Comprehensive Scanning Mutagenesis of Human Retrotransposon LINE-1 Identifies Motifs Essential for Function.

Long Interspersed Nuclear Element-1 (LINE-1, L1) is the only autonomous active transposable element in the human genome. The L1-encoded proteins ORF1p and ORF2p enable the element to jump from one locus to another via a "copy-and-paste" mechanism. ORF1p is an RNA-binding protein, and ORF2p has endonuclease and reverse transcriptase activities.

KLF4 as a rheostat of osteolysis and osteogenesis in prostate tumors in the bone.

We previously showed that KLF4, a gene highly expressed in murine prostate stem cells, blocks the progression of indolent intraepithelial prostatic lesions into aggressive and rapidly growing tumors. Here, we show that the anti-tumorigenic effect of KLF4 extends to PC3 human prostate cancer cells growing in the bone. We compared KLF4 null cells with cells transduced with a DOX-inducible KLF4 expression system, and find KLF4 function inhibits PC3 growth in monolayer and soft agar cultures.

Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation.

Wolbachia is an intracellular bacterium that infects a remarkable range of insect hosts. Insects such as mosquitos act as vectors for many devastating human viruses such as Dengue, West Nile, and Zika. Remarkably, Wolbachia infection provides insect hosts with resistance to many arboviruses thereby rendering the insects ineffective as vectors.

Functional Interactions Between /S6K, /Notch, and Regulators of Fertility and Germline Stem Cell Maintenance.

The proper accumulation and maintenance of stem cells is critical for organ development and homeostasis. The Notch signaling pathway maintains stem cells in diverse organisms and organ systems. In , GLP-1/Notch activity prevents germline stem cell (GSC) differentiation.

LINE-1 protein localization and functional dynamics during the cell cycle.

LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology.

Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention.

Characterization and molecular profiling of PSEN1 familial Alzheimer's disease iPSC-derived neural progenitors.

Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations.

β-cell dysfunction due to increased ER stress in a stem cell model of Wolfram syndrome.

Wolfram syndrome is an autosomal recessive disorder caused by mutations in WFS1 and is characterized by insulin-dependent diabetes mellitus, optic atrophy, and deafness. To investigate the cause of β-cell failure, we used induced pluripotent stem cells to create insulin-producing cells from individuals with Wolfram syndrome. WFS1-deficient β-cells showed increased levels of endoplasmic reticulum (ER) stress molecules and decreased insulin content.

Engineering bone tissue substitutes from human induced pluripotent stem cells.

Congenital defects, trauma, and disease can compromise the integrity and functionality of the skeletal system to the extent requiring implantation of bone grafts. Engineering of viable bone substitutes that can be personalized to meet specific clinical needs represents a promising therapeutic alternative. The aim of our study was to evaluate the utility of human-induced pluripotent stem cells (hiPSCs) for bone tissue engineering.

Improved methods for reprogramming human dermal fibroblasts using fluorescence activated cell sorting.

Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes.

Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants.

Mitochondrial DNA mutations transmitted maternally within the oocyte cytoplasm often cause life-threatening disorders. Here we explore the use of nuclear genome transfer between unfertilized oocytes of two donors to prevent the transmission of mitochondrial mutations. Nuclear genome transfer did not reduce developmental efficiency to the blastocyst stage, and genome integrity was maintained provided that spontaneous oocyte activation was avoided through the transfer of incompletely assembled spindle-chromosome complexes.

A functionally characterized test set of human induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate.

B-lymphoid cells with attributes of dendritic cells regulate T cells via indoleamine 2,3-dioxygenase.

A discrete population of splenocytes with attributes of dendritic cells (DCs) and coexpressing the B-cell marker CD19 is uniquely competent to express the T-cell regulatory enzyme indoleamine 2,3-dioxygenase (IDO) in mice treated with TLR9 ligands (CpGs). Here we show that IDO-competent cells express the B-lineage commitment factor Pax5 and surface immunoglobulins. CD19 ablation abrogