutoffs CD4+ T-cell count 200 cells/μl) showed relevant contributions of nadir, recent thymic emigrants  and PD-1 expression, activation (CD38) and cell death in CD4+ T cells to immunodiscordance. However, at higher cutoff values, the parameters related to activation (HLA-DR expression) and death of CD4+ T cells as well as nadir increased their relevance for classification. Activation and death of CD4+ T cells also showed importance for classification in ΔCD4 classification, although the absolute CD8+ T-cell count was unexpectedly relevant for lower and intermediate ΔCD4 values, and the coexpression of CD95 and HLA-DR in CD4+ T cells were highly relevant for ΔCD4 values above 250 cells/μl. In summary, immunological parameters seem to better classify participants according to the value of circulating CD4+ T cells achieved, rather than to the global increase of CD4+ T-cell counts.The analysis of relevant variables that might contribute to the immunodiscordant phenotype in each definition is shown in Fig. + T-cell count 400 cells/μl) random forest approach (highest Gini score) were used in an unsupervised hierarchical clustering analysis  CD4+ T cells, and a larger cluster including all concordant individuals with intermingled immunodiscordant participants. This cluster can be subdivided in two groups, one of them (Group II) encompassing mostly immunodiscordant participants and the other one (Group III) that included mostly immunoconcordant individuals. Main differences between these latter groups were lower naïve CD4+ T cells, higher activation and death of CD4+ T cells in Group II compared with Group III. A similar cluster structure was obtained using parameters of importance for the ΔCD4 350 cells/μl classification .Variables of importance obtained from the optimal cutoff value , with a low segregation performance for the frequency of naïve cells or the differentiation status of T cells. In contrast, the frequency of Tregs and specifically the proliferation of this subset appears as a major contributor to segregation.We further explored whether information on T-cell differentiation, proliferation and Treg frequency  could modify the classification of immunodiscordant individuals. The analysis of variables of importance and the resulting heatmap for the cutoff CD4+ T-cell counts or CD4+ T-cells increases were observed among immunoconcordant subgroups. A detailed analysis of immunological features of each subset confirmed previous data associated with poor CD4+ T-cell recovery but also revealed surprising features in some groups. Among immunoconcordant participants, Group C-II showed lower CD4/CD8 ratio, lower RTE frequency but higher activation in both CD4+ and CD8+ T cells compared with Group C-III. Among immunodiscordant groups, Group D-I showed the expected profile with lowest RTE frequency, highest levels of CD4+ T-cell activation (HLA-DR+), PD-1 expression and cell death, both caspase-dependent or caspase-independent . Immunodiscordant individuals clustered in Group D-II showed an intermediate pattern and were characterized by low RTE frequency, intermediate cell death and a relatively low activation in the CD4+ T-cell compartment . Importantly, from a clinical point of view, a simple combination of three parameters measuring CD45RA, HLA-DR and PD-1 expression in CD4+ T cells is able to classify immunodiscordant participants in Groups D-I, D-II and D-III with an overall 81.4% accuracy, suggesting that a much simpler strategy using a single panel of flow cytometry staining could be easily implemented in the routine clinical follow-up . In summary, our data provide immunological evidence for a subclassification of HIV-infected individuals (both immunodiscordant and immunoconcordant) and underscores the existence of a subset of immunodiscordant individuals with a paradoxical immune profile comparable with immunoconcordant , suggesting that the immunodiscordant phenotype defined by CD4+ T-cell counts below 400 cells/μl may accommodate very different, even opposite immunological profiles.To evaluate the immunological and clinical differences among clustered individuals, we defined and compared immunoconcordant and immunodiscordant groups in each cluster Fig. . Data suent Fig. . ClusterIII Fig. . Importaent Fig. . The ful+ T-cell counts and VL data from sampling time (2007–2008) to April 2015. Follow-up criteria were similar to inclusion criteria; individuals analyzed showed undetectable VL  and did not receive pegylated Interferon (PEG-IFN) or chemotherapy treatment. Data were available for 67 immunodiscordant individuals . Collected data showed a median follow-up time of 6.4 years and were similar for all three subgroups , 8.3 (0.3–23.0) and 2.6 (−4.3 to 21.7) cells/year for Groups D-I, D-II and D-III, respectively. Taking all together, our data demonstrate that a similar blunted CD4+ T-cell recovery may be observed in different immunological scenarios, suggesting that different causes could be behind a poor response to ART.To address the clinical evolution of the different subgroups of participants, we collected CD4+ T-cell immune activation, immunological checkpoint molecules and CD4+ T-cell homeostasis to the pathogenesis of HIV infection [Few reports have attempted a comprehensive analysis of the contribution of CD8nfection –28. In t+ T-cell recovery, is a relevant clinical concern. Immunodiscordance is associated with adverse events occurring before ART initiation such as rapid progression [The lack of proper immune recovery in immunodiscordant individuals, defined by a poor CD4gression  or late gression , two phegression ,32. Furtgression  and seemgression .+ T-cell count 400 cells/μl cutoff segregates better immunological profiles of immunoconcordant and immunodiscordant individuals, highlighting differences principally in CD4+ T-cell death and activation between groups. In several studies, a cutoff of CD4+ T-cell count 200 cells/μl has been defined as a critical threshold for the risk of AIDS events or death [+ T-cell count gradient in ART-treated participants, with clear risk for counts below 200 cells/μl but still some benefit for those individuals with a CD4+ T-cell count at least 500 cells/μl [+ T cells in participants with lower CD4+ T-cell counts might explain this increased risk. In our analysis, the segregation of immunoconcordant and immunodiscordant individuals is better achieved by cutoff definitions rather than by increases in CD4+ T cells. This is probably associated with the structure of our cohort in which participants starting therapy a relatively high CD4+ T-cell count may show low cell increase, despite showing a fully immunoconcordant phenotype [+ T-cell counts, or including a low nadir count as inclusion criteria would make both definitions (cutoff and increase) much more similar.Our results reveal that the CD4or death –36. Howecells/μl . The perhenotype . Adjusti+ T cells, platelets, hematies, leukocytes and lymphocytes . However, the well described association of immunological alterations and CD4+ T-cell counts with clinical evolution [+ T-cell recovery and early CD4+ T-cell redistribution events.Our data also underscore the previously unnoticed existence of immunodiscordant participants with different immunological pattern. In fact, our data indicate that only a subgroup of immunodiscordant individuals  fulfill all the previously defined features of these participants, that is low RTE frequency, high activation/cell death and high frequency and turnover of Treg cells ,37–42. Ovolution ,25 might+CD45RA+, CD4+HLA-DR+ and CD4+PD-1+ cells) with an overall 81% accuracy. ART intensification or immunomodulating approaches have been already tested to improve CD4+ T-cell recovery with limited effects [Classifying immunodiscordance could be particularly relevant in clinical settings, as distinct clinical approaches could be needed to reverse immunodiscordance in these subgroups. Noteworthy, such subclassification can be easily achieved with only three parameters  may help to segregate participants, especially those with immunodiscordant responses to optimize treatments aimed to increase their CD4+ T-cell counts. However, our data also suggest that the quality of CD4+ T cells is relevant. Therefore, increasing CD4+ T-cell counts may not be sufficient, in the absence of functional improvement, to impact clinical output as shown for IL-2 treatment [In conclusion, our data show that complementing absolute cell count follow-up with measures of naive T-cell production and immune activation  and by amfAR grant 109316 with support from FAIR. J.B. and C.C. are researchers from Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol supported by the ISCIII and the Health Department of the Catalan Government . M.M. is supported by BP-DGR AGAUR Postdoctoral Fellowship.Author's contributions: M.M., J.C. and C.C. designed and performed immunophenotyping, cell death and ELISA assays. J.P.S., D.O., V.U. and J.V.F. performed statistical analyses. E.N., J.P. and B.C. recruited participants and collected clinical data. M.M., R.P., E.N., B.C. and J.B. interpreted the data. J.P.S., M.M. and J.B. coordinated the work and wrote the manuscript. All authors read and approved the final version of the manuscript.There are no conflicts of interest.


