Blockade of trans PD-L1 interaction with CD80 augments antitumor immunity.

PD-L1 has two receptors: PD-1 and CD80. Previous reports assumed that PD-L1 and CD80 interacted in trans, but recent reports showed that only cis PD-L1/CD80 interactions existed, and prevention of cis PD-L1/CD80 interactions on antigen-presenting cells (APCs) reduced antitumor immunity via augmenting PD-L1/PD-1 and CD80/CTLA4 interactions between T and APCs. Here, using tumor-bearing mice capable of cis and trans or trans only PD-L1/CD80 interactions, we show that trans PD-L1/CD80 interactions do exist between tumor and T cells, and the effects of trans PD-L1/CD80 interactions require tumor cell expression of MHC-I and T cell expression of CD28. The blockade of PD-L1/CD80 interactions in mice with both cis and trans interactions or with only trans interactions augments antitumor immunity by expanding IFN-γ-producing CD8+ T cells and IFN-γ-dependent NOS2-expressing tumor-associated macrophages. Our studies indicate that although cis and trans PD-L1/CD80 interactions may have opposite effects on antitumor immunity, the net effect of blocking PD-L1/CD80 interactions in vivo augments CD8+ T cell-mediated antitumor immunity.

Rare, Tightly-Bound, Multi-Cellular Clusters in the Pancreatic Ducts of Adult Mice Function Like Progenitor Cells and Survive and Proliferate After Acinar Cell Injury.

Pancreatic ductal progenitor cells have been proposed to contribute to adult tissue maintenance and regeneration after injury, but the identity of such ductal cells remains elusive. Here, from adult mice, we identify a near homogenous population of ductal progenitor-like clusters, with an average of 8 cells per cluster. They are a rare subpopulation, about 0.1% of the total pancreatic cells, and can be sorted using a fluorescence-activated cell sorter with the CD133highCD71lowFSCmid-high phenotype. They exhibit properties in self-renewal and tri-lineage differentiation (including endocrine-like cells) in a unique 3-dimensional colony assay system. An in vitro lineage tracing experiment, using a novel HprtDsRed/+ mouse model, demonstrates that a single cell from a cluster clonally gives rise to a colony. Droplet RNAseq analysis demonstrates that these ductal clusters express embryonic multipotent progenitor cell markers Sox9, Pdx1, and Nkx6-1, and genes involved in actin cytoskeleton regulation, inflammation responses, organ development, and cancer. Surprisingly, these ductal clusters resist prolonged trypsin digestion in vitro, preferentially survive in vivo after a severe acinar cell injury and become proliferative within 14 days post-injury. Thus, the ductal clusters are the fundamental units of progenitor-like cells in the adult murine pancreas with implications in diabetes treatment and tumorigenicity.

Donor programmed cell death 1 ligand 1 is required for organ transplant tolerance in major histocompatibility complex-mismatched mixed chimeras although programmed cell death 1 ligand 1 and major histocompatibility complex class II are not required for inducing chimerism.

Induction of major histocompatibility complex (MHC) human leukocyte antigen (HLA)-mismatched mixed chimerism is a promising approach for organ transplantation tolerance; however, human leukocyte antigen-mismatched stable mixed chimerism has not been achieved in the clinic. Tolerogenic dendritic cell (DC) expression of MHC class II (MHC II) and programmed cell death 1 ligand 1 (PD-L1) is important for immune tolerance, but whether donor-MHC II or PD-L1 is required for the induction of stable MHC-mismatched mixed chimerism and transplant tolerance is unclear. Here, we show that a clinically applicable radiation-free regimen can establish stable MHC-mismatched mixed chimerism and organ transplant tolerance in murine models. Induction of MHC-mismatched mixed chimerism does not require donor cell expression of MHC II or PD-L1, but donor-type organ transplant tolerance in the mixed chimeras (MC) requires the donor hematopoietic cells and the organ transplants to express PD-L1. The PD-L1 expressed by donor hematopoietic cells and the programmed cell death 1 expressed by host cells augment host-type donor-reactive CD4+ and CD8+ T cell anergy/exhaustion and differentiation into peripheral regulatory T (pTreg) cells in association with the organ transplant tolerance in the MC. Conversely, host-type Treg cells augment the expansion of donor-type tolerogenic CD8+ DCs that express PD-L1. These results indicate that PD-L1 expressed by donor-type tolerogenic DCs and expansion of host-type pTreg cells in MHC-mismatched MCs play critical roles in mediating organ transplant tolerance.

Tolerogenic anti-IL-2 mAb prevents graft-versus-host disease while preserving strong graft-versus-leukemia activity.

Donor T cells mediate both graft-versus-leukemia (GVL) activity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT). Development of methods that preserve GVL activity while preventing GVHD remains a long-sought goal. Tolerogenic anti-interleukin-2 (IL-2) monoclonal antibody (JES6-1) forms anti-IL-2/IL-2 complexes that block IL-2 binding to IL-2Rß and IL-2Rγ on conventional T cells that have low expression of IL-2Rα. Here, we show that administration of JES6 early after allo-HCT in mice markedly attenuates acute GVHD while preserving GVL activity that is dramatically stronger than observed with tacrolimus (TAC) treatment. The anti-IL-2 treatment downregulated activation of the IL-2-Stat5 pathway and reduced production of granulocyte-macrophage colony-stimulating factor (GM-CSF). In GVHD target tissues, enhanced T-cell programmed cell death protein 1 (PD-1) interaction with tissue-programmed cell death-ligand 1 (PD-L1) led to reduced activation of protein kinase-mammalian target of rapamycin pathway and increased expression of eomesodermin and B-lymphocyte-induced maturation protein-1, increased T-cell anergy/exhaustion, expansion of Foxp3-IL-10-producing type 1 regulatory (Tr1) cells, and depletion of GM-CSF-producing T helper type 1 (Th1)/cytotoxic T cell type 1 (Tc1) cells. In recipient lymphoid tissues, lack of donor T-cell PD-1 interaction with tissue PD-L1 preserved donor PD-1+TCF-1+Ly108+CD8+ T memory progenitors and functional effectors that have strong GVL activity. Anti-IL-2 and TAC treatments have qualitatively distinct effects on donor T cells in the lymphoid tissues, and CD8+ T memory progenitor cells are enriched with anti-IL-2 treatment compared with TAC treatment. We conclude that administration of tolerogenic anti-IL-2 monoclonal antibody early after allo-HCT represents a novel approach for preventing acute GVHD while preserving GVL activity.

Reversal of autoimmunity by mixed chimerism enables reactivation of ß cells and transdifferentiation of α cells in diabetic NOD mice.

Type 1 diabetes (T1D) results from the autoimmune destruction of ß cells, so cure of firmly established T1D requires both reversal of autoimmunity and restoration of ß cells. It is known that ß cell regeneration in nonautoimmune diabetic mice can come from differentiation of progenitors and/or transdifferentiation of α cells. However, the source of ß cell regeneration in autoimmune nonobese diabetic (NOD) mice remains unclear. Here, we show that, after reversal of autoimmunity by induction of haploidentical mixed chimerism, administration of gastrin plus epidermal growth factor augments ß cell regeneration and normalizes blood glucose in the firmly established diabetic NOD mice. Using transgenic NOD mice with inducible lineage-tracing markers for insulin-producing ß cells, Sox9+ ductal progenitors, Nestin+ mesenchymal stem cells, and glucagon-producing α cells, we have found that both reactivation of dysfunctional low-level insulin expression (insulinlo) ß cells and neogenesis contribute to the regeneration, with the latter predominantly coming from transdifferentiation of α cells. These results indicate that, after reversal of autoimmunity, reactivation of ß cells and transdifferentiation of α cells can provide sufficient new functional ß cells to reach euglycemia in firmly established T1D.

Frequent monoallelic or skewed expression for developmental genes in CNS-derived cells and evidence for balancing selection.

