Caenorhabditis eleganslet-7 miRNA promotes the juvenile-to-adult transition through repression of LIN41 alone, thereby activating four LIN41 targets that function in pairs to execute distinct developmental functions.Although miRNAs may act through regulation of numerous targets, this study shows that the  let-7 miRNA temporally regulates J/A transition events in nematodes and mammals. Here, we uncover the targets and downstream pathways through which Caenorhabditis elegans let-7 controls male and female sexual organ morphogenesis and skin progenitor cell fates. We find that let-7 directs all three processes by silencing a single target, the post-transcriptional regulator lin-41. In turn, the RNA-binding protein LIN41/TRIM71 regulates these processes by silencing only four target mRNAs. Thus, by silencing LIN41, let-7 activates LIN-29a and MAB-10  to terminate progenitor cell self-renewal and to promote vulval integrity. By contrast, let-7 promotes development of the male sexual organ by up-regulating DMD-3 and MAB-3, two Doublesex/MAB-3 domain–containing transcription factors. Our results provide mechanistic insight into how a linear chain of post-transcriptional regulators diverges in the control of a small set of transcriptional regulators to achieve a coordinated J/A transition.The juvenile-to-adult (J/A) transition, or puberty, is a period of extensive changes of animal body morphology and function. The onset of puberty is genetically controlled, and the  These so-called seam cells divide asymmetrically once in each larval stage, but exit the cell cycle during transition into adulthood  To understand how the down-regulation of LIN41 triggers the transition to adulthood, we characterized LIN41 targets. Although LIN41 proteins can function as E3 ubiquitin ligases and structural proteins  animals. These worms harbor a G-to-A point mutation in the let-7 seed sequence that abrogates let-7 activity at 25°C  for let-7 point mutation. Second, we tested if preventing let-7–mediated silencing of only lin-41 is sufficient to recapitulate a phenotype. To this end, we used lin-41(xe8)–mutant animals that lack a segment of the lin-41 3′ UTR that contains the two let-7 target sites. We refer to lin-41(xe8) in the following as lin-41(∆LCS), where LCS stands for let-7 complementary sites ; let-7(n2853) double mutant animals, in which both let-7 target sites on the lin-41 3′ UTR contain a compensatory point mutation (CPM) to allow base pairing to the let-7(PM)–mutant miRNA  single mutant animals (lin-41(xe11) single mutant animals, for which lin-41 silencing is also incomplete because binding of wild-type let-7 to the mutant LCS’s is impaired (lin-41(xe11) as lin-41(CPM).We previously showed that regulation of LIN41 alone accounted for the function of elopment . Given t-41 only   animals and rescued in lin-41(CPM); let-7(PM) animals (lin-41(CPM) single mutant animals (Table S1).This set of mutant strains allowed us to test which of the developmental events triggered by  targets . Specifirvations , the faiVulval bursting phenotypes. Numbers are given in percentage of burst animals in each of three independent experiments (exp) with n ≥ 400 scored animals.Table S1 let-7–mediated control of seam cell proliferation. Loss of let-7 activity causes a failure of seam cells to exit the cell cycle (let-7(PM) and lin-41(∆LCS) animals died. At the L4 stage, just before the final molt, both lin-41(∆LCS)– and let-7(PM)–mutant animals exhibited a wild-type number of seam cells (lin-41 in lin-41(CPM)–mutant animals caused no extra seam cell divisions (Table S2), a complete uncoupling of lin-41 from let-7 in lin-41(∆LCS)–mutant animals recapitulated the let-7(PM) phenotype both qualitatively and quantitatively ; let-7(PM) double mutant animals sufficed to revert seam cell numbers to the lower, wild-type level  as well as in lin-41 gain-of-function (gf) males (let-7(PM) mutants were analyzed only at 15°C, a semi-permissive temperature for survival and presumably other phenotypes. Moreover, the lin-41gf alleles used were considered weak, and the mechanistic basis of their hyperactivation, involving mutations in the first coding exon of lin-41, is unknown. Strikingly, let-7(PM) animals grown at 25°C and lin-41(∆LCS)–mutant animals exhibited phenotypes that were much more severe than those caused by a mere delay in tail cell retractions. As analyzed in more detail in the following section, almost all let-7(PM) and all lin-41(∆LCS)–mutant adult males had “unretracted” tails with a long, pointed shape, resembling those of hermaphrodites (lin-41(CPM); let-7(PM) double mutant animals were of normal, wild-type appearance  males . HoweverMale tail retraction phenotypes. Numbers are given in percentage of animals, n = number of scored animals.Table S3 lin-41(∆LCS) and let-7(PM)–mutant animals were much more severe than what was reported earlier for lin-41gf–mutant animals and let-7(PM)–mutant animals grown at 15°C  and lin-41(bx42)gf mutations on male tail development. Both weak gf alleles caused a “leptoderan” (Lep) phenotype, which we confirmed (lin-41(CPM) (lin-41(∆LCS) and in let-7(PM) males (onfirmed . We obseM) males .lin-41(bx37)– and lin-41(bx42)–mutant males had a partially retracted tail at the late L4 stage, with a spike protruding from the otherwise normal tail (lin-41(∆LCS)–mutant males displayed any cell retraction (lin-41(bx37) or lin-41(bx42) mutants, whereas lin-41(∆LCS) and let-7(PM) animals had a distinct and more severe “unretracted” phenotype (let-7(PM) males grown at 25°C, male tail development was fully restored in lin-41(CPM); let-7(PM) double mutant animals (lin-41 by let-7. We propose that the previously reported Lep phenotypes of lin-41gf– and let-7(PM)–mutant animals grown at 15°C reflects the fact that, under these conditions, let-7 will still eventually silence lin-41, but with a delay and/or less extensively. By contrast, let-7–mediated silencing is altogether lost in let-7(PM)–mutant animals at 25°C and in lin-41(∆LCS) animals. We further note that male tail morphogenesis appears particularly sensitive to LIN41 activity levels, since the weak gf alleles lin-41(bx37), lin-41(bx42), and lin-41(CPM) cause delayed, albeit largely functional male tail retraction  on a mixture of L3- and L4-stage animals. We used an anti-FLAG antibody to enrich for mRNAs bound by LIN41, which we expressed from a rescuing flag::gfp::lin-41 transgene in the lin-41(n2914)–mutant background (flag::gfp::sart-3 (him-5(e1490) genetic background, which increases the incidence of males in a population  and lin-41(∆LCS)–mutant animals.Previously, we showed that all four targets accumulate shortly before wild-type worms turn into adults, when targets  domain–containing transcription factors, proposed to act at least partially redundantly on common targets due to similar binding motifs -type transcription factor of the l family . MAB-3 ag motifs .mab-10 and mab-3 by removing almost the entire coding region using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9  allele, which is considered a null allele for its function in male tail development (dmd-3(lf).To explore the effects of losing the activity of LIN41 targets, we created deletion alleles of PR)-Cas9 . lin-29 d MAB-10 . Since w lin-29b , we crea mutants with those of the LIN41 target pairs, we observed and quantified the male tail phenotypes of mab-10(0) lin-29(Δa) as well as of mab-3(0); dmd-3(lf) double mutant animals. mab-10(0) lin-29(Δa) double mutant males exhibited normal tail cell retraction, although they displayed shorter fan and ray structures than wild-type animals (mab-3(0); dmd-3(lf) males were completely deficient for cell retraction at any stage during development  and the let-7(PM) single mutations ::h2b from putative mab-3 and dmd-3 promoters, that is, the 4-kb region upstream of their start codons. For each promoter, we created two reporters, one with a promoter-orthologous 3′ UTR that harbors LIN41 target sites and one with the heterologous unc-54 3′ UTR that is not regulated by LIN41  or lin-41(∆LCS) mutants by mutating lin-29a and/or mab-10. Seam cell numbers in young adults of either lin-29(Δa) or mab-10(0) single mutants were unchanged from the wild-type situation (mab-10(0) adults displayed a few additional seam cells (data not shown and mab-10(0) lin-29(Δa) double mutant animals displayed additional seam cell divisions to a comparable extent as let-7(PM)– or lin-41(∆LCS)–mutant animals  and lin-29(∆a) mutations is most parsimoniously explained by MAB-10 functioning with both LIN-29a and LIN-29b to control seam cell self-renewal. Indeed, lin-29(xe37)–mutant animals, which lack both LIN-29a and LIN-29b (lin-29(0), display highly penetrant extra seam cell divisions at the young adult stage (Table S2), consistent with an earlier report for putative lin-29(0)–mutant animals ; mab-10(0) lin-29(∆a); dmd-3(lf) quadruple mutant animals, only ∼15% burst, a bursting frequency greatly below the ∼90% penetrance seen with let-7(PM) and lin-41(∆LCS) single mutations. Examination of LIN41 target single mutations as well as of the relevant mutation pairs, that is, mab-3(0); dmd-3(lf) and mab-10 lin-29(∆a), showed that the bursting frequency of 15% was caused by the combined lack of LIN-29a and MAB-10.Our findings thus far show that continued silencing of two distinct target pairs of LIN41 explains let-7–LIN41 promote vulval integrity through additional, currently unknown LIN41 targets or functions. However, we favored a different explanation, namely, that highly penetrant vulval bursting required residual or tissue-specific activity of LIN-29 and/or its cofactor MAB-10, whereas a complete loss of their activity caused by gene deletions leads to a much lower penetrance. This is because mab-10(0) lin-29(∆a); let-7(PM) triple mutant animals phenocopied the mab-10(0) lin-29(∆a) double mutant rather than the let-7(PM) single mutant phenotype (mab-10 and lin-29a suppress the let-7(PM)–mutant phenotype. Moreover, loss of both lin-29a and lin-29b in lin-29(0)–mutant animals caused bursting in only < 2% of animals, and it was not further enhanced by additional loss of mab-10 (let-7(PM) mutation to < 2% in lin-29(0); let-7(PM) double mutant animals (These results might suggest that lin-29(Δa) mutation alone reduced the bursting frequency of let-7(PM) animals to about 15%, whereas the mab-10(0) mutation did not reduce the bursting frequency  worms, we specifically tagged the endogenous LIN-29a isoform by placing a GFP::3xFLAG tag at the N terminus (lin-29a expression in the epidermis of wild-type but not let-7(PM)–mutant late L4-stage animals (lin-29a expression was lost in the let-7(PM) mutant. Specifically, we observed GFP::LIN-29a accumulation in the distal tip cell, the anchor cell (AC), and most vulval cells. The strongest GFP signal was that in the AC  but not mab-10(0) lin-29(∆a) double mutant and lin-29(0) single mutant animals could explain why the former, but not the two latter, exhibited a highly penetrant bursting phenotype. To investigate this, we produced LIN-29a specifically in the AC of mab-10(0) lin-29(∆a)– and lin-29(0)–mutant animals. We used a single-copy integrated xeSi417 transgene that combines the ∆pes-10 basal promoter  to exprpression , althougmab-10(0) lin-29(Δa) double mutant animals, and to >90% in lin-29(0)–mutant animals (let-7(PM)–mutant animals die because of a lack of LIN-29 activity in some tissues while retaining LIN-29a activity in the AC.Wild-type animals expressing the transgene did not reveal any overt defects. However, consistent with our hypothesis, expression of this transgene increased the frequency of bursting to about 50% in  animals . We conclin-29(0) mutants, π cell fates are not specified and the utse does not form  single mutant and in mab-10(0) lin-29(Δa) double mutant animals. We further hypothesized that re-expression of lin-29a in the AC would restore utse formation, and thereby render animals that lack LIN-29 activity in other tissues prone to bursting.Given the above results, we sought to examine the role