PARVA oncogene and inhibits its expression, thus preventing the progression of melanoma towards the malignant phenotype. Indeed, edited miR-378a-3p but not its WT form inhibits melanoma metastasis in vivo. These results further emphasize the role of RNA editing in melanoma progression.Previously we have reported that metastatic melanoma cell lines and tumor specimens have reduced expression of ADAR1 and consequently are impaired in their ability to perform A-to-I microRNA (miRNA) editing. The effects of A-to-I miRNAs editing on melanoma growth and metastasis are yet to be determined. Here we report that miR-378a–3p is undergoing A-to-I editing only in the non-metastatic but not in metastatic melanoma cells. The function of the edited form is different from its wild-type counterpart. The edited form of miR-378a-3p preferentially binds to the 3′-UTR of the  PARVA oncogene, inhibiting its expression and preventing melanoma progression and metastasis.In melanoma, reduced ADAR1 impairs A-to-I microRNA editing. Here, the authors show that miR-378a-3p undergoes this editing in non-metastatic cells and the edited form of miR-378a-3p binds to the  Previously we have identified the CREB transcription factor as a “master switch” in melanoma metastasis by regulating genes involved in survival, angiogenesis and invasion5. CREB also regulates the expression of other important transcription factor involved in melanoma progression such as AP-2α and MITF7. Recently, we reported that CREB negatively regulates the expression of the ADAR1 enzyme, which is involved in A-to-I RNA editing of mRNAs and microRNAS (miRNAs)9. Indeed, we reported that metastatic melanoma cell lines and tumor specimens have reduced expression of ADAR1 and consequently are deficient in their ability to perform miRNAs A-to-I editing. We identified three miRNAs  to undergone A-to-I editing only in the non-metastatic but not in the metastatic melanoma cells that lack ADAR1 expression8. A-to-I miRNAs editing can affect melanoma progression. For example, the function of miR-455-5p WT is different from its edited counterpart as they recognize different set of genes. Indeed, miR-455-5p WT but not the edited form specifically targets the tumor suppressor gene CPEB1, thus contributing to melanoma metastasis.Melanoma is the most aggressive type of skin cancer with an estimated 87,000 annual new cases in the United States, and close to 9,700 will result in death mostly due to metastasisPARVA thus preventing the progression of melanoma towards the malignant phenotype. We demonstrate that A-to-I editing in regions other than the canonical seed regions can affect miR-378a-3p binding to the 3′-UTR of PARVA. PARVA is over expressed in metastatic melanoma cell lines and tumor specimens which have reduced expression of ADAR1. Alpha parvin, also known as actopaxin/CH-ILKB, is a member of the ILK, PINCH and parvin complex involved in the integrin-mediated signaling11. Its oncogenic roles have been previously described in breast cancer cells invasion12 and colorectal tumor progression13. In addition, α-parvin promotes lung adenocarcinoma by regulating the ILK signaling pathway14. Regulation of ILK pathway will result in regulation of AKT and GSK3-β14. However, the role of PARVA in melanoma has not been described. Here, we report on a novel epigenetic mechanism regulating the expression of PARVA, and thus affecting melanoma progression.Here we focused our study on the relevance of miR-378a-3p editing on melanoma progression. We found that the edited form (expressed in non-metastatic cells) preferentially targets the oncogene ADAR1 expression is reduced in metastatic melanoma cell lines and in clinical metastatic melanoma specimens9. Loss of ADAR1 directly contributes to melanoma growth and metastasis, by affecting A-to-I miRNAs editing8. We showed that the function of the edited miR-455-5p (expressed in non-metastatic melanoma cells) is different from its WT counterpart (expressed in metastatic melanoma cells lacking ADAR1). Indeed, WT miR-455-5p preferentially binds and inhibits the expression of the tumor suppressor gene CPEB1, thus contributing to melanoma metastasis. One of the three previously reported miRNAs undergoing A-to-I editing only in non-metastatic but not in the metastatic melanoma cells is miR-378a-3p  after silencing ADAR18, to prevent endogenous editing. These cells were subjected to gene expression profiling. The generated heat map depicted in Fig. OAS1, NF2, C21orf33, IFI27, IFI6, PARVA, PARP9, YOD1, STAT1, SAMHD1). Binding prediction analysis demonstrated the predicted binding site of edited or wild-type miR-378a-3p on the 3′-UTR of PARVA  and in two highly metastatic melanoma cell lines (TXM-18 and C8161). Alpha-parvin expression is higher in metastatic melanoma cells when compared to melanocytes and early primary low-metastatic cells  and silenced its expression in SB2 cells  and tested for α-parvin expression. Figure ADAR1 and α-parvin expression. Silencing ADAR1 in SB2 cells resulted with a 2.4-fold increase in α-parvin expression while overexpression of ADAR1 in C8161 cells resulted with 0.5-fold reduction in α-parvin expression. To further confirm the regulation of PARVA by the edited form of miR-378a-3p, we transfected the WT and the edited forms into the C8161 and SB2 parental cells. α-parvin expression was reduced 0.5-fold in both cell lines by the edited miR-378a-3p but not by the WT or miR-378a-3p antago-miR . In contrast, overexpression of PARVA in SB2 cells resulted with a significant increase in tumor growth . To study the role of PARVA on in vivo metastasis, C8161 KD- PARVA cells were injected intravenously for experimental lung metastasis assay. Silencing of PARVA in C8161 cells significantly reduced their ability to form lung metastases . Taken together, these data validate the oncogenic role of PARVA in melanoma. We did observe a repeated trend that treatment with the edited miRNA, reduced the number of lung metastasis although not statistically significant due to the relatively small number of animal used, which involved a complicated and highly expensive injections of encapsulated miRNAs. The results obtained with WT miR-378a-3p is almost identical to the results when the mice were injected with the scramble control.To study the role of In the last set of experiments, we wanted to elucidate the role of WT mir-378a-3p in comparison to its edited counterpart on melanoma metastasis in vivo. To that end, C8161-luciferase labelled cells were injected intravenously into nude mice. Three days later, the mice were treated 3 times weekly by intra-peritoneum injection with neutral nanoliposomes (DOPC) encapsulated with different miR-378a-3p sequences . Mice treated with the edited miR-378a-3p showed a significant reduction in their ability to form lung metastases when compared to mice treated with scrambled control or liposomes containing edited antago-miR at 36 days after injections Fig. .Representative mouse from each group is shown in Fig. 8. Indeed, re-expression of ADAR1 in metastatic melanoma cells inhibited their tumor growth and metastatic capabilities. More importantly, we previously reported that the function of the edited miR-455-5p is different from its WT counterpart8. For example, only the WT form of miR-455-5p binds and inhibits the expression of the tumor suppressor gene CPEB1, thus contributing to melanoma metastasis.Previously we have reported that metastatic melanoma cells have reduced expression of the ADAR1 enzyme. Consequently, metastatic melanoma cells are impaired in their ability to perform A-to-I editing on microRNAs. We have identified three miRNAs: miR-455-5p, miR-324-5p and miR-378a-3p undergoing A-to-I editing only in the non-metastatic cells that express ADAR1 but not in metastatic melanoma cells lacking ADAR1 expression15. Indeed, only the edited form (found in non-metastatic cells) preferentially binds and inhibits the expression of the PARVA oncogene. These results demonstrate a previously unrecognized role of miRNA editing (epigenetic mechanism) in melanoma progression. In this model  melanoma cells, and accumulation of the WT form in the metastatic cells lacking ADAR1 expression. The edited form of miR-378a-3p preferentially binds and inhibits the expression of the PARVA oncogene, thus preventing the progression of melanoma cells towards the malignant phenotype. It should be noted, however, that the PARVA mRNA is not the only target for the edited form of miR-378a-3p. Indeed, we have identified several target genes that their regulations are influenced by miRs editing contributing to melanoma growth and metastasis. This concept is supported by our in vivo studies, demonstrating that a delivery of the edited but not the WT form of miR-378a-3p by means of nanoparticles inhibited the metastatic capabilities of melanoma cells. Other studies have reported that miR-378a-3p plays a role in diverse cancers19. Hyun et al. showed that miR-378a-3p reduced the activation of hepatic stellate cells by binding to the 3′-UTR of Gli320, while in lung adenocarcinoma, miR-378a-3p reversed the chemo-resistance to cisplatin by targeting clusterin17. These studies, however, did not distinguish between the edited and non-edited forms of miR-378a-3p.In the present study, we sought to determine the relevance of A-to-I editing on miR-378a-3p on melanoma progression. Unlike the case with miR-455-5p, in which we identified two edited sites within the seed sequences, the A-to-I editing in miR-378a-3p occurs at position 18, which is considered a non-canonical seed region. Nevertheless, our