Cellular mosaicism due to monoallelic autosomal expression (MAE), with cell selection during development, is becoming increasingly recognized as prevalent in mammals, leading to interest in understanding its extent and mechanism(s). We report here use of clonal cell lines derived from the CNS of adult female [Formula: see text] hybrid (C57BL/6 X JF1) mice to characterize MAE as neural stem cells (nscs) differentiate to astrocyte-like cells (asls). We found that different subsets of genes show MAE in the two populations of cells; in each case, there is strong enrichment for genes specific to the respective developmental state. Genes that exhibit MAE are 22% of nsc-specific genes and 26% of asl-specific genes. Moreover, the promoters of genes with MAE have reduced CpG dinucleotides but increased CpG differences between the two parental mouse strains. Extending the study of variability to wild populations of mice, we found evidence for balancing selection as a contributing force in evolution of those genes showing developmental specificity (i.e., expressed in either nsc or asl), not just for genes showing MAE. Furthermore, we found that genes showing skewed allelic expression (SKE) were similarly enriched among cell type-specific genes and also showed a heightened probability of balancing selection. Thus, developmental stage-specific genes and genes with MAE or SKE seem to make up overlapping classes subject to selection for increased diversity. The implications of these results for development and evolution are discussed in the context of a model with stochastic epigenetic modifications taking place only during a relatively brief developmental window.

MHC-mismatched mixed chimerism restores peripheral tolerance of noncross-reactive autoreactive T cells in NOD mice.

Autoimmune type 1 diabetes (T1D) and other autoimmune diseases are associated with particular MHC haplotypes and expansion of autoreactive T cells. Induction of MHC-mismatched but not -matched mixed chimerism by hematopoietic cell transplantation effectively reverses autoimmunity in diabetic nonobese diabetic (NOD) mice, even those with established diabetes. As expected, MHC-mismatched mixed chimerism mediates deletion in the thymus of host-type autoreactive T cells that have T-cell receptor (TCR) recognizing (cross-reacting with) donor-type antigen presenting cells (APCs), which have come to reside in the thymus. However, how MHC-mismatched mixed chimerism tolerizes host autoreactive T cells that recognize only self-MHC-peptide complexes remains unknown. Here, using NOD.Rag1-/-BDC2.5 or NOD.Rag1-/-BDC12-4.1 mice that have only noncross-reactive transgenic autoreactive T cells, we show that induction of MHC-mismatched but not -matched mixed chimerism restores immune tolerance of peripheral noncross-reactive autoreactive T cells. MHC-mismatched mixed chimerism results in increased percentages of both donor- and host-type Foxp3+ Treg cells and up-regulated expression of programmed death-ligand 1 (PD-L1) by host-type plasmacytoid dendritic cells (pDCs). Furthermore, adoptive transfer experiments showed that engraftment of donor-type dendritic cells (DCs) and expansion of donor-type Treg cells are required for tolerizing the noncross-reactive autoreactive T cells in the periphery, which are in association with up-regulation of host-type DC expression of PD-L1 and increased percentage of host-type Treg cells. Thus, induction of MHC-mismatched mixed chimerism may establish a peripheral tolerogenic DC and Treg network that actively tolerizes autoreactive T cells, even those with no TCR recognition of the donor APCs.

Rlip depletion prevents spontaneous neoplasia in TP53 null mice.