of LIN-29a in the AC. During wild-type development, the AC induces and coordinates cell fates of both the vulva and the uterus. In uterine development, the AC specifies its lateral neighbors as π cells, and some of the π daughter cells fuse with each other and eventually with the AC to form the uterine seam cell (utse). The utse is important for the structure of the egg-laying apparatus, as it anchors the adult uterus to the epidermal seam. It also forms a thin cytoplasmic extension that separates the lumens of vulva and uterus until it is broken when the first embryo is laid. In not form . Insteadnot form  Fig 7A)lin-29(0) animals . Insteadlin-29(0) single mutant hermaphrodites and in >50% of mab-10(0) lin-29(Δa) double mutant hermaphrodites. Consistent with an impairment of vulval function, these animals displayed highly penetrant egg-laying deficient (Egl) and protruding vulvae (Pvl) phenotypes  lin-29(Δa) double mutant and lin-29(0) single mutant animals, like let-7–mutant animals, are prone to rupturing, but observation of this phenotype is obscured when lack of lin-29a expression in the AC prevents normal utse formation. Hence, the let-7 bursting phenotype can be explained by sustained LIN41-mediated repression of mab-10 and lin-29a in tissues other than the AC.We conclude that expression of C. elegans heterochronic pathway is arguably one of the best-characterized developmental timing pathways, and recent evidence has suggested that its function in controlling the onset of J/A transition might be evolutionarily conserved  mutants lead to fully penetrant phenotypes. Thus, to control events for the transition to adulthood, a cascade of post-transcriptional regulators eventually times the expression of two pairs of transcriptional regulators.Within each pair, the individual factors have partially redundant functions, as individual mutations cause either no, or only partially penetrant phenotypes. By co-regulating partially redundant genes within each pathway, LIN41 itself assumes a more unique function and, as a consequence, elevated LIN41 levels as in dmd-3 and mab-3 in male tail cell retraction had been reported previously  lin-29(∆a) animals that produced LIN-29a in the AC burst, but 100% exhibited overtly normal utse morphology, a thin utse alone appears insufficient to permit bursting, and it remains an open question what prevents a fully penetrant phenotype in these animals. However, the thick cell layer that separates uterus and vulva in lin-29(0)– or mab-10(0) lin-29(∆a)–mutant animals may be sufficient to prevent bursting, as it is presumably more resistant than a wild-type utse to the internal pressure in the worm. Taken together, our findings reveal an important role of MAB-10 and LIN-29a as effectors of let-7–LIN41 in its function to ensure vulval integrity, and they explain why lin-29 and let-7 mutations were previously found to yield incompatible phenotypes  animals could be detected  animals, re-expression of let-7 in the epidermis, uterus, and vulva sufficed to prevent vulval bursting, whereas re-expression in the epidermal hyp7 syncytium only was sufficient to restore some degree of epidermal differentiation but was insufficient to prevent vulval bursting  and let-7(lf) mutations were reported to delay the onset of puberty  per gene were injected to generate large deletions spanning almost the entire coding region. The following pairs were used: (i) lin-29 sgRNA1: gctggaaccaccactggctc, lin-29 sgRNA2: atattatttatcagtgattg; (ii) mab-10 sgRNA1: gatgatgatgatgaagaggt, mab-10 sgRNA2: gctcccggaatcttgaagct; and (iii) mab-3 sgRNA1: aggagctctaatgctcaccg, mab-3 sgRNA2: agctcagctcaatttgggcg.To obtain mutant alleles for lin-29, we generated a large deletion spanning exons 2–11 and thus most of the coding region of both lin-29a and lin-29b. Specifically, the resulting lin-29(xe37) = lin-29(0) allele is a 14,801-bp deletion with a 2-bp insertion with the following flanking sequences: 5′ ggactctggaatagctggaa—xe37 deletion—xe37 insertion (aa)—aatatgaaaaatcattccta 3′. Translation of xe37 yields only a short stretch of 28 amino acids (MDQTVLDSAFNSPVDSGIAG-KNMKNHSY*), containing the N-terminal 20 and the C-terminal 7 amino acids of LIN-29a. The small insertion leads to translation of an additional lysine (K).For mab-10, the resulting mab-10(xe44) = mab-10(0) allele spans exons 3–9 and is a 2,901-bp deletion with a 4-bp insertion with the following flanking sequences: 5′ ttatcatctcttacaactca—xe44 deletion—xe44 insertion (ctct)—tattttttgttttcctcgtga 3′. Translation of xe44 yields a 58-amino acid stretch (MSSSSSSSLPTSSASTTTSSITSRPSASHHLESILSSSSSSPSILSSLTT-HSYFLFSS*) containing the N-terminal 50 amino acids of MAB-10, followed by eight additional amino acids, translated from the small insertion and the mab-10 3′ UTR, and a stop codon (underlined in the flanking sequence above).For mab-3, the resulting mab-3(xe49) = mab-3(0) allele is a 4,291-bp deletion starting in exon 2, just downstream of the ATG start codon of the longer isoform, and ending downstream of the stop codon. The flanking sequences are: 5′ tttgcagaggagctctaatg—xe49 deletion—ctccgcccacactttcccag 3′. Translation of xe49 is presumably initiated at the normal ATG start codon, but then translates a sequence that is normally noncoding, yielding a stretch of 31 amino acids (MLRPHFPRITVLFLALRLSFSFPLSLFYLGK*) unrelated to the MAB-3 protein.For lin-29a without affecting expression of lin-29b, we deleted part of the coding exons specific to lin-29a, at the same time introducing a frameshift in the downstream lin-29a reading frame. By injecting two sgRNAs , we obtained the lin-29a(xe40) = lin-29(∆a) allele, a 1,102-bp deletion covering exons 2–4, introducing a frameshift in the lin-29a reading frame with a predicted stop codon in exon 6. The deletion has the following flanking sequences: 5′ ctctggaatagctggaaccac—xe40 deletion—attctctcctgccacatcat 3′. Translation of xe40 yields a protein with the N-terminal 22 amino acids of LIN-29A (MDQTVLDSAFNSPVDSGIAGTT), followed by a stretch of 69 out-of-frame amino acids and a stop codon.To specifically mutate To specifically tag LIN-29a at the N terminus, the following mix was injected into wild-type worms : 50 ng/μPlasmids used in this study.Table S6 lin-41(xe8) = lin-41(∆LCS) allele is not temperature sensitive like let-7(n2853) and, therefore, causes lethality at any temperature (lin-41(xe8) animals, a balancer null allele, lin-41(bch28), was previously created by inserting an expression cassette driving ubiquitous nuclear GFP from the eft-3 promoter into the lin-41 coding sequence (lin-41 copy—and a recombined lin-41(bch28 xe8) allele—through intragenic recombination of lin-41(bch28) with lin-41(xe8), we additionally deleted a large part of the lin-41 coding sequence together with the part of the lin-41 3′ UTR containing the let-7 complementary sites within the lin-41(bch28) allele. To this end, lin-41(bch28) heterozygous worms were injected with a mix containing 50 ng/μl pIK155, 100 ng/μl of each pIK198 with a cloned sgRNA, 5 ng/μl pCFJ90, and 5 ng/μl pCFJ104, as previously described  by PCR. The lin-41(bch28 xe70) allele thus obtained was further validated by DNA sequencing and outcrossed three times to the wild-type strain before crossing it with lin-41(xe8) heterozygous animals. The final lin-41(bch28 xe70) allele consists of the inserted expression cassette, as described in xe70 deletion —accattgaaaccttctccc 3′.The perature . To mainsequence . To avoiescribed . We injettTi5605 locus), using a protocol for injection with low DNA concentration , synchronized arrested L1 stage larvae were grown for 20 h at 25°C on RNAi-inducing plates with HT115 bacteria. The bacteria either contained the L4440 parental RNAi vector without insert (denoted “mock RNAi”) or with an insert targeting lin-41 (xeSi417 transgene expression), a Zeiss Axio Observer Z1 microscope was used. Differential Interference Contrast and fluorescent images were acquired with a 40×/1.3 oil immersion objective . Using the Fiji software  homozygous animals within a population of balanced lin-41(xe8)/lin-41(bch28 xe70) animals, all eft-3p::gfp::h2b expressing  animals were excluded from the analysis. Images were acquired on a Zeiss Axio Observer Z1 microscope using the AxioVision SE64 software. Animals were mounted on a 2% (wt/vol) agarose pad and immobilized in 10 mM levamisole. Selection of regions and processing of images was performed with Fiji –mutant worms in different genetic backgrounds. Images were acquired after growing males to early, mid, or late L4 larvae as well as to young adults. To quantify tail retraction defects, male tail phenotypes were scored in mounted worms at the late L4 and at the young adult stage. At the late L4 stage, tail retraction defects were categorized as unretracted (no cell retraction visible), partially retracted (cells less retracted than in wild-type) or over-retracted tails (cells more retracted than in wild-type). At the young adult stage, abnormal tails were categorized into over-retracted, Lep, and unretracted  phenotypes. Tails with a spike extending beyond the fan were counted as “Lep” and tails with smaller spikes were considered wild-type . Whereas most lin-41(bx37)– or lin-41(bx42)–mutant males had a Lep tail, all lin-41(xe8) animals showed the distinct and more severe “unretracted” phenotype. 85% of let-7(n2853)–mutant males also displayed a completely unretracted tail. In 15% of the scored let-7(n2853)–mutant males, the tail cell started retracting at the time point of analysis (young adult stage), still resulting in a similarly elongated tail, but with a rounded tip. This phenotype was categorized as “unretracted,” as it was more similar to fully unretracted tails than to Lep tails. The observed delayed start of the retraction program may be due to residual let-7 activity in the let-7(n2853)ts mutant.To examine male tail retraction defects, we used let-7(n2853) or lin41(xe8) animals burst through their vulva. Animals with part of the intestine protruding through the vulva were counted as “burst,” all other animals as “non-burst.” Pvl phenotypes were only scored in non-burst animals. Egl phenotypes were scored for non-burst worms in synchronized populations grown at 25°C for 60 h. At least 400 worms were scored for each genotype in each of these experiments to quantify vulval phenotypes.To examine vulval phenotypes , worm phenotypes were scored directly on the nematode growth medium agar plates using a dissecting microscope. Vulval bursting and Pvl phenotypes were quantified in synchronized worm populations grown at 25°C for 45 h; a time ∼5 h after the first To quantify uterine-vulval connection phenotypes, worms were grown in synchronized populations at 25°C for 36–38 h to reach the late L4 larval stage. Phenotypes of mounted worms were assigned to one of two categories, worms with a normal thin utse structure and worms with an abnormal thicker cell layer, and counted.lin-41(n2914); him-5(e1490)–mutant worms expressing flag::gfp::lin-41 (him-5(e1490)–mutant worms expressing flag::gfp::sart-3  and with the parameter “splicedAlignment=TRUE,” which calls the SpliceMap aligner with default parameters (ftp://ftp.wormbase.org/pub/wormbase/releases/WS190/). Reads overlapping all annotated exons for each gene were counted. For plotting, samples were normalized by the mean number of counts mapping to exons in all samples and then log2-transformed after adding a pseudocount of 8. To determine genes significantly bound by LIN-41, a model was constructed using edgeR (v. 3.22.3)  nta Cruz . Alignmenta Cruz , with thrameters . The resrameters . Gene co 3.22.3)  containiGSE120405.All RIP-seq data generated in this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO)  under GE


This article has been corrected: A concentration in the Case Presentation section was corrected. The proper sentence is shown below.…“In May 2017, 40 mg propranolol was administered daily and PET-CT scans were performed at regular intervals to assess the response of the tumor to propranolol.”Original article: Oncoscience. 2018 Oct 11;5(9-10):264-8PMCID: PMC6231448 PMID: 30460329DOI:10.18632/oncoscience.472