data support the notion that this editing event could cause a differential gene regulation when compared to its WT form, as it was recently suggestedPARVA plays a role in the degradation of extracellular matrix leading to cell invasion via regulation of Rho GTPase signaling in breast cancer21, and in hepatocellular carcinoma (HCC) progression and metastasis22. The current study identified the PARVA gene to be a direct target for miR-378a-3p (its edited form) and outlined the contribution of PARVA to melanoma growth and metastasis. Overall, these results provide a key missing link in the mechanistic epigenetic pathway of A-to-I miRNAs editing in the acquisition of the melanoma metastatic phenotype. At this stage, further clinical studies to determine the relevance of micro-RNA editing in human melanoma progression must be conducted.Previous publications have shown that 23, C8161 and TXM18 are highly metastatic melanoma cell lines that were originated from patient’s lymph nodes24. All cell lines used in our studies were established at the University of Texas M. D. Anderson Cancer Center and tested before their usage for authentication by DNA fingerprinting using the short tandem repeat method. Cell lines were routinely tested to confirm the absence of Mycoplasma.Human melanoma cell lines were culture in MEM media supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% non-essential amino acids, and 1% antibiotic antimycotic. 293T cells (Invitrogen) were used to generate lentivirus for overexpression or knockdown of the genes of interest. These cells were grown in DMEM supplemented with 10% FBS. Primary normal human melanocytes cells were obtained from Promo Cell (Cat. # C-12403). The following human melanoma cells were used: SB2 cells poorly metastatic that was originated from early primary cutaneous melanoma patientADAR1 cells transfected using RNA iMAX transfection reagent in Opti-MEM I Gluta MAX reduced serum media with either wild-type or edited miR-378a-3p, in triplicates. Cells were incubated overnight and fresh media was added. After 24 h, the media was removed and RNA was purified using RNA mini-prep (Zymo Research). Gene Chip Human Genome U133 plus 2.0 array analysis was performed. Gene expression profile was determined using Nexus and uploaded to the Gene Expression Omnibus (GEO) repository with the accession number GSE107088.SB2 KD-Protein samples from human melanoma cells were subjected to SDS-PAGE and Western Blot analysis. Briefly, cells were lysed using RIPA B buffer containing proteases and phosphatases inhibitors (ROCHE) to extract the proteins. Protein concentration was determined using Bio-Rad protein assay (Bio-Rad) and the Epoch spectrophotometer (BioTek). SDS-poly-acrylamide gels were used to separate the proteins by molecular weight. Western blots were performed to detect the following proteins of interest: ADAR1 , α-tubulin , actin , c-Jun Ser 63/73 , α-parvin , AKT Ser 473  and MMP2 . Supplementary Fig. Bio-coat growth factor reduced, matrigel invasion chambers with 8.0 µm membrane (Corning) were used to perform cell invasion assay. Either 20,000 (C8161) or 50,000 (SB2) cells were plated on the top chamber in serum-free media. The bottom chamber contained complete media supplemented with 10% FBS. After 16 h, the top cells were wiped out with a cotton tip and the membranes were stain with HEMA 3 (Fisher Scientific). Membranes were cut from the chamber and mounted on slides. The numbers of invaded cells were counted under the microscope. Triplicates of each condition were performed.Cells were plated on 96-wells plates at concentrations of 2000 or 5000 cells per well. MTT assay were performed for 5 consecutive days. MTT was dissolved at a concentration of 5 µg/mL in sterile PBS. To each well, 20 µL of MTT were added and incubated for 2 h. After 2 h media was removed and 200 µL of DMSO was added into each well. O.D. was measured using Epoch (Bio Tek) spectrophotometer. Each sample has 12 replicas.ADAR1 cells were infected with Mission PARVA 3′-UTR Lenti GoClone, Mission PARVA 3′-UTR-mutated Lenti GoClone or non-target control1 (Sigma) and selected with puromycin. Micro-RNAs against WT miR-378a-3p, edited miR-378a-3p or miR-378a-3p antago-miR (Ambion) were transfected using RNA iMAX transfection reagent (Invitrogen). Luciferase Assay (Promega) was used to determine the firefly luciferase activity and each sample was normalized with its non-targeting control. Each sample has six biological replicas.SB2 KD-PARVA was cloned from A375 melanoma cells cDNA into pCDH-CMV-MCS-EF1-puro expression vector  with the following primers. PARVA-XbaI-F: 5′-GCTCTAGAATGGCCACCTCCCCGCAGAAGTC-3′ and PARVA-NotI-R: 5′-ATAAGAATGCGGCCGCTCACTCCACGTTACGGTACTTGG-3′. All