TP53 (p53) is a tumor suppressor whose functions are lost or altered in most malignancies. p53 homozygous knockout (p53-/-) mice uniformly die of spontaneous malignancy, typically T-cell lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic acid pathway transporter protein that also functions as a Ral effector involved in clathrin-dependent endocytosis. In stark contrast to p53-/- mice, Rlip-/- mice are highly resistant to carcinogenesis. We report here that partial Rlip deficiency induced by weekly administration of an Rlip-specific phosphorothioate antisense oligonucleotide, R508, strongly inhibited spontaneous as well as benzo(a)pyrene-induced carcinogenesis in p53-/- mice. This treatment effectively prevented large-scale methylomic and transcriptomic abnormalities suggestive of inflammation found in cancer-bearing p53-/- mice. The remarkable efficiency with which Rlip deficiency suppresses spontaneous malignancy in p53-/- mice has not been observed with any previously reported pharmacologic or genetic intervention. These findings are supported by cross-breeding experiments demonstrating that hemizygous Rlip deficiency also reduces the spontaneous malignancy phenotype of p53+/- mice. Rlip is found on the cell surface, and antibodies directed against Rlip were found to inhibit growth and promote apoptosis of cell lines as effectively as Rlip siRNA. The work presented here investigates several features, including oxidative DNA damage of the Rlip-p53 association in malignant transformation, and offers a paradigm for the mechanisms of tumor suppression by p53 and the prospects of suppressing spontaneous malignancy in hereditary cancer syndromes such as Li-Fraumeni.

Elimination of human folypolyglutamate synthetase alters programming and plasticity of somatic cells.

Folates are vital cofactors for the regeneration of S-adenosyl methionine, which is the methyl source for DNA methylation, protein methylation, and other aspects of one-carbon (C1) metabolism. Thus, folates are critical for establishing and preserving epigenetic programming. Folypolyglutamate synthetase (FPGS) is known to play a crucial role in the maintenance of intracellular folate levels. Therefore, any modulation in FPGS is expected to alter DNA methylation and numerous other metabolic pathways. To explore the role of polyglutamylation of folate, we eliminated both isoforms of FPGS in human cells (293T), producing FPGS knockout (FPGSko) cells. The elimination of FPGS significantly decreased cell proliferation, with a major effect on oxidative phosphorylation and a lesser effect on glycolysis. We found a substantial reduction in global DNA methylation and noteworthy changes in gene expression related to C1 metabolism, cell division, DNA methylation, pluripotency, Glu metabolism, neurogenesis, and cardiogenesis. The expression levels of NANOG, octamer-binding transcription factor 4, and sex-determining region Y-box 2 levels were increased in the mutant, consistent with the transition to a stem cell-like state. Gene expression and metabolite data also indicate a major change in Glu and GABA metabolism. In the appropriate medium, FPGSko cells can differentiate to produce mainly cells with characteristics of either neural stem cells or cardiomyocytes.-Srivastava, A. C., Thompson, Y. G., Singhal, J., Stellern, J., Srivastava, A., Du, J., O'Connor, T. R., Riggs, A. D. Elimination of human folypolyglutamate synthetase alters programming and plasticity of somatic cells.

Differences in silencing of mismatched targets by sliced versus diced siRNAs.

It has been reported that the two major types of RNA interference triggers, the classical Dicer-generated small RNAs (siRNAs), which function with all members of the Argonaute (Ago) protein family in mammals, and the Ago2-sliced small RNAs (sli-siRNAs), which function solely through Ago2, have similar potency in target cleavage and repression. Here, we show that sli-siRNAs are generally more potent than siRNAs in silencing mismatched targets. This phenomenon is usually more apparent in targets that have mismatched nucleotides in the 3' supplementary region than in targets with mismatches in the seed region. We demonstrate that Ago2 slicer activity is a major factor contributing to the greater silencing efficiency of sli-siRNA against mismatched targets and that participation of non-slicing Agos in silencing mismatched siRNA targets may dilute the slicing ability of Ago2. The difference in length of the mature guide RNA used in sli-RISCs and si-RISCs may also contribute to the observed difference in knockdown efficiency. Our data suggest that a sli-siRNA guide strand is likely to have substantially stronger off-target effects than a guide strand with the same sequence in a classical siRNA and that Dicer and non-slicing Agos may play pivotal roles in controlling siRNA target specificity.

Analysis of high-resolution 3D intrachromosomal interactions aided by Bayesian network modeling.