As a hallmark of metabolic reprogramming, aerobic glycolysis contributes to tumorigenesis and aggressiveness. However, the mechanisms and therapeutic strategies regulating aerobic glycolysis in neuroblastoma (NB), one of leading causes of cancer-related death in childhood, still remain elusive.Methods: Transcriptional regulators and their downstream glycolytic genes were identified by a comprehensive screening of publicly available datasets. Dual-luciferase, chromatin immunoprecipitation, real-time quantitative RT-PCR, western blot, gene over-expression or silencing, co-immunoprecipitation, mass spectrometry, peptide pull-down assay, sucrose gradient sedimentation, seahorse extracellular flux, MTT colorimetric, soft agar, matrigel invasion, and nude mice assays were undertaken to explore the biological effects and underlying mechanisms of transcriptional regulators in NB cells. Survival analysis was performed by using log-rank test and Cox regression assay.Results: Transcription factor myeloid zinc finger 1 (MZF1) was identified as an independent prognostic factor , and facilitated glycolysis process through increasing expression of hexokinase 2 (HK2) and phosphoglycerate kinase 1 (PGK1). Meanwhile, a 21-amino acid peptide encoded by upstream open reading frame of MZF1, termed as MZF1-uPEP, bound to zinc finger domain of Yin Yang 1 (YY1), resulting in repressed transactivation of YY1 and decreased transcription of MZF1 and downstream genes HK2 and PGK1. Administration of a cell-penetrating MZF1-uPEP or lentivirus over-expressing MZF1-uPEP inhibited the aerobic glycolysis, tumorigenesis and aggressiveness of NB cells. In clinical NB cases, low expression of MZF1-uPEP or high expression of MZF1, YY1, HK2, or PGK1 was associated with poor survival of patients.Conclusions: These results indicate that therapeutic targeting of YY1/MZF1 axis by MZF1-uPEP inhibits aerobic glycolysis and NB progression. HK2), phosphoglycerate kinase 1 (PGK1), and enolase 1 (ENO1) Neuroblastoma (NB) is the most common extracranial solid malignancy in pediatric population, and accounts for approximately 15% of all childhood cancer deaths GLUT1) and lactate dehydrogenase A (LDHA) HK2 and LDHA expression GLUT1 and GLUT4LINC01554 inhibits aerobic glycolysis via promoting degradation of pyruvate kinase isozyme M2 (PKM2) in hepatocellular carcinoma cells Recent studies show that aerobic glycolysis is driven by activation of oncogenes or inactivation of tumor suppressors. For example, hypoxia inducible factor 1 alpha, a key mediator of hypoxic response, contributes to aerobic glycolysis by up-regulating glucose transporters 1 (MZF1) and its upstream open reading frame (uORF)-derived peptide (uPEP) as crucial regulators of aerobic glycolysis and NB progression. We demonstrate that MZF1 is up-regulated in NB tissues and cells, and facilitates the aerobic glycolysis, growth, and aggressiveness of NB cells by up-regulating HK2 and PGK1. Meanwhile, MZF1-uPEP interacts with Yin Yang 1 (YY1) to repress its transactivation, resulting in transcriptional inhibition of MZF1 and downstream glycolytic genes. Pre-clinically, administration of a cell-penetrating MZF1-uPEP or lentivirus over-expressing MZF1-uPEP significantly suppresses aerobic glycolysis, tumorigenesis and aggressiveness, indicating the crucial roles of MZF1-uPEP in repressing YY1/MZF1 axis during NB progression.In this study, through an integrative screening approach, we identify myeloid zinc finger 1 , or LY294002 as indicated (Sigma).Human non-transformed mammary epithelial MCF 10A (CRL-10317) cells, embryonic kidney HEK293 (CRL-1573) cells, NB cell lines SH-SY5Y (CRL-2266), SK-N-AS (CRL-2137), BE(2)-C (CRL-2268), and IMR-32 (CCL-127), and cervical cancer HeLa (CCL-2) cells were purchased from American Type Culture Collection . Cell lines were authenticated by short tandem repeat profiling, and used within 6 months after resuscitation of frozen aliquots. Mycoplasma contamination was regularly examined using Lookout Mycoplasma PCR Detection Kit . Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum  at 37°C in a humidified atmosphere of 5% COTotal RNA was isolated with RNeasy Mini Kit . Reverse transcription reactions were conducted with Transcriptor First Strand cDNA Synthesis Kit . Real-time PCR was performed with SYBR Green PCR Master Mix  and primers .The peptide corresponding to MZF-uPEP (METRWGTDGVLMTAVIGAGSC) was synthesized, and coupled to keyhole limpet hemocyanin using chemical crosslinker glutaraldehyde. Rabbit anti-MZF1-uPEP polyclonal antibody was prepared by immunizing New Zealand rabbit with synthesized peptide, purified by persulfate, Sephadex G25 and DEAE-Sephadex G100 , and validated by antigen peptide or fusion protein recognition. Tissue or cellular protein was extracted with 1× cell lysis buffer . Western blot was performed as previously described MZF1 and promoters of MZF1 (-1530/+30), HK2 (-1813/+424), or PGK1 (-882/+246) were amplified from genomic DNA by PCR  of A by PCR  and subcng sites  and inseng sites . Dual-luChIP assay was performed according to instructions of EZ-ChIP kit  MZF1 coding sequence , MZF1 cDNA (2920 bp), YY1 cDNA (1245 bp) and corresponding truncations were obtained from NB tissues by PCR primers  and primers  to target upstream or downstream region relative to transcription start site of MZF1  into HEK293T cells. Infectious lentivirus was harvested and filtered through 0.45 μm PVDF filters. Recombinant lentiviruses were concentrated 100-fold by ultracentrifugation .Tumor cells were plated on coverslips and incubated with antibodies specific for YY1  or MZF1-uPEP (1:200 dilution). Then, cells were incubated with Alexa Fluor 594 goat anti-rabbit IgG or Alexa Fluor 488 goat anti-rabbit IgG (1:1000 dilutions), and stained with 4',6-diamidino-2-phenylindole .Co-IP was performed as previously described Cell-penetrating peptide of MZF1-uPEP was designed and synthesized . The 11-amino acid long peptide (YGRKKRRQRRR) from Tat protein transduction domain served as a cell-penetrating peptide. Thus, inhibitory peptides were chemically synthesized by linking with biotin-labeled cell-penetrating peptide at N-terminus and conjugating with fluorescein isothiocyanate (FITC) at C-terminus, with purity larger than 95%.Cellular proteins were isolated using 1× cell lysis buffer (Promega), and incubated with biotin-labeled peptide at 4°C overnight. Then, incubation of cell lysates with streptavidin-agarose was undertaken at 4°C for 2 hrs. Beads were extensively washed, and proteins pulled down were measured by western blot assay.-1), oligomycin (2 μmol·L-1), and 2-DG (100 mmol·L-1) using a Seahorse Biosciences XFe24 Flux Analyzer  Cellular glucose uptake, lactate production, and adenosine triphosphate (ATP) levels were detected as previously described Tumor cells were treated with 100 μg/ml of cycloheximide (Sigma) for 5-10 min. Cell extracts were layered on top of 15-30% (w/v) linear sucrose gradient. After centrifugation at 40,000 ×g for 2 hrs at 4°C, fractions were collected using a piston-gradient fractionator . The polysome profiles were monitored by absorbance of light with a wavelength of 260 nm (A260). The polysome-bound transcripts were extracted and detected by real-time qRT-PCR.TM Matrix, BD Biosciences, Franklin Lakes, NJ) invasion The 2--2,5-diphenyl tetrazolium bromide  colorimetric In vivo tumor growth (1×106 tumor cells per mouse) and experimental metastasis (0.4×106 tumor cells per mouse) studies were performed with blindly randomized four-week-old female BALB/c nude mice as previously described in vivo therapeutic studies, tumor cells (1×106 or 0.4×106) were injected into dorsal flanks or tail vein of nude mice, respectively. One week later, mice were blindly randomized and treated by tail vein injection of synthesized cell-penetrating peptide  or lentivirus-mediated MZF1-uORF . Animals were imaged using the In-Vivo Xtreme II small animal imaging system .All animal experiments were carried out in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, and approved by the Animal Care Committee of Tongji Medical College . The Institutional Review Board of Tongji Medical College approved the human tissue study . All procedures were undertaken in accordance with guidelines set forth by Declaration of Helsinki. Written informed consent was obtained from all legal guardians of patients. Patients with a history of preoperative chemotherapy or radiotherapy were excluded. Human normal dorsal root ganglia tissues were collected from therapeutic abortion. Fresh specimens were collected at surgery, validated by pathological diagnosis, and stored at -80°C.Immunohistochemical staining was performed as previously described t test, one-way analysis of variance (ANOVA), and χ2 analysis were applied to compare the difference in tumor cells or tissues. Fisher's exact test was applied to analyze the statistical significance of overlap between two gene lists. Pearson's correlation coefficient was applied for analyzing the relationship among gene expression. Log-rank test and Cox regression analysis were used to assess survival difference and hazard ratio. All statistical tests were two-sided and considered statistically significant when false discovery rate-corrected P values less than 0.05.All data were shown as mean ± standard error of the mean (SEM). Cutoff values were determined by average gene expression levels. Student's P<0.05) differentially expressed in NB specimens with varied status of age, death, and international neuroblastoma staging system (INSS) stages, respectively , 7 genes were found to be consistently associated with age, death, and advanced INSS stage of NB . The results revealed that two transcription factors, MZF1 and E2F transcription factor 3 (E2F3), might regulate expression of these glycolytic genes , death (P=1.3×10-6), or advanced INSS stage (P=2.1×10-4), and was associated with poor survival of patients . In addition, higher MZF1 expression was observed in NB cell lines, than that in normal dorsal root ganglia -C, and IMR-32  cells as models. Sable over-expression or knockdown of MZF1 increased and decreased the levels of HK2 or PGK1, but not of fructose-bisphosphate C (ALDOC), ENO1, glucose-6-phosphate isomerase (GPI), or LDHA, in these NB cells, respectively -C, and IMR-32 cells, respectively, while mutation of MZF1 binding site abolished these effects  or PGK1 (P=5.3×10-8 and P=3.0×10-23) levels were associated with poor survival of NB patients  6476 P<0.B Figure . SimilarB Figure , which ws Figure  and chos ganglia B. To fury Figure C. The MZs Figure D. Consispatients E, and wearcinoma . These dMZF1 in NB cells, we applied dCas9-based clustered regularly interspaced short palindromic repeats (CRISPR) MZF1. As shown in Figure MZF1 in NB cells, respectively. Stable transfection- or dCas9a-induced up-regulation of MZF1 increased the ECAR, an indicator of glycolysis, in SH-SY5Y and SK-N-AS cells, while shRNA- or dCas9i-induced knockdown of MZF1 significantly attenuated glycolytic process in BE(2)-C and IMR-32 cells -C cells to restore their expression, glucose uptake, lactate production, and ATP levels altered by stable over-expression or knockdown of MZF1 -C cells decreased the growth, weight, Ki-67 proliferation index, and expression levels of HK2 and PGK1 of subcutaneous xenograft tumors in nude mice -C cells resulted in less lung metastatic colonies and greater survival probability in nude mice , which revealed the existence of an uORF within this region  was decreased, while MZF1 antisense RNA 1 (MZF1-AS1), a noncoding transcript containing complementary sequence of uORF, was not enriched in heavy polysomes -C and SH-SY5Y cells, respectively -C cells led to increase in MZF1 promoter activity, while mutation of YY1 binding site or stable transfection of MZF1-uORF abolished these effects  in BE(2)-C and SH-SY5Y cells -C cells. Administration of YIP-21 resulted in decreased growth and weight of subcutaneous xenograft tumors in nude mice -C cells Figure A. Biotine Figure G. The Kit Figure G-H. In et Figure I. ConseqMZF1-uORF over-expression on tumor progression, nude mice were treated with subcutaneous or tail vein injection of IMR-32 cells stably expressing red fluorescent protein. One week later, mice were randomly divided into groups, and received intravenous administration of lentivirus carrying empty vector (mock) or MZF1-uORF. Administration of lentivirus-mediated MZF1-uORF dramatically reduced the growth and weight of xenograft tumors , poor differentiation (P=0.024), higher mitosis karyorrhexis index , or advanced INSS stages , breast cancer (P=3.1×10-3), endometrial carcinoma (P=6.1×10-3), glioma (P=1.6×10-11), head and neck carcinoma (P=1.2×10-2), lung cancer (P=5.5×10-3), lymphoma (P=1.0×10-3), pancreatic cancer (P=1.4×10-4), or renal clear cell carcinoma , death (P=6.5×10-4), or advanced INSS stage  promoter and increase of migratory, invasive, and metastatic potential of colorectal cancer cells PKCα), thus facilitating the migration and invasion of cancer cells MMP-2) MZF1 exerts oncogenic or tumor suppressive roles via transcriptional changes associated with malignant cell migration and invasiveness in a context-dependent manner. However, the roles of MZF1 in aerobic glycolysis during tumor progression still remain elusive. In this study, MZF1 was identified as an independent prognostic factor for poor outcome of NB patients, while HK2 and PGK1 were direct target genes of MZF1. Our gain- and loss-of-function studies indicated that MZF1 promoted aerobic glycolysis, growth, and invasiveness of NB cells, suggesting the oncogenic roles of MZF1 in NB progression.cis-regulatory element within uORF, and imposes a negative effect on protein translation in DrosophilaCHOP) contains a conserved uORF which encodes a 31-amino acid peptide that inhibits the translation of CHOPMZF1 5'-UTR, which encoded a small peptide that bound to YY1 protein. Notably, MZF1-uPEP inhibited YY1-facilitated transcription of MZF1, indicting a novel action mode of uORF-encoded peptide in regulating gene transcription rather than protein translation. In addition, tumor suppressive functions of MZF1-uPEP were mediated, at least in part, through interacting with YY1 protein in NB cells.The widespread presence of uORF within 5'-UTR is one of the mechanisms regulating gene expression YY1 is a transcription factor of GLI-Kruppel family, and plays a regulatory role in cellular growth, oncogenic transformation, epithelial-mesenchymal transition, and metastasis YY1 is highly expressed in many types of cancerous tissues, including prostate cancer, colon cancer, liver cancer, and lung cancer YY1 promotes the growth and Wnt signaling pathway of cancer cells through inhibiting p53YY1 facilitates the transcription of p-glycoprotein in acute lymphoblastic lekeumia, and is associated with poor survival of patients YY1 promoted the expression of MZF1 in NB cells, resulting in facilitated glycolytic gene expression and tumor progression. In addition, MZF1-uPEP bound to zinc finger domain of YY1, resulting in repression of YY1 transactivation in NB cells. Importantly, administration of a cell-penetrating MZF1-uPEP or lentivirus over-expressing MZF1-uPEP was able to suppress aerobic glycolysis, tumorigenesis, and aggressiveness of NB cells, suggesting the crucial roles of MZF1-uPEP in repressing YY1/MZF1 axis in aerobic glycolysis and tumor progression.MZF1 is associated with poor outcome of NB, and exerts oncogenic roles in aerobic glycolysis and tumor progression. Meanwhile, MZF1-uORF-encoded peptide suppresses the MZF1 expression, aerobic glycolysis, growth, and aggressiveness of NB cells. Mechanistically, MZF1 promotes the expression of glycolytic genes HK2 and PGK1, while MZF1-uPEP binds to YY1 to repress its transactivation, resulting in transcriptional suppression of MZF1 and downstream glycolytic genes. Administration of a cell-penetrating MZF1-uPEP or lentivirus over-expressing MZF1-uPEP suppresses the aerobic glycolysis, tumorigenesis, and aggressiveness of NB cells. Since MZF1 expression is negatively regulated by microRNAs let-7e and let-7d in breast cancer cells MZF1-AS1 in regulating MZF1-uPEP expression in NB. We believe that this study extends our knowledge about the regulation of aerobic glycolysis by transcription factor and its derived uPEP, and suggests that MZF1 and YY1 may be potential therapeutic targets for tumor progression.In summary, we demonstrate that Supplementary figures and tables.Click here for additional data file.


KLF1, are common within certain populations of the world. Heterozygous missense mutations in KLF1 mostly lead to benign phenotypes, but a heterozygous mutation in a DNA-binding residue (E325K in human) results in severe Congenital Dyserythropoietic Anemia type IV (CDA IV); i.e. an autosomal-dominant disorder characterized by neonatal hemolysis.Mutations in the transcription factor, Klf1−/− genetic background. Nuclear translocation of wild type KLF1 results in terminal erythroid differentiation, whereas mutant KLF1 results in hemolysis without differentiation. The E to K variant binds poorly to the canonical 9 bp recognition motif (NGG-GYG-KGG) genome-wide but binds at high affinity to a corrupted motif (NGG-GRG-KGG). We confirmed altered DNA-binding specificity by quantitative in vitro binding assays of recombinant zinc-finger domains. Our results are consistent with previously reported structural data of KLF-DNA interactions. We employed 4sU-RNA-seq to show that a corrupted transcriptome is a direct consequence of aberrant DNA binding.To investigate the biochemical and genetic mechanism of CDA IV, we generated murine erythroid cell lines that harbor tamoxifen-inducible (ER™) versions of wild type and mutant KLF1 on a Since all KLF/SP family proteins bind DNA in an identical fashion, these results are likely to be generally applicable to mutations in all family members. Importantly, they explain how certain mutations in the DNA-binding domain of transcription factors can generate neomorphic functions that result in autosomal dominant disease.The online version of this article (10.1186/s12864-019-5805-z) contains supplementary material, which is available to authorized users. Klf1−/− mice die in utero from severe anemia  was kept constant and the [protein] was altered in different reactions. The [protein] at which 50% is bound represents the KD. Curves were fit individually using Specific Binding with Hill Slope. Averaged KD and its standard error are reported.Electromobility shift assays were performed as previously described with some modification . Bindingwww.graphpad.com. Alignments of human and mouse KLF1 proteins was performed using the program Geneious version R7 [http://easeq.net. Proportional Venn Diagrams were produced using EulerAPE [swapaa, tug and minimize functions within the OpenMM dynamics environment. Structural images were then prepared in ChimeraX.Statistics analysis was performed with GraphPad Prism version 6.04 for Windows, GraphPad Software, La Jolla California USA, rsion R7 . Densityrsion R7 , availabEulerAPE . ModelinEulerAPE . StructuEulerAPE , 35. MutAdditional file 1:Figure S1. Inducible cell lines to study human CDA type IV. Full length western blot of nuclear extracts from cell lines generated in this study as shown in Fig. Additional file 2:Figure S2. Central enrichment of identified PWM. CentriMo analysis of PWM (motif) identified by MEME is found closest to the summit of ChIP-seq peaks for KLF1-E339K-ER (A) and KLF1-ER (B). (JPG 302 kb)Additional file 3:Figure S3. 4sU-labeled RNA Enrichment. Validation by qRT-PCR of 4sU-RNA isolation from total RNA. Samples used for RNA-seq analysis were validated for enrichment of primary transcript (4sU-labeled) relative to mature Hprt (A) and E2f2 (B) transcripts. (JPG 305 kb)Additional file 4:Figure S4. Gene Set Analysis of KLF1-E339K-ER DEGs. Gene signatures from the analysis of K1-E339K-ER cells versus K1 were tested using the gene set analysis toolkit on the KEGG pathway using Entrez IDs Additional file 6:Table S2. K1-E339K-ER4sU-RNA-seq DEGs. Gene expression changes following activation of KLF1-E339K-ER compared to K1 (Klf1−/−) cells. Differentially Expressed Genes (DEGs) using a false discovery rate (FDR) of 0.05 with the treat method employing a minimum log-fold-change of 2. (XLSX 1979 kb)


Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved  Immunohistochemistry (IHC) allows the visualization of specific antigens in tissue using the binding of fluorophore-labeled antibodies. Although IHC is a relatively simple technique and has been widely used for decades ,2, this e.g., HaloTag, SNAP-tag) expressed in a specific cellular location + 667.3, found 666.9.6-Carboxy-JF549   to provide 4.8 mg  of the title compound as a dark blue gum. Analytical HPLC: tR = 14.6/14.9 min (mixture of diastereomers), >99% purity ; MS (ESI) calculated for C52H72ClN4O4 [M]+ 851.5, found 851.1.ATTO 647N NHS ester  and HaloTag amine (O2) ligand  were combined in DMF (1 mL), and DIEA  was added. The reaction was stirred at room temperature for 18 h while being shielded from light. It was subsequently purified by reverse phase HPLC  to provide 2.6 mg  of the title compound as a violet solid. Analytical HPLC: tR = 11.1 min, 98.7% purity ; MS (ESI) calculated for C45H55ClN3O12S2 [M]+ 928.3, found 927.8.Alexa Fluor 594 NHS ester  and HaloTag amine (O2) ligand  were combined in DMF (1 mL), and DIEA  was added. The reaction was stirred at room temperature for 18 h while being shielded from light. It was subsequently purified by reverse phase HPLC . After stirring for 1 h at room temperature, additional BG-NH2  was added. The reaction was stirred at room temperature for 1 h, concentrated in vacuo, and purified by reverse phase HPLC  to provide 2.5 mg of the Cy2(mono-NHS)-SNAP-tag ligand as an orange solid. This intermediate was combined with glycine  and DIEA  in DMF (1 mL). The reaction was stirred at room temperature for 18 h, concentrated in vacuo, and purified by reverse phase HPLC  to provide 0.5 mg  of the title compound as an orange solid. Analytical HPLC: tR = 8.6 min, >99% purity ; MS (ESI) calculated for C44H48N9O13S2 [M]+ 974.3, found 974.1.Cy2 bis-NHS ester  was taken up in DMF (1 mL); DIEA  was added, followed by a solution of BG-NHSS02565: Stable split GAL4 stock 02565 consists of 55C09-p65ADZp in VK00027 and VT040566-ZpGDBD in attP2 attP1 [brp-SNAP [UAS-myr::4xCLIPf in VK00005 [UAS-Syt::Halo7 in VK0027 [57C10-Flp2 in attp18;; pJFRC201-10XUAS>STOP>myr::smGFP-HA in VK00005, pJFRC240-10XUAS>STOP>myr::smGFP-V5-THS-10XUAS>STOP>myr::smGFP-FLAG in su(Hw)attP1 [20XUAS-Cs-Chrimson-mVenus trafficked in attP18 [UAS-7xHalo7::CAAX in VK00005 [UAS-myr-Halo2 in attP2 [Flies were raised on standard corn meal molasses food. The stocks used in this paper included the following: 2_AV_01; –16); SS0in attP2 ,16; 5XUAHw)attP1 ; brp-SNAn attP18 ; UAS-7xH2, briefly submerged in cold 70% ethanol, briefly submerged in cold S2 medium , then held in additional cold S2 medium for up to 20 minutes until dissection. Brains and ventral nerve cords were dissected in cold S2 medium, then fixed in room temperature 2% paraformaldehyde in S2 medium for 55 minutes while rotating on a nutator. Samples were washed 1–4 times  in phosphate-buffered saline plus 0.5% Triton X-100 (PBT) for 10–20 minutes each.For a detailed protocol, see IHC samples were held for up to two days at 4°C in PBT after dissection. Polarity labeling followed , with a 549 SNAP-tag ligand [Samples were tagged following 1–4 post-fixation washes plus in some cases being held up to two hours in PBT, otherwise following the approach of . Chemicag ligand .https://dx.doi.org/10.17504/protocols.io.nycdfsw; MCFO hybrid protocol https://dx.doi.org/10.17504/protocols.io.nyhdft6). The processing required for IHC appeared to diminish the chemical tag signal somewhat when compared to pure chemical tagging.The hybrid IHC/chemical tag protocol combined Cy2 SNAP-tag ligand labeling of