constructs were fully sequenced in both direction.PARVA mutant luciferase analysis, the two binding site for miR-378a-3 promoter region were deleted: 5′-CCCAAGAGTTCTTGCTGTTGGCGTACTGGACCCTCCTCCGAACTGCCTTACCC TGCTTATTCCTGTCTCTTGCACTGTGCTCTCCCACAAGT CCAGCTGCAACCCAGAGATAGTGGAAACTGAAATTAGGAAGGAAATCATCAATAA CTCAGTGGGCTGACCCATCC CTCCCAGGCGCTGGGGACCAACCTAGCAATGAAGGTTGGGAAGGT TGTTCCCTTCCCGGTGCCAGGTCCAGATTTCC CTCCATGATTTGGGAACCAGC TTAGGCAAAAGAGTCCCCACAAGATGAAAATAAAGATCCTAGTTACCAT TCAAAGGATGCT-3′ (highlighted “bold” area shows the binding region with the deleted region in grey font). Light-grey fonts indicate the deleted region. The fragment was digested with HindIII and SepI and ligated into the pGL3-basic vector (Promega), digested with KpnI and XhoI and ligated into the pGL3-basic vector (Promega). Site-directed mutagenesis of the PARVA sites (deletion), was carried out using the QuikChange II XL site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions.For the PARVA expression or empty vector and with pGreenFire positive control vector containing GFP and Luciferase (TR011PA-1. SBI). After transduction, cells were selected by puromicin and sorted after 10 days by flow cytometry using GFP expression. C8161 cells were stably transfected with shRNA for PARVA KD. PARVA expression levels were subjected to western blot analyses to verify protein overexpression or KD.SB-2 cells were stably transduced with Lentivirus-based PARVA and miR-378a- 3p editing. First, the in vivo effect of PARVA regulation was studied. 1 × 106 of C8161 NT-control or KD PARVA and SB2 EV-control or OE-PARVA were subcutaneously injected. Tumor volume was recorded three times a week using a caliper. Tumor volume was determined by (wide2) × (length) / 2.Athymic female nude mice Nu/Nu with an age range of 8–12 weeks were used to determine the in vivo effect of PARVA manipulated cells was determined using 1.0 × 106 C8161 NT-control or KD-PARVA cells by tail vein intra-venous (i.v.) injection. After i.v., we waited 3 days before starting treatment with encapsulated miRs including WT, edited or antagomir. Mice were monitored daily for 36 days. Lungs were collected, fixed in Bouin fixative  and lung colonies were counted.The metastatic potential of the 25. After addition of Tween-20, the mixture was frozen in an acetone-dry ice bath and dried. Liposomes were reconstituted by adding 200 µL water into the vial and mixing briefly.The miRNA oligos were incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine  by lyophilization. Briefly, 5 µg RNA oligo and DOPC were mixed in the presence of excess t-butanol at a ratio of 1:10 (w/w) as described previously5 luciferase labelled C8161 cells.DOPC liposomes  were injected via intra-peritoneum three times a week, starting 3 days after i.v. tail vein injections of 2.0 × 10In vivo imaging was performed to monitor mice lung tumor burden. Mice were anesthetized using 2% inhaled isofluorane. Bioluminescence imaging were obtained, using IVIS 100 series system (Xenogen), 12 min after i.p. injection of 0.2 mL D-luciferin . After killing, lungs were fixed in Bouin solution and the number of lung colonies were counted using a dissecting microscope (Nikon). Statistical significances were analyzed by the Tukey’s multiple comparisons test. All animal experiments were conducted in accordance with American Association for Laboratory Animal Science regulations and the approval of The University of Texas MD Anderson Cancer Center Institutional Animal Care & Use Committee.PARVA for the TCGA Skin Cutaneous Melanoma cohort is the RNASeqv2 level 3 data sets downloaded from Broad GDAC Firehose http://gdac.broadinstitute.org/. The clinical information for this cohort was obtained from cBioPortal .The mRNA expression of PARVA mRNA were significant in the univariate analysis and a multivariate Cox proportional hazard model was fitted to this data. PARVA expression in metastatic melanomas is an independent poor prognostic marker.Survival analysis was performed in R (version 3.2.5). The relationship between overall survival and covariates  was examined using a Cox proportional hazard model. Age at diagnosis, stage and PARVA levels, we used the log-rank test to find the point (cut-off) with the most significant  split into high and low mRNA level groups. The Kaplan-Meier plots were generated for these cutoffs. The median survival time of each group is presented in brackets. The numbers of patients at risk in low and high mRNA groups at different time points are presented at the bottom of the graph. For overall survival, the cut-off to optimally separate the patients in high, low  group was 0.63.In order to visualize survival differences between groups of patients based on PARVA expression in metastatic melanomas and clinical information for the cohort.Supplementary Table https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE107088].The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. Any other relevant data that support the findings of this study are available from the corresponding author upon reasonable request. The normalized gene expression profile data generated from Gene Chip Human Genome U133 plus 2.0 array analysis supporting the opposite effect mediated by the two forms of miR-378a-3p (Fig. Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