Long-range intrachromosomal interactions play an important role in 3D chromosome structure and function, but our understanding of how various factors contribute to the strength of these interactions remains poor. In this study we used a recently developed analysis framework for Bayesian network (BN) modeling to analyze publicly available datasets for intrachromosomal interactions. We investigated how 106 variables affect the pairwise interactions of over 10 million 5-kb DNA segments in the B-lymphocyte cell line GB12878. Strictly data-driven BN modeling indicates that the strength of intrachromosomal interactions (hic_strength) is directly influenced by only four types of factors: distance between segments, Rad21 or SMC3 (cohesin components),transcription at transcription start sites (TSS), and the number of CCCTC-binding factor (CTCF)-cohesin complexes between the interacting DNA segments. Subsequent studies confirmed that most high-intensity interactions have a CTCF-cohesin complex in at least one of the interacting segments. However, 46% have CTCF on only one side, and 32% are without CTCF. As expected, high-intensity interactions are strongly dependent on the orientation of the ctcf motif, and, moreover, we find that the interaction between enhancers and promoters is similarly dependent on ctcf motif orientation. Dependency relationships between transcription factors were also revealed, including known lineage-determining B-cell transcription factors (e.g., Ebf1) as well as potential novel relationships. Thus, BN analysis of large intrachromosomal interaction datasets is a useful tool for gaining insight into DNA-DNA, protein-DNA, and protein-protein interactions.

SMC1A is associated with radioresistance in prostate cancer and acts by regulating epithelial-mesenchymal transition and cancer stem-like properties.

Prostate cancer is one of the most commonly diagnosed cancers and a pressing health challenge in men worldwide. Radiation therapy (RT) is widely considered a standard therapy for advanced as well as localized prostate cancer. Although this primary therapy is associated with high cancer control rates, up to one-third of patients undergoing radiation therapy becomes radio-resistant and/or has tumor-relapse/recurrence. Therefore, focus on new molecular targets and pathways is essential to develop novel radio-sensitizing agents for the effective and safe treatment of prostate cancer. Here, we describe functional studies that were performed to investigate the role of structural maintenance of chromosome-1 (SMC1A) in radioresistance of metastatic prostate cancer cells. Short hairpin RNA (shRNA) was used to suppress SMC1A in metastatic castration-resistant prostate cancer cells, DU145 and PC3. Clonogenic survival assays, Western blot, RT-PCR, and γ-H2AX staining were used to assess the effect of SMC1A knockdown on radiation sensitivity of these prostate cancer cells. We demonstrate that SMC1A is overexpressed in human prostate tumors compared to the normal adjacent tissue. SMC1A knockdown limits the clonogenic potential, epithelial-mesenchymal transition (EMT), and cancer stem-like cell (CSC) properties of DU145 and PC3 cells and enhanced efficacy of RT in these cells. Targeted inhibition of SMC1A not only plays a critical role in overcoming radio-resistance in prostate cancer cells, but also suppresses self-renewal and the tumor-propagating potential of x-irradiated cancer cells. We propose that SMC1A could be a potential molecular target for the development of novel radio-sensitizing therapeutic agents for management of radio-resistant metastatic prostate cancer.

Growth factors and medium hyperglycemia induce Sox9+ ductal cell differentiation into ß cells in mice with reversal of diabetes.

We previously reported that long-term administration of a low dose of gastrin and epidermal growth factor (GE) augments ß-cell neogenesis in late-stage diabetic autoimmune mice after eliminating insulitis by induction of mixed chimerism. However, the source of ß-cell neogenesis is still unknown. SRY (sex-determining region Y)-box 9(+) (Sox9(+)) ductal cells in the adult pancreas are clonogenic and can give rise to insulin-producing ß cells in an in vitro culture. Whether Sox9(+) ductal cells in the adult pancreas can give rise to ß cells in vivo remains controversial. Here, using lineage-tracing with genetic labeling of Insulin- or Sox9-expressing cells, we show that hyperglycemia (>300 mg/dL) is required for inducing Sox9(+) ductal cell differentiation into insulin-producing ß cells, and medium hyperglycemia (300-450 mg/dL) in combination with long-term administration of low-dose GE synergistically augments differentiation and is associated with normalization of blood glucose in nonautoimmune diabetic C57BL/6 mice. Short-term administration of high-dose GE cannot augment differentiation, although it can augment preexisting ß-cell replication. These results indicate that medium hyperglycemia combined with long-term administration of low-dose GE represents one way to induce Sox9(+) ductal cell differentiation into ß cells in adult mice.