the brp-SNAP reference with antibody labeling of specific neurons. The procedure followed the full chemical tag procedure and then an IHC protocol modified to remove nc82 reference labeling  to measure the mean intensity inside the circle. Without changing depth, 30-pixel diameter mean intensities were measured from the brightest part of each medial optic lobe. The three measurements were averaged together for each sample. Intensity values were measured in arbitrary units of intensity between 0 and 4095.SS02565 neurons were measured similarly to the neuropil reference, but moving to the brightest z slice of expression in prominent projections to the anterior optic tubercle for each hemisphere. Mean intensity for a 30-pixel diameter region around each was measured and the two measurements were averaged together.Intensity values for 549 SNAP-tag ligand, unfortunately could not be meaningfully quantitatively performed.Samples that had been initially imaged with both 20X and 63X objectives had approximately 50% lower average intensity than those imaged only at 20X, presumably due to photo-bleaching, and were excluded from the reported averages. Raw intensity comparisons between fluorophores imaged with different lasers and detectors, e.g. Cy2 SNAP-tag ligand and JF549 SNAP-tag ligands [Fig 2). The higher brightness of the IHC protocol likely stems from the inherent amplification at both the tag and primary-to-secondary antibody stages (see https://www.janelia.org/project-team/flylight/protocols).We first investigated chemical tagging to label the Drosophila neuropil as a reference for nervous system morphology. We evaluated two different dye ligands for the Brp-SNAP-tag neuropil marker: a novel 488 nm-excited Cy2 SNAP-tag and the known 560 nm-excited JF ligands ,11. AlthFig 3); and (2) ‘MultiColor FlpOut’ (MCFO), to characterize the morphology of individual neurons by stochastic labeling . The current Polarity scheme consists of neuropil reference, a myristoylated reporter for neuronal membrane, and a Synaptotagmin-fused reporter for presynaptic terminals: 5XUAS-IVS-myr::smFLAG in VK00005, pJFRC51-3XUAS-IVS-Syt::smHA in su(Hw)attP1 [pJFRC201-10XUAS>STOP>myr::smGFP-HA in VK00005, pJFRC240-10XUAS>STOP>myr::smGFP-V5-THS-10XUAS>STOP>myr::smGFP-FLAG in su(Hw)attP1 [We tested the new Cy2 SNAP-tag ligand in combination with previously described chemical tag and antibody reagents to determine the optimal approach for two labeling schemes: (1) ‘Polarity’, to label neuronal membrane and presynaptic sites .Hw)attP1 . The MCFHw)attP1 .Fig 3A); (2) a hybrid approach where the neuropil reference was labeled with brp-SNAP/Cy2-SNAP-tag ligand and specific neurons labeled with antibodies ; and (3) a pure chemical tag approach using brp-SNAP, UAS-myr::4xCLIPf in VK00005, and UAS-Syt::Halo7 in VK0027 transgenes . As in the case of neuropil labeling alone, the Polarity protocol signal quality of the chemical tagging systems was worse than for pure IHC. Moreover, in the hybrid protocol, the Cy2 chemical tag reference labeling exhibited an average intensity of 114.0 ± 20.0 a.u., standard deviation, n = 11, suggesting that the requisite processing steps for IHC lowered the chemical tag signal quality (see below). The results for the MCFO hybrid protocol were similar, with lower signal levels than the pure IHC MCFO protocol .For the Polarity protocol, we compared three different labeling strategies: (1) the existing pure IHC approach . This ‘bleed-through’ may be due to reported impurities in these cyanine-based dyes and/or due to fluorescence changes when embedded in DPX [Fig 4C and 4D).In the pure IHC Polarity & MCFO protocols, and especially in the hybrid approaches, we observed signal from Cy5 (Polarity) or Alexa Fluor 647 (MCFO) in the 488 reference channel . To enhance the signal, we replaced tetramethylrhodamine (TMR) CLIP-tag ligand  with newly synthesized JF549 CLIP-tag ligand , which improved signal substantially, but still was not as bright as IHC. We also compared the effectiveness of Alexa Fluor 594 HaloTag, and ATTO 647N HaloTag ligands in a two-color reference plus membrane label protocol . Both performed similarly well when paired with brp-SNAP and Cy2 SNAP-tag ligand.Closer examination of the pure chemical tag Polarity protocol showed that membrane-labeling was suboptimal at high resolution Click here for additional data file.S2 Protocol(PDF)Click here for additional data file.S3 Protocol(PDF)Click here for additional data file.S4 Protocol(PDF)Click here for additional data file.


When CD44v3-10high/CD44slow PDAC cell lines, which exhibited an epithelial phenotype before the onset of epithelial–mesenchymal transition (EMT), were cocultured with a monolayer of HEK293T cells overnight, they formed Ad-MCAs on the feeder layer and acquired gemcitabine resistance. CD44v8-10 expression was dramatically increased and Ki-67 staining decreased, suggesting that PDAC cells forming Ad-MCAs acquired cancer stem cell (CSC)-like intractable properties. We found that highly downregulated genes in PDAC cells cocultured with HEK293T cells were significantly upregulated in malignant lesions from pancreatic cancer patients. Our work implies that PDAC cells forming Ad-MCAs partially return to a normal tissue gene profile before the onset of EMT. The collective cell behavior like Ad-MCA formation by PDAC cells may mimic critical events that occur in cancer cells at the very early phase of metastatic colonization.Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal refractory cancers. Aggressive features in PDAC cells have been well studied, but those exhibited by a population of PDAC cells are largely unknown. We show here that coculture with epithelial-like feeder cells confers more malignant phenotypes upon PDAC cells forming anchorage-dependent multicellular aggregates ,  Current evidence indicates that CD44, a CSC marker, directly reprograms stem cell properties in colon cancer cells [Pancreatic ductal adenocarcinoma (PDAC), which constitutes approximately 90% of pancreatic cancers, is one of the most lethal malignant tumors . Once PDer cells .Epithelial–mesenchymal transition (EMT) generates cells with properties of CSCs . A recenin vitro in follicular lymphoma [Previously, we reported that when cocultured with epithelial-like feeder cells, lymphoma cells form anchorage-dependent multicellular aggregates (Ad-MCAs) and that a fraction of aggregate-forming lymphoma cells acquire quiescent CD44-high CSC-like phenotypes. This observation suggests that the intractability of tissue-infiltrating lymphoma cells may be partly accounted for by the acquisition of CSC-like properties , 19. Indlymphoma , suggestlymphoma , 22. Howhigh/CD44slow PDAC cells into GM-resistant quiescent CSC-like cells. Furthermore, our work shows that the transcriptomes of PDAC cells are very rapidly and significantly changed by coculture with HEK293T cells. The rapid phenotypic changes of PDAC cells observed in this coculture system appear to mimic those occurring at the early phase of metastatic colonization of PDAC cells. This coculture system should be useful for understanding the molecular mechanisms underlying the emergence of intractable PDAC cells and the true nature of collective cell behavior.In this study, we demonstrate that direct coculture with epithelial-like feeder cells induces Ad-MCA formation in PDAC cells before the onset of EMT, and Ad-MCA formation converts GM-sensitive CD44v3-10high/CD44slow), of which PCI-55, PCI-24, and PCI-43 showed high expression of CD44v3-10 mRNA (CD44v3-10high/CD44slow), and PCI-6 and PCI-35 cells showed high expression of CD44v8-10 mRNA (CD44v8-10high/CD44slow). These CD44 variants were confirmed by direct sequencing of PCR products. In contrast, MIA-PaCa-2 and PANC-1 cells showed a mesenchymal cell phenotype, exhibiting low expression of CD44v mRNA and high expression of CD44s mRNA (CD44vlow/CD44shigh). Next, we evaluated GM sensitivity in each PDAC cell line by measuring the percentage of apoptotic cells induced by treatment with 0.8 μM GM for 48 h. PCI-55, PCI-24, and PCI-43 were more sensitive to GM (∼30% and ∼20% of apoptotic cells) than PCI-6, PCI-36, MIA-PaCa-2, and PANC-1 (less than 6% of apoptotic cells)  Figure . Interes) Figure . CD44v3-M Figure . TreatmeM Figure  nor PCI-Trans-axial images of cocultured cells captured by confocal microscopy revealed that CD44 was expressed exclusively by Ad-MCA-forming PCI-55 cells Figure . Next, whigh/CD44slow PDAC cells form Ad-MCAs in vivo using a mouse model for peritoneal dissemination. To determine whether colonizing microtumors were formed by clonal proliferation or cellular aggregation of PCI-55 cells, a mixture of CFSE- or PKH26-labeled PCI-55 cells were injected intraperitoneally into SCID mice , GDS4102 , and GDS4336 . Interestingly, we found that genes downregulated in PDAC cells cocultured with HEK293T cells were frequently upregulated in malignant lesions resected from pancreatic cancer patients when compared with their normal adjacent tissue , 5′-nucleotidase (NT5E), keratin 6A (KRT6A), transmembrane protein 154 (TMEM154), tumor necrosis factor receptor superfamily member 10A (TNFRSF10A), HEN methyltransferase 1 (HENMT1), amphiregulin (AREG), ladinin-1 (LAD1), zinc finger BED domain-containing protein 1 (ZBED1), matrix metalloproteinase-1 (MMP1), insulin receptor substrate 1 (IRS1), fermitin family member 1 (FERMT1), ovarian carcinoma immunoreactive antigen-like protein (OCIAD2), interferon-induced protein 44 (IFI44), schlafen family member 13 (SLFN13), heparan sulfate glucosamine 3-O-sulfotransferase 1 (HS3ST1), claudin 23 (CLDN23), dual adaptor of phosphotyrosine and 3-phosphoinositides 1 (DAPP1), NAD(P)H quinone dehydrogenase 1 (NQO1), serpin family B member 5 (SERPINB5), macrophage stimulating 1 receptor (MST1R), pleckstrin-2 (PLEK2), integrin alpha-2 (ITGA2), and laminin subunit gamma 2 (LAMC2) , and compared with genes downregulated by the coculture. Specifically, three independent pancreatic cancer datasets obtained from surgically excised tissues were curated from GEO. These data sets consist of a total of 220 microarrays: GDS4103 (tumors ) Figure . These g) Figure . To clarhigh/CD44slow PDAC cells, and that MCA-forming PDAC cells undergo phenotypic conversion into GM-resistant, CD44v8-10+, quiescent CSC-like PDAC cells . This might be because they have acquired a mesenchymal phenotype. On the other hand, PCI-6 cells were GM-resistant, but still kept the ability to form Ad-MCAs and markedly increased CD44s expression when cocultured with HEK293T cells. These observations suggest that PCI-6 cells cultured on HEK293T feeder cells might be poised to undergo EMT. Coculture with HEK293T cells induced CD44v8-10 expression and cellular quiescence in CD44v3-10high/CD44slow PDAC cells , rat anti-human CD44v9 , and mouse anti-human Ki-67  antibodies were used for immunofluorescence staining. GM was purchased from Sigma-Aldrich . 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester , and PKH26  were used for live-cell labeling.5 PDAC cells were directly cocultured with feeder cells growing as monolayers in 24-well plates.PDAC cells were cocultured with HEK293T cells or NHDFs using a direct coculture system that allowed for cell-cell contact. PDAC cells or feeder cells were labeled with 1 μmol/L of CFSE. A total of 2 × 10Apoptotic PDAC cells were evaluated by the percentage of sub G0/G1 phase cells in CFSE-negative cells using the propidium iodide staining method, as previously described . Analysi6 cells/mL in PBS supplemented with 3% FBS. Cells were then analyzed and sorted by FACSAria .PDAC cells cocultured with CFSE-labeled HEK293T cells overnight were trypsinized and washed in PBS. Cocultured cells were resuspended at a concentration of 5 × 10This was performed as previously described . Images C.B-17/lcr-scid/scidJcl mice were purchased from CLEA Japan, Inc. . Mice were bred and housed under specific pathogen-free conditions in our animal facility. All of the animal experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals at Hokkaido University Faculty of Medicine and approved by the Institutional Review Committee of Hokkaido University.6 cells in total) were suspended in 200 μl of PBS and injected intraperitoneally into 6- to 8-week-old male mice with severe combined immunodeficiency (SCID). Peritonea were harvested at indicated times after injection and prepared for frozen sections with Tissue-Tek O.C.T. Compound .CFSE- or PKH26-labeled PCI-55 cells  labeled cRNA was prepared from 0.1 μg total RNA using the Low Input Quick Amp Labeling Kit  according to the manufacturer's instructions. The labeled cRNAs were hybridized onto the microarray. After washing, the arrays were scanned by the Agilent SureScan Microarray Scanner (G2600D). The scanned images were analyzed with Feature Extraction Software 11.5.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended processed signal intensities.t-test. P values less than 0.05 were considered significant.Data were analyzed using the Student's