Betaherpesvirinae.Quantitative PCR is a diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of human herpesvirus 6A/B (HHV-6A/B) is important for detection of viral reactivation and inherited chromosomally integrated HHV-6A/B in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies Inc. Eleven of 17 (65%) HHV-6A/B candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1,254 bp and 983 bp, respectively. The average copy number measured was between 5 and 10 times the number of copies of the rest of the genome. We also report the first interspecies recombinant HHV-6A/B strain with a HHV-6A backbone and a >5.5-kb region from HHV-6B, from U41 to U43, that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at U1/U2, U87, and U89, as well as deletion in the U12-to-U24 region and the U94/U95 genes. HHV-6A/B strains derived from cord blood mononuclear cells from different laboratories on different continents with fewer passages revealed no copy number differences throughout the viral genome. These data indicate that large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within the IMPORTANCE Anything in science that needs to be quantitated requires a standard unit of measurement. This includes viruses, for which quantitation increasingly determines definitions of pathology and guidelines for treatment. However, the act of making standard or reference material in virology can alter its very accuracy through genomic duplications, insertions, and rearrangements. We used deep sequencing to examine candidate reference strains for HHV-6, a ubiquitous human virus that can reactivate in the immunocompromised population and is integrated into the human genome in every cell of the body for 1% of people worldwide. We found large tandem repeats in the origin of replication for both HHV-6A and HHV-6B that are selected for in culture. We also found the first interspecies recombinant between HHV-6A and HHV-6B, a phenomenon that is well known in alphaherpesviruses but to date has not been seen in betaherpesviruses. These data critically inform HHV-6A/B biology and the standard selection process. Human herpesvirus 6B (HHV-6B) is a ubiquitous human virus with human exposure levels of >90% by the age of 2 years as measured by serological assays performed worldwide , 2. Both7–Several prototypic strains of HHV-6A/B have been identified and utilized, including GS, U1102, SIE, LHV, Z29, and HST –16. Unti–Accurate and sensitive real-time PCR assays that detect and quantify HHV-6A/B are critical to diagnosis and monitoring of the variety of manifestations of HHV-6A/B-associated disease. Recently, several quantitative cutoffs have been proposed that are associated with end-organ disease or iciHHV-6A/B status –23. A re–26–in vitro . These strains had tandem repeats that were similar in size at the origin of replication present in the GS strain obtained from the HHV-6 Foundation and Z29 strains obtained from the NIH AIDS repository . HoweverPrevious analysis of 125 HHV-6B genomes obtained from clinical specimens revealed no tandem repeats across the origin of replication . To confWhole-genome sequencing of the DA strain revealed a hybrid genome indicative of interspecies recombination between HHV-6A and HHV-6B strains. The DA strain genome overall showed closer sequence identity to HHV-6A than to HHV-6B strains but included an oriLyt repeat that measured the exact length of the Z29 repeat at 983 bp . The DA NC_001664) that were detected by six of seven recombination analysis programs  (NC_000898) between the 5′ end of U41 and the 3′ end of U43 (bp 68535 to 73824 of NC_000898) (NC_000898) in KC465951) between genes U3 and U41, U44 and U85, and U94 and U100 outside the repeat regions and the oriLyt repeat.Analysis of the DA strain sequence yielded consensus recombination breakpoints at bp 67314 and bp 72667 of the HHV-6A U1102 reference genome (nd 3Seq) . Reads m_000898) . Only a _000898) . These sKJ123690.1; 99.2% pairwise nucleotide identity). Both isolates CO4 and CO7 had 60% decreased coverage in a 13.5-kb region from U12 to U24 relative to the CO1 to CO3 isolates (NC_001664)  . No otheTo find the best standard for HHV-6A/B clinical testing and to better understand the source of the origin tandem repeat, we sequenced four HHV-6B strains and one HHV-6A strain that were isolated from phytohemagglutinin (PHA)-stimulated cord blood mononuclear cells (CBMC). Four HHV-6B strains  were isolated from Japanese exanthem subitum patients, one HHV-6B strain (MAR) was isolated from an asymptomatic French child, and one HHV-6A strain (SIE) was isolated from an Ivory Coast patient with adult T-cell leukemia –39. Inte–We showed the presence of copy number heterogeneity at multiple genomic loci across HHV-6A and HHV-6B culture isolates that are used as reference material for clinical assay development and normalization, as well as basic-science virology work. The most prominent copy number difference was a large tandem repeat in the origin of replication that was present in both HHV-6A and HHV-6B strains. The presence of large copy number differences in HHV-6A and HHV-6B strains was associated with passaging in immortalized cell lines, as strains passaged in primary cell lines and cord blood mononuclear cells did not carry genomic duplications and deletions and early-passage virus contained fewer tandem repeats than late-passage virus.in vitro and that tandem HHV-6B origin elements were more efficiently replicated in vitro  in the HHV-6B Z29 ABI reference material and CO4-7 strains are notable, as none of these genes have been shown to be essential for HHV-6A/B replication , 42, 43.in vitro . The bouin vitro . HHV-6A in vitro . Replicain vitro  gave the same rearrangement. As commercial material, its passage history is not as completely clear. Although not previously formally tested in HHV-6A/B, the helicase-primase complex (containing U74 and U77 in HHV-6A/B) is essential for HSV and HCMV replication, as is the HSV UL9 gene, encoding the homolog of the HHV-6A/B U73 origin-binding protein . The HSVin vitro . HHV-6A/in vitro . The HCMin vitro .Interestingly, no low-passage-number HHV-6A/B isolate grown in cord blood mononuclear cells showed tandem repeats, despite growing to high titer. These low-passage-number cord blood isolates may provide the best material for HHV-6A/B standard development. Instead, the isolates showed direct-repeat copy numbers that were approximately 60 to 70% that of the unique region. This copy number difference was present in the majority of strains sequenced in this study and suggests that the majority of HHV-6A/B strains sequenced contained only one direct-repeat region repeat, which has previously been seen in HHV-6A/B replication , 48, 49.in vitro, before the discovery of clinical strain recombinants, which are widespread  T-antigen-immortalized cell lines , 34. WhiHHV-6A/B culture isolates were obtained from the HHV-6 Foundation. The original HHV-6A isolate GS was first isolated at the National Cancer Institute (NCI), NIH, in 1986 from an AIDS patient . The HHVhttp://jgi.doe.gov/data-and-tools/bbtools/) and de novo assembled using SPAdes (NC_001664) and HHV-6B (NC_000898) genomes and visualized using Geneious v9.1. Read mapping for copy number analysis was performed against the HHV-6A (NC_001664) and HHV-6B (NC_000898) reference genomes, using the Geneious read mapper with 10% allowed gaps per read, word length of 18, and 20% maximum mismatches per read and with structural variant, insertion, and gap finding allowed.DNA was extracted from culture isolates using the Zymo viral DNA kit. DNA-sequencing libraries were prepared from 50 ng of genomic DNA using quarter volumes of the Kapa HyperPrep kit with 7 min of fragmentation time and 12 cycles of dual-indexed TruSeq adapter PCR . The libg SPAdes , and conQuantitative PCR to estimate copy numbers between the origin of replication and the HHV-6A/B U32 locus was performed in 20-μl reaction mixtures using the SsoAdvanced Universal SYBR green SuperMix (Bio-Rad). Three 10-fold dilutions of DNA template from HHV-6A/B strains were tested using quantU32 F-R and species-specific origin primers  with cycPCR across the origin tandem repeat was performed using 1 ng of template genomic DNA in 20-μl total volume reaction mixtures using 10 pmol of each primer and the NEB HiFi Q5 DNA polymerase according to the manufacturer's instructions. PCR primer sequences are listed in NC_000898) using the LASTZ and Geneious aligners to screen for origin-containing reads . Amplification-free tagmented libraries were run according to Oxford Nanopore protocols v1.3.24 on a singular Mk1 (R9.4) FLO-MIN106 flow cell. Nanopore reads were mapped to the HHV-6B Z29 reference genome  and associated with NCBI BioProject 338014.The sequences from this study are available in NCBI GenBank (accession numbers