Epigenomic profiling reveals an association between persistence of DNA methylation and metabolic memory in the DCCT/EDIC type 1 diabetes cohort.

We examined whether persistence of epigenetic DNA methylation (DNA-me) alterations at specific loci over two different time points in people with diabetes are associated with metabolic memory, the prolonged beneficial effects of intensive vs. conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) Study. We compared DNA-me profiles in genomic DNA of whole blood (WB) isolated at EDIC Study baseline from 32 cases (DCCT conventional therapy group subjects showing retinopathy or albuminuria progression by EDIC Study year 10) vs. 31 controls (DCCT intensive therapy group subjects without complication progression by EDIC year 10). DNA-me was also profiled in blood monocytes (Monos) of the same patients obtained during EDIC Study years 16-17. In WB, 153 loci depicted hypomethylation, and 225 depicted hypermethylation, whereas in Monos, 155 hypomethylated loci and 247 hypermethylated loci were found (fold change ≥1.3; P < 0.005; cases vs. controls). Twelve annotated differentially methylated loci were common in both WB and Monos, including thioredoxin-interacting protein (TXNIP), known to be associated with hyperglycemia and related complications. A set of differentially methylated loci depicted similar trends of associations with prior HbA1c in both WB and Monos. In vitro, high glucose induced similar persistent hypomethylation at TXNIP in cultured THP1 Monos. These results show that DNA-me differences during the DCCT persist at certain loci associated with glycemia for several years during the EDIC Study and support an epigenetic explanation for metabolic memory.

MHC-mismatched mixed chimerism augments thymic regulatory T-cell production and prevents relapse of EAE in mice.

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system with demyelination, axon damage, and paralysis. Induction of mixed chimerism with allogeneic donors has been shown to not cause graft-versus-host disease (GVHD) in animal models and humans. We have reported that induction of MHC-mismatched mixed chimerism can cure autoimmunity in autoimmune NOD mice, but this approach has not yet been tested in animal models of MS, such as experimental autoimmune encephalomyelitis (EAE). Here, we report that MHC-mismatched mixed chimerism with C57BL/6 (H-2(b)) donor in SJL/J (H-2(s)) EAE recipients eliminates clinical symptoms and prevents relapse. This cure is demonstrated by not only disappearance of clinical signs but also reversal of autoimmunity; elimination of infiltrating T, B, and macrophage cells in the spinal cord; and regeneration of myelin sheath. The reversal of autoimmunity is associated with a marked reduction of autoreactivity of CD4(+) T cells and significant increase in the percentage of Foxp3(+) Treg among host-type CD4(+) T cells in the spleen and lymph nodes. The latter is associated with a marked reduction of the percentage of host-type CD4(+)CD8(+) thymocytes and an increase of Treg percentage among the CD4(+)CD8(+) and CD4(+)CD8(-) thymocytes. Thymectomy leads to loss of prevention of EAE relapse by induction of mixed chimerism, although there is a dramatic expansion of host-type Treg cells in the lymph nodes. These results indicate that induction of MHC-mismatched mixed chimerism can restore thymic negative selection of autoreactive CD4(+) T cells, augment production of Foxp3(+) Treg, and cure EAE.

SAIC: an iterative clustering approach for analysis of single cell RNA-seq data.

BACKGROUND: Research interests toward single cell analysis have greatly increased in basic, translational and clinical research areas recently, as advances in whole-transcriptome amplification technique allow scientists to get accurate sequencing result at single cell level. An important step in the single-cell transcriptome analysis is to identify distinct cell groups that have different gene expression patterns. Currently there are limited bioinformatics approaches available for single-cell RNA-seq analysis. Many studies rely on principal component analysis (PCA) with arbitrary parameters to identify the genes that will be used to cluster the single cells. RESULTS: We have developed a novel algorithm, called SAIC (Single cell Analysis via Iterative Clustering), that identifies the optimal set of signature genes to separate single cells into distinct groups. Our method utilizes an iterative clustering approach to perform an exhaustive search for the best parameters within the search space, which is defined by a number of initial centers and P values. The end point is identification of a signature gene set that gives the best separation of the cell clusters. Using a simulated data set, we showed that SAIC can successfully identify the pre-defined signature gene sets that can correctly separated the cells into predefined clusters. We applied SAIC to two published single cell RNA-seq datasets. For both datasets, SAIC was able to identify a subset of signature genes that can cluster the single cells into groups that are consistent with the published results. The signature genes identified by SAIC resulted in better clusters of cells based on DB index score, and many genes also showed tissue specific expression. CONCLUSIONS: In summary, we have developed an efficient algorithm to identify the optimal subset of genes that separate single cells into distinct clusters based on their expression patterns. We have shown that it performs better than PCA method using published single cell RNA-seq datasets.

Enhanced evolution by stochastically variable modification of epigenetic marks in the early embryo.

Evolution by gene duplication is generally accepted as one of the crucial driving forces for the gain of new complexity and functions, but the formation of pseudogenes remains a problem for this mechanism. Here we expand on earlier ideas that epigenetic modifications can drive neo- and subfunctionalization in evolution by gene duplication. We explore the effects of stochastic epigenetic modifications on the evolution (and thus development) of complex organisms in a constant environment. Modeling is done both using a modified genetic drift analytical treatment and computer simulations, which were found to agree. A transposon silencing model is also explored. Some key assumptions made include (i) stochastic, incomplete removal (or addition) of repressive epigenetic marks takes place during a window(s) of opportunity in the zygote and early embryo; (ii) there is no statistical variation of the marks after the window closes; and (iii) the genes affected are sensitive to dosage. Our genetic drift treatment takes into account that after gene duplication the prevailing case upon which selection operates is a duplicate/singlet heterozygote; to the best of our knowledge, this has not been considered in previous treatments. We conclude from our modeling that stochastic epigenetic modifications, with rates consistent with experimental observation, can both increase the rate of gene fixation and decrease pseudogenization, thus dramatically improving the efficacy of evolution by gene duplication. We also find that a transposon silencing model is advantageous for fixation of recessive genes in diploid organisms, especially with large effective population sizes.

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells.

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity.

Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel.

The study of hematopoietic colony-forming units using semisolid culture media has greatly advanced the knowledge of hematopoiesis. Here we report that similar methods can be used to study pancreatic colony-forming units. We have developed two pancreatic colony assays that enable quantitative and functional analyses of progenitor-like cells isolated from dissociated adult (2-4 mo old) murine pancreas. We find that a methylcellulose-based semisolid medium containing Matrigel allows growth of duct-like "Ring/Dense" colonies from a rare (∼1%) population of total pancreatic single cells. With the addition of roof plate-specific spondin 1, a wingless-int agonist, Ring/Dense colony-forming cells can be expanded more than 100,000-fold when serially dissociated and replated in the presence of Matrigel. When cells grown in Matrigel are then transferred to a Matrigel-free semisolid medium with a unique laminin-based hydrogel, some cells grow and differentiate into another type of colony, which we name "Endocrine/Acinar." These Endocrine/Acinar colonies are comprised mostly of endocrine- and acinar-like cells, as ascertained by RNA expression analysis, immunohistochemistry, and electron microscopy. Most Endocrine/Acinar colonies contain beta-like cells that secrete insulin/C-peptide in response to D-glucose and theophylline. These results demonstrate robust self-renewal and differentiation of adult Ring/Dense colony-forming units in vitro and suggest an approach to producing beta-like cells for cell replacement of type 1 diabetes. The methods described, which include microfluidic expression analysis of single cells and colonies, should also advance study of pancreas development and pancreatic progenitor cells.