Secondly, we evaluated whether cholesterol-lowering adeno-associated viral serotype 8 (AAV8)-mediated LDLr gene transfer prevents HFpEF. AAV8-LDLr gene transfer strongly (p < 0.001) decreased plasma cholesterol in standard chow (SC) mice (66.8 ± 2.5 mg/dl versus 213 ± 12 mg/dl) and in HSHF mice (84.6 ± 4.4 mg/dl versus 464 ± 25 mg/dl). The HSHF diet induced cardiac hypertrophy and pathological remodeling, which were potently counteracted by AAV8-LDLr gene transfer. Wet lung weight was 19.0% (p < 0.001) higher in AAV8-null HSHF mice than in AAV8-null SC mice, whereas lung weight was normal in AAV8-LDLr HSHF mice. Pressure–volume loop analysis was consistent with HFpEF in AAV8-null HSHF mice and showed a completely normal cardiac function in AAV8-LDLr HSHF mice. Treadmill exercise testing demonstrated reduced exercise capacity in AAV8-null HSHF mice but a normal capacity in AAV8-LDLr HSHF mice. Reduced oxidative stress and decreased levels of tumor necrosis factor-α may mediate the beneficial effects of cholesterol lowering. In conclusion, AAV8-LDLr gene therapy prevents HFpEF.Hypercholesterolemia may be causally related to heart failure with preserved ejection fraction (HFpEF). We aimed to establish a HFpEF model associated with hypercholesterolemia and type 2 diabetes mellitus by feeding a high-sucrose/high-fat (HSHF) diet to C57BL/6J low-density lipoprotein receptor (LDLr) Overwhelming epidemiological evidence shows that diabetes mellitus is independently associated with heart failure incidence, with the risk being increased by more than twofold in men and by more than fivefold in women . The mosDiabetes mellitus is associated with dyslipidemia, which may also result in pro-inflammatory, pro-oxidative, and pro-fibrotic effects contributing to HFpEF. Dyslipidemia increases the incidence rate of heart failure that is independent of its association with myocardial infarction . In the Given the profound difference in the metabolism of fructose and glucose , the hyp−/− mice with the control vector AAV8-null or with AAV8-LDLr containing hepatocyte-specific transcriptional regulatory sequences to induce expression of the murine LDLr in parenchymal liver cells was performed at the age of 11 weeks. The high-sugar/high-fat (HSHF) diet was initiated in two groups one week later. The two other groups received a standard chow (SC) diet. Endpoint analyses in all groups were performed at the age of 28 weeks.The global study design evaluating the effect of AAV8-LDLr gene transfer on the development of HFpEF in HSHF diet mice is illustrated in −/− mice, wild-type C57BL/6J mice, and C57BL/6J LDLr−/− mice treated with 3 × 1012 genome copies/kg of AAV8-LDLr is illustrated in −/− mice. Murine LDLr expression 17 weeks after gene transfer with AAV8-LDLr in C57BL/6J LDLr−/− mice was 4.26-fold (p < 0.05) higher compared to wild-type C57BL/6J mice.Murine LDLr expression in the liver of C57BL/6 LDLrp < 0.0001) higher at 4 weeks, 1.28-fold (p < 0.0001) higher at 8 weeks, 1.38-fold (p < 0.0001) higher at 12 weeks, and 1.49-fold (p < 0.0001) higher at 16 weeks. AAV8-LDLr gene transfer did not affect body weight in SC diet mice but attenuated weight gain in HSHF diet mice. Body weight in AAV8-LDLr HSHF diet mice was reduced by 10.7% (p < 0.001) at 4 weeks, by 15.3% (p < 0.0001) at 8 weeks, by 16.6% (p < 0.0001) at 12 weeks, and by 18.7% (p < 0.0001) at 16 weeks compared to AAV8-null HSHF diet mice. Daily food intake measured during the entire duration of the experiment was not significantly different between AAV8-null HSHF diet mice (5.93 ± 0.37 g/mouse/day) and AAV8-LDLr HSHF diet mice (6.23 ± 0.33 g/mouse/day). The HSHF diet induced diabetes mellitus (p < 0.0001) higher at 8 weeks and 1.26-fold (p < 0.0001) higher at 16 weeks compared to AAV8-null SC diet mice. Blood glucose levels in AAV8-LDLr HSHF diet mice were significantly lower at 8 weeks (p < 0.05), at 12 weeks (p < 0.05), and at 16 weeks (p < 0.0001) compared to AAV8-null HSHF diet mice.The time course of the body weight in SC-diet and HSHF-diet mice is shown in mellitus B. Blood p < 0.001) higher in AAV8-null HSHF diet mice than in AAV8-null SC diet mice. AAV8-LDLr gene transfer abrogated adipocyte hypertrophy induced by the HSHF diet (p < 0.001) in AAV8-LDLr HSHF diet mice compared to AAV8-null HSHF diet mice. Adipocyte density was reduced by 59.5% (p < 0.001) in AAV8-null HSHF diet mice compared to AAV8-null SC diet mice and was not reduced at all in AAV8-LDLr HSHF diet mice.To evaluate the effect of AAV8-LDLr gene transfer on adiposity induced by the HSHF diet, a histological analysis of the gonadal fat pad was performed . The adiSHF diet A. The adp < 0.001) and 3.98-fold (p < 0.001) higher in AAV8-null HSHF diet mice and AAV8-LDLr HSHF diet mice, respectively, compared to respective SC diet groups (p < 0.01) lower in AAV8-LDLr HSHF diet mice than in AAV8-null HSHF diet mice (The HSHF diet induced hyperinsulinemia. Plasma insulin levels were 4.93-fold (t groups A. Plasmaiet mice B. No sigiet mice C.p < 0.001) higher in AAV8-null HSHF diet mice compared to AAV8-null SC diet mice (p < 0.001) and by 81.8% (p < 0.001) in SC diet mice and in HSHF diet mice, respectively. Plasma non-HDL cholesterol levels were increased by 2.32-fold (p < 0.001) in AAV8-null HSHF diet mice compared to AAV8-null SC diet mice (p < 0.001) and 89.8% (p < 0.001) lower in AAV8-LDLr SC diet mice and AAV8-LDLr HSHF diet mice, respectively, compared to respective AAV8-null groups. Plasma HDL cholesterol was 19.1% (p < 0.01) lower in AAV8-LDLr HSHF diet mice than in AAV8-null HSHF diet mice (Lipid levels in SC diet mice and in HSHF diet mice are represented in iet mice A. AAV8-Liet mice B. Plasmaiet mice C. Plasmaiet mice D.p < 0.001) increase of the wet heart weight and a 1.18-fold increase (p < 0.001) increase of heart weight normalized to tibia length. Wet heart weight was 16.0% (p < 0.001) lower in AAV8-LDLr HSHF diet mice compared to AAV8-null HSHF diet mice. Left ventricular weight and right ventricular weight in AAV8-null HSHF diet mice were 1.23-fold (p < 0.001) and 1.35-fold (p < 0.01) higher, respectively, than in AAV8-null SC diet mice (p < 0.001) and by 24.1% (p < 0.01), respectively, compared to AAV8-null HSHF diet mice. Wet lung weight was increased 1.19-fold (p < 0.001) in AAV8-null HSHF diet mice compared to AAV8-null SC diet mice indicating the presence of heart failure. In contrast, wet lung weight was not higher in AAV8-LDLr HSHF diet mice than in AAV8-LDLr SC diet mice and was 12.6% (p < 0.001) lower than in AAV8-null HSHF diet mice. Moreover, liver weight, kidney weight, and spleen weight were significantly (p < 0.001) increased in AAV8-null HSHF diet mice compared to AAV8-null SC diet mice, whereas no increase was observed in AAV8-LDLr HSHF diet mice. The degree of liver steatosis was 1.98-fold (p < 0.01) higher in AAV8-null HSHF diet mice than in AAV8-null SC diet mice (p < 0.01) in AAV8-LDLr HSHF diet mice compared to AAV8-null HSHF diet mice. Representative photomicrographs illustrating hematoxylin- and eosin-stained liver tissue are shown in Organ and tissue weights in AAV8-null SC diet mice, AAV8-LDLr SC diet mice, AAV8-null HSHF diet mice, and AAV8-LDLr HSHF diet mice are represented in iet mice . Left veiet mice A. Liver p < 0.001) and 1.27-fold (p < 0.001) larger, respectively, than in AAV8-null SC diet mice (p < 0.01) and by 15.6% (p < 0.001), respectively, compared to AAV8-null HSHF diet mice. This was paralleled at the microscopic level by a significant (p < 0.001) increase of the cardiomyocyte cross-sectional area in AAV8-null HSHF diet mice compared to AAV8-null SC diet mice and a significantly (p < 0001) lower cardiomyocyte cross-sectional area in AAV8-LDLr HSHF diet mice than in AAV8-null HSHF diet mice. Moreover, the hypertrophy in HSHF diet mice was pathological as evidenced by the decrease of capillary density (p < 0.01) and relative vascularity (p < 0.01) and by the increased myocardial fibrosis (p < 0.001). Capillary density and relative vascularity were prominently (p < 0.01) increased in AAV8-LDLr HSHF diet mice compared to AAV8-null HSHF diet mice. Moreover, the degree of interstitial fibrosis and of perivascular fibrosis in AAV8-LDLr HSHD diet mice were 37.6% (p < 0.001) and 22.0% (p < 0.05) lower, respectively, in AAV8-LDLr HSHF diet than in AAV8-null HSHF diet mice (p < 0.001). Representative Sirius Red-stained cross-sections of SC diet and HSHF diet mice are shown in Morphometric analysis demonstrated that left ventricular wall area and anterior wall thickness in AAV8-null HSHF diet mice were 1.24-fold (iet mice . Left vep < 0.001) decrease in the maximal rate of isovolumetric contraction (dP/dtmax), a 19.7% (p < 0.05) reduction of the preload recruitable stroke work (PRSW), a 29.7% (p < 0.05) decrease of end-systolic elastance (Ees), and a 25.7% reduction (p < 0.01) of the absolute value of the peak emptying rate (dV/dtmax) compared to AAV8-null SC diet mice. AAV8-null HSHF diet mice were characterized by a 20.1% (p < 0.01) reduction of the absolute value of the maximal rate of isovolumetric relaxation (dP/dtmin), a 1.27-fold (p < 0.001) increase in the time constant of isovolumetric relaxation tau, a 1.78-fold (p < 0.01) increase in the slope of the end-diastolic pressure–volume relationship (EDPVR), and a 28.5% (p < 0.01) reduction of the peak filling rate (dV/dtmin). These alterations led to a 27.9% (p < 0.001) and a 28.8% (p < 0.001) reduction of stroke volume and of cardiac output, respectively, in AAV8-null HSHF diet mice compared to AAV8-null SC diet mice. Ejection fraction was above 50% in AAV8-null HSHF diet mice, indicating that these mice were affected by HFpEF. Consistently, the end-diastolic volume (EDV) was reduced by 20.3% (p < 0.05) in AAV8-null HSHF diet mice compared to AAV8-null SC diet mice. Ventriculo–arterial coupling was impaired in AAV8-null HSHF diet mice, as evidenced by a 2.07-fold (p < 0.05) increase in the ventriculo–arterial coupling ratio. AAV8-LDLr gene transfer induced a supernormal cardiac function in SC diet mice, as evidenced by a 19.3% (p < 0.01) increase in dP/dtmax, a significantly increased ejection fraction (p < 0.01), and a 1.26-fold (p < 0.05) increase in the absolute value of the peak emptying rate (dV/dtmax). More importantly, AAV8-LDLr gene transfer in HSHF diet mice normalized systolic function  and diastolic function . Moreover, stroke volume, cardiac output, and ventriculo–arterial coupling were completely normalized. Taken together, cholesterol-lowering gene therapy completely prevents diabetic HFpEF in C57BL/6J LDLr−/− mice fed the HSHF diet.Pressure–volume loop data obtained in AAV8-null SC diet mice, AAV8-LDLr SC diet mice, AAV8-null HSHF diet mice, and AAV8-LDLr HSHF diet mice are summarized in p < 0.001) in AAV8-null HSHF diet compared to AAV8-null SC diet mice (p < 0.001) higher than in AAV8-null HSHF diet mice. Lactate post-exercise levels were 1.60-fold (p < 0.01) higher in AAV8-null HSHF diet than in AAV8-null SC diet mice (p < 0.001) reduction of lactate post-exercise levels was observed in AAV8-LDLr HSHF diet mice compared to AAV8-null HSHF diet mice.Exercise treadmill testing was applied to evaluate exercise capacity in the two SC diet groups and in the two HSHF diet groups A. The diiet mice B. In coniet mice C. A 46.9p < 0.001) higher in AAV8-null HSHF diet than in AAV8-null SC diet mice (p < 0.001) reduction of plasma TNF-α was observed in AAV8-LDLr HSHF diet mice compared to AAV8-null HSHF diet mice  cholesterol-lowering gene therapy potently counteracted structural remodeling and normalized cardiac function in HSHF diet mice; 3) HFpEF in HSHF diet mice was completely prevented by cholesterol-lowering gene therapy, as evidenced by the normal wet lung weight and by the normal exercise capacity; 4) cholesterol-lowering gene therapy strongly reduced nitro-oxidative stress and plasma TNF-α levels in HSHF diet mice, which may be important mediators of the beneficial effects of this treatment on cardiac structure and function.The principal findings of the current study are that 1) feeding an HSHF diet in female C57BL/6J LDLrPreviously, we demonstrated that cholesterol-lowering gene therapy improves survival, counteracts left ventricular hypertrophy, attenuates metabolic remodeling, and improves cardiac function in a HFrEF model . In this−/− mice are characterized by a preserved oxidative phosphorylation efficiency but have at the same time a higher net production of reactive oxygen species [Oxidative stress, which reflects an excess production of reactive oxygen species relative to antioxidant defense systems, contributes to cardiac remodeling and the development of heart failure . Mitocho species . When th species  and cell species ,19,20,21 species . The redEvidence for a causal role of TNF-α in diabetic heart failure has been provided in a rat model of streptozotocin-induced diabetes mellitus . ElevateThe HSHF diet resulted in marked hyperglycemia and marked hyperinsulinemia, indicating that insulin resistance and type 2 diabetes mellitus were induced by this diet. A limitation of the current study is that the weights of fat pads were not directly determined. However, the quantitative data on adipocyte hypertrophy provide evidence for the presence of an enlargement of adipose tissue . Type 2 A surprising finding in this study is that AAV8-LDLr gene therapy attenuated the weight gain in mice receiving the HSHF diet. This difference is not explained by a difference in food intake since daily food intake was not different between AAV8-null HSHF diet mice and AAV8-LDLr HSHF diet mice. In general, lipoprotein metabolic pathways are a pivotal contributor to the development of obesity. Modulation of lipolysis, receptor-mediated clearance of triglyceride-rich lipoproteins, and cross-talk between the liver and adipose tissue are major regulators of energy expenditure, whole-body homeostasis, and body weight. Apolipoprotein E is a key protein in the clearance of chylomicron remnants, LDL, and very low-density lipoproteins (VLDL). Apolipoprotein E-deficient mice are resistant to diet-induced obesity, insulin resistance, and glucose intolerance ,33,34. A−/− mice may attenuate the increase in adiposity when placed on an HSHF diet. Indeed, body weight in AAV8-null C57BL/6J LDLr−/− HSHF diet mice was 9.38% (p < 0.05) lower than in wild-type C57BL/6J HSHF diet mice with exactly the same genetic background (unpublished data). This difference is also consistent with observations in humans showing that body mass index in patients with heterozygous familial hypercholesterolemia is significantly lower compared to unaffected relatives [−/− HSHF diet mice may further attenuate weight gain by potentiation of postprandial catabolism of triglyceride-rich lipoproteins and may explain the significantly lower body weight and the absence of adipocyte hypertrophy in AAV8-LDL C57BL/6J LDLr−/− HSHF diet mice compared to AAV8-null C57BL/6J LDLr−/− HSHF diet mice.Modulation of body weight by the LDLr may be tissue-dependent. On the one hand, absence of the LDLr in adipocytes of C57BL/6J LDLrelatives . On the −/− mice compared to AAV8-null C57BL/6J LDLr−/− HSHF diet mice, but this observation is confounded by the difference in body weight of both conditions.Several lines of evidence indicate the existence of a link between cholesterol levels and diabetes risk. Major randomized clinical trials and meta-analyses of these trials have indicated that statin therapy, which inhibits 3-hydroxy-3-methylglutaryl-coenzyme A reductase, is associated with a modestly higher risk of developing type 2 diabetes mellitus in a dose-dependent fashion ,42. MoreThe impact of cholesterol-lowering therapies on clinical heart failure is an area full of controversy. The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins), which constitute the major class of hypolipidemic drugs, reduce heart failure events in primary and secondary prevention trials . MoreoveIn conclusion, cholesterol-lowering gene therapy prevents cardiac remodeling, cardiac dysfunction, and HFpEF in a model of obesity and type 2 diabetes mellitus induced by an HSHF diet. This study corroborates the view that hypercholesterolemia may causally contribute to HFpEF.1-antitrypsin promoter fused together with 2 copies of the 160 bp α1-microglobulin enhancer [12 genome copies/kg of AAV8-LDLr. Control mice were treated with an equivalent dose of AAV8-null. The AAV8-null control vector contains the same transcriptional regulatory sequences compared to AAV8-LDLr but no insert. AAV vector production was performed as described [Cholesterol-lowering gene therapy was performed using an adeno-associated viral (AAV) serotype 8 vector containing a hepatocyte-specific expression cassette to induce expression of the murine LDLr (AAV8-LDLr) ,50. The escribed .−/− mice, originally purchased from Jackson Laboratories , were locally bred at the semi-specific pathogen free facility of the Catholic University of Leuven at Gasthuisberg. The study design is illustrated in All experimental procedures in animals were executed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee of the Catholic University of Leuven  . C57BL/6Group assignment at the start of the study was performed randomly. No mice died during the study. No mice were excluded from the analysis. Endpoint analyses were performed by investigators who were blinded to the group allocation of the mice. Unblinding of animal numbers corresponding to specific allocation groups was performed at the completion of measurements.Liver tissue samples were isolated and immediately frozen in liquid nitrogen and stored at –80 °C. The extraction, blotting, and protein quantification procedures have been described in detail before . The pri® ultra-miniature pressure-volume (PV) loop catheter PVR-1035 (1.0 French polyimide catheter), the MPVS Ultra Single Segment Pressure-Volume Unit, and a PowerLab 16/35 data acquisition system .Invasive hemodynamic measurements were performed before sacrifice following anesthesia induced by intraperitoneal administration of 1.2 g/kg urethane (Sigma) . Measureg for 10 min and stored at –20 °C [Blood was obtained by puncture of the retro-orbital plexus. Anticoagulation was performed with 0.1 volume of 136 mmol/L trisodium citrate. Subsequently, plasma was immediately isolated by centrifugation at 1100× t –20 °C ,55,56.−/− mice were determined using the Cholesterol Quantification kit from Sigma-Aldrich . HDL and non-HDL lipoproteins were separated by ultracentrifugation as described [Plasma cholesterol and lipoprotein cholesterol levels in C57BL/6J LDLrescribed . Plasma Plasma free fatty (FFA) levels were determined using the Free Fatty Acids Quantification Kit  according to the instructions of the manufacturer .−/− mice were measured using the Ultra Sensitive Mouse Insulin enzyme-linked immunosorbent assay (ELISA) Kit . Murine plasma adiponectin was determined by ELISA according to the instructions of the manufacturer . Plasma tumor necrosis factor (TNF)-α levels were quantified using the Mouse TNF-α Platinum ELISA .Plasma insulin levels in C57BL/6J LDLr−/− mice were perfused via the abdominal aorta with phosphate-buffered saline and hearts were arrested in diastole by KCl , followed by perfusion fixation with 1% paraformaldehyde in phosphate-buffered saline. Thereafter, hearts were post-fixated overnight in 1% paraformaldehyde and embedded in paraffin. Cross-sections of 6 μm thickness at 130 μm spaced intervals were made, extending from the apex to the basal part of the left ventricle. Comparative sections were analyzed for all histological analyses by using the same slide numbers (1–40 from apex to base) and cross-section numbers (1–10).Histological analyses were performed as described before ,55,56. ATo measure collagen content in the interstitium, Sirius Red staining was performed as described by Junqueira et al. . Sirius 2) of at least 200 randomly selected cardiomyocytes in the left ventricular myocardium. Capillary density in the myocardium was determined on CD31-stained sections using rat anti-mouse CD31 antibodies . Relative vascularity was calculated as the ratio of capillary density to cardiomyocyte density divided by the cardiomyocyte cross-sectional area [−2. Two mid-ventricular cross-sections were analyzed per mouse [Cardiomyocyte hypertrophy was analyzed on paraffin sections stained with rabbit anti-mouse laminin  by measuring the cardiomyocyte cross-sectional area .2) and density (number/mm2) were determined on images taken at 100x magnification. The computerized image analysis was performed using KS300 software (Zeiss).Hematoxylin and eosin staining was performed on paraffin sections with a thickness of 10 μm. The gonadal adipocyte cross-sectional area  for ≥5 s. At this point, the mouse was immediately removed from the treadmill. The total exercise time was recorded as the elapsed time to exhaustion (min) and was then converted to distance (m), which was the end-point. Mice were subjected to tail snip before and after exercise the tolerance test for a lactate analysis .A motor-driven treadmill  was applied to evaluate the exercise capacity in mice ,61. C57BNo mice died during the study. At the end of the study, data of all surviving mice were included in the analysis. Investigators who performed endpoint analyses were blinded to group allocation. Unblinding of animal numbers corresponding to specific allocation groups was performed at the completion of measurements.n = 13) for proving the effect of AAV8-LDLr gene transfer on hemodynamic parameters in HSHF diet mice was based on a statistical power of 90%, a two-sided cut-off value of statistical significance of 0.05, a difference of main hemodynamic parameters at the population level of 20%, and a standard deviation at population level at 16% of the average of population means. Parameters between four groups were compared by one-way analysis of variance followed by Bonferroni multiple comparisons post-test for comparing SC diet groups, HSHF diet groups, and SC diet groups versus the respective HSHF diet groups using GraphPad InStat . When the assumption of sampling from populations with identical standard deviations was not met, a logarithmic transformation was performed. When the assumption of sampling from populations with Gaussian distributions was not met, a Kruskal-Wallis test was performed, followed by a Dunn’s multiple comparisons post-test. A two-sided p-value of less than 0.05 was considered statistically significant.Statistical analysis was performed as outlined before ,56. Data


Malt1 protease-dead knock-in  mice suffer from autoimmune disease have somewhat tempered the initial enthusiasm. Although it has been proposed that an imbalance between immune suppressive regulatory T cells (Tregs) and activated effector CD4+ T cells plays a key role in the autoimmune phenotype of Malt1-PD mice, the specific contribution of MALT1 proteolytic activity in T cells remains unclear. Using T cell-conditional Malt1 protease-dead knock-in  mice, we here demonstrate that MALT1 has a T cell-intrinsic role in regulating the homeostasis and function of thymic and peripheral T cells. T cell-specific ablation of MALT1 proteolytic activity phenocopies mice in which MALT1 proteolytic activity has been genetically inactivated in all cell types. The Malt1-PDT mice have a reduced number of Tregs in the thymus and periphery, although the effect in the periphery is less pronounced compared to full-body Malt1-PD mice, indicating that also other cell types may promote Treg induction in a MALT1 protease-dependent manner. Despite the difference in peripheral Treg number, both T cell-specific and full-body Malt1-PD mice develop ataxia and multi-organ inflammation to a similar extent. Furthermore, reconstitution of the full-body Malt1-PD mice with T cell-specific expression of wild-type human MALT1 eliminated all signs of autoimmunity. Together, these findings establish an important T cell-intrinsic role of MALT1 proteolytic activity in the suppression of autoimmune responses.MALT1 is a central signaling component in innate and adaptive immunity by regulating NF-κB and other key signaling pathways in different cell types. Activities of MALT1 are mediated by its scaffold and protease functions. Because of its role in lymphocyte activation and proliferation, inhibition of MALT1 proteolytic activity is of high interest for therapeutic targeting in autoimmunity and certain lymphomas. However, recent studies showing that  MALT1 (PCASP1 or paracaspase-1) is ubiquitously expressed in multiple cell types, where it is involved in signaling leading to pro-inflammatory gene expression. The best known role of MALT1 is the activation of NF-κB signaling in response to T cell and B cell antigen receptor triggering, which is critical for the adaptive immune system. At the border between the innate and adaptive immune system, MALT1 acts downstream of several innate immune receptors on antigen-presenting dendritic cells (DC), which in turn regulate cytokine production that can influence the polarization of the adaptive immune system. MALT1 is also critical for NF-κB induction, but not for degranulation responses, downstream of several ITAM-coupled natural killer cell (NK) receptors and the Fcε receptor on mast cells –8. A MALMalt1-PD mice  mice. In order to better predict safety risks upon pharmacological MALT1 protease inhibition and get further insight into the role of T cells in the autoimmune pathology of Malt1-PD mice, we employed conditional KI mice lacking MALT1 proteolytic activity specifically in T cells , and compared their phenotype with mice lacking MALT1 proteolytic activity in all cell types . To verify that only MALT1 protease deficiency in T cells can cause the disease, we also reconstituted Malt1-PD mice with transgenic T cell-specific expression of human wild-type (WT) MALT1. Taken together, we show that T cell-specific inactivation of MALT1 proteolytic activity phenocopies inactivation of MALT1 in all cell types, resulting in a disruption in T cell immune homeostasis and development of autoimmunity, indicating an important T cell-intrinsic role of MALT1 proteolytic activity in keeping normal immune homeostasis.MALT1 belongs to the type 1 paracaspase family, an ancient protein family that originated before the last common ancestor of planulozoa (bilaterians and cnidarians) , 23. The-PD mice –6, 47, w-PD mice . The gennd Tregs . Much of disease . HoweverMalt1-PD mice, all previous studies have relied on crosses with other knock-out (KO) lines , bone marrow transplants and injection of WT Tregs  all behaved as WT and were consequently all used as phenotypic controls  of mice already suffering from ataxia. We decided to analyze cLN as they are located close to known inflamed organs. Both Malt1-PD and -PDT mice have a slight reduction in total CD4+ T cells and an increase in CD4+ T cells that express CD44, so-called effector CD4+ T cells compared to WT control mice . To assess the effect of MALT1 proteolytic activity on the percentage of Tregs (Foxp3+CD4+ T cells) with surface CTLA-4 expression, we isolated cells from cLN of young disease-free Malt1-PDT mice and analyzed CTLA-4 surface expression by flow cytometry. This revealed that the percentage of surface CTLA-4 expressing Tregs was also reduced in Malt1-PDT mice compared to control mice (+ T cells (CD44+CD4+ T cells), where reduced expression is associated with elevated tissue infiltration and suppression of autoreactivity  were previously shown to suffer from severe autoimmunity    may alsoeg cells , 66.Malt1-PD and Malt1-PDT mice develop equally severe disease symptoms, disease development cannot be solely explained by reduced peripheral Treg levels. Therefore, we also assessed the Treg surface expression of CTLA-4, which is a known Treg functionality marker and whose deficiency partially leads to similar disease symptoms as those seen in Malt1-PD and -PDT mice  or is removed during early T cell differentiation . In contrast, upon pharmacological inhibition with for example small compound inhibitors of MALT1, Tregs are MALT1 protease proficient before the treatment is started, which may result in a different outcome. While the current paper was written, however, several studies using Malt1FL/PDFoxp3-CreTg/+ mice that specifically express catalytically inactive MALT1 in Tregs indicated that post-development loss of MALT1 proteolytic activity in Tregs leads to severe autoimmune disease . Human MALT1  was cloned into pENTR221 together with a genotype-specific barcode sequence downstream of the stop codon for genotyping. The ENTR clones were integrated in the pRMCE_DV1 (LMBP 8870) destination vector to generate RMCE targeting vectors for MALT1 .Plasmids of the cloned genes were deposited in the BCCM/GeneCorner plasmid collection along with detailed descriptions of cloning strategy and plasmid sequence Hmgu/+ ES cells  Hmgu/+ mice were also back-crossed with a germline Cre-line and back crossed to generate Cre-negative co-isogenic Malt1LacZ/−LacZ− full-body KO mice. Malt1PD/+ (the mouse MALT1 C472A mutation that disrupts protease activity) mice were generated in a C57/BL6 background by CRISPR/Cas9  in breeding to avoid suffering from the autoimmune phenotype. Rosa26LSL-MALT1-WT transgenic mice were crossed with CD4-Cre and subsequently back-crossed with full-body Malt1 KO mice, and later back-crossed to Malt1PD/+ mice to generate rescued Malt1PD/−Rosa26LSL−MALT1−WTCD4-Cre and Malt1PD/PDRosa26LSL−MALT1−WTCD4-Cre mice where all cells except T cells are MALT1 protease deficient. The transgenes were confirmed by western blot . All experiments were done according to the UGent ethical guidelines with the local ethical committee approval number: EC 2015-031.In order to generate conditional ES cells , which wES cells  to gener CD4-Cre  line andCD4-Cre) –91. The SPR/Cas9  by Cyageern blot  for specMalt1 Flox-allele or KO allele from Eucomm was monitored with the primers MALTcKO-F (GTTTCTCAGGTCTTTAGTTCATGTC), CoMLT-3-R (TATACTCTACATCTCCATGGT), MALTcKO-R (TTGTTTTGCAGATCTCTGCC), and MLT-LacZ-F (TCGCTACCATTACCAGTTGGT) resulting in 280 bp (WT), 400 bp (FL), 345 (KO), or 514 bp (KO-LacZ) PCR products. SNP-specific primers were designed as described in Staal et al. , R-MALT1-KICA-GT (TGCTCTAGATCCACAGGTGTGGTT), KI-MALT-CA-F (AATGTGTTCCTGTTGGATATGGCCAG), and KI-MALT-WT-R (GAGACATTTTACCTTTTCCGACAC) resulting in 461 bp , 304 bp (PD) and 201 bp (WT) PCR fragments. The Rosa26LSL-MALT1-WT transgenes were genotyped with the primers RMCE-MALT-F (GCAGCTACTTGGTATCAAAGGATCT), RMCE-MALT-R (TTTCCACAACTATCCATGTCGC), generating an allele-independent 896 bp PCR fragment, and the allele-specific primer RMCE-MALT-WT-R (CCAATTTCTGAAAAATAGTGTCCAAC) generating an allele specific band of 537 bp. Flp was detected with the primers Flp-F (TTAGTTCAGCAGCACATGATG) and Flp-R (GGAGGATTTGATATTCACCTG), resulting in a 370 bp PCR fragment. Cre was detected with the primers Cre-F (TGCCACGACCAAGTGACAGCAATG) and Cre-R (AGAGACGGAAATCCATCGCTCG) producing a 374 bp PCR fragment. All genotypings were done using the GoTaq Green Hot Start (Promega) master mix, with a typical PCR program: 5 min 95°C denaturation, 35–40 cycles [30 s 95°C|30 s 52–60°C|60 s 72°C] and 10 min 72°C final elongation.The l et al. , with thData were obtained with a LSRII flow cytometer (BD Biosciences) and FlowJo Software  was used for data analysis.Cells from thymus and cLN were surfaced stained with Fixable Viability Dye eFluor506 (eBioscience), anti-CD16/CD32 Fc block , anti-CD3-eFluor450 , anti-CD4-FITC , anti-CD25-PercPcy5.5  for 20 min. Next, cells were permeabilized for 30 min, followed by 30 min of intracellular staining for anti-Foxp3-PE . For the intracellular staining, the Foxp3 buffer set (eBioscience) was used and all incubation steps were done on ice.Cells from cLN cultured in complete medium  were stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 4 h at 37°C. The cells were stained as mentioned above, but anti-CD44-APC-efluor780  and anti-CTLA-4 PE-eFluor610  were also included in the surface staining. For the other Treg effector markers, non-stimulated cells from cLN were stained as mentioned above, but anti-CD44-AlexaFluor700 , anti-CD62L-eFluor450  and anti-KLRG1-APC  or anti-TNFR2-BV421  and anti-ICOS-PE-Cy5  were also included in the surface stain.Cells from cLN were cultured in complete medium and stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) and brefeldin A (1 μg/ml) for 4–5 h at 37°C. Stimulated cells were washed, surface stained with anti-CD16/CD32, Fixable Viability Dye eFluor 506, anti-CD3-eFluor450, anti-CD4-FITC, anti-CD44-APCeFluor780, for 20 min. Next, cells were fixed and permeabilized for 30 min using the Foxp3 buffer set, followed by intracellular staining with anti-IFNγ-PE-Cy7  for 30 min.Spleen cells were surface stained with Fixable Viability Dye eFluor506 (eBioscience), anti-CD16/CD32 Fc block , anti-CD3-PE-Cy5 , anti-CD45R/B220 , anti-CD21/CD35-eFluor450 , and anti-CD23-PE-Cy7  for 20 min.Peripheral blood samples were collected for serum preparation. The levels of IL-2 (171-G5003M), IL-4 (171-G5005M), IL-6 (171-G5007M), IL-17 (171-G5013M), IFN-γ (171-G5017M), and TNF (171-G5023M) were determined by Bio-Plex (Biorad) according to the manufacturer's conditions. B cells were purified from spleen with the MagniSort Mouse B cell Enrichment Kit (eBioscience), cultured in complete medium  and stimulated overnight with 20 μM CpG1826 (Invivogen) as described . The levStomach, lacrimal gland and salivary gland were fixed with 4% paraformaldehyde and imbedded in paraffin. Sections (5 μm) were stained with haematoxylin and eosin. Images (100× magnification) were acquired with a BX51 discussion microscope (Olympus) with PixeLink camera under 100× magnification.All datasets generated for this study are included in the manuscript/All experiments were done according to the UGent ethical guidelines with the local ethical committee approval number: EC 2015-031.Malt1PD/+ C472A PD mice were generated by MB. TH generated the Rosa26LSL−hMALT1−WT transgenic mice and the Malt1tm1a(EUCOMM)Hmgu/+ mice from EUCOMM ES cells. AD, JS, MB, and RB contributed to the scientific discussion. AD, JS, and RB wrote the manuscript.AD and JS designed the experiments. AD performed all experiments, except for the weighing and ataxia scoring done by JS, YD, and AD. IS did experiments for MZ B cells. YD and MK assisted with genotyping of mice. The The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Part of the work was done during a research collaboration between VIB, CD3, and AstraZeneca on MALT1 inhibitors .


Scientific Reports 10.1038/s41598-019-55034-9, published online 27 December 2019Correction to: The Acknowledgements section in this Article is incomplete.“This project was supported by NSF-CREST center for Complex Material Design for Multidimensional Additive processing (CoManD) award # 1735968.”should read:“This project was supported by NSF-CREST center for Complex Material Design for Multidimensional Additive processing (CoManD) award # 1735968, and FAMU center of Health disparities research grant, RCMI 5U54MD007582-35 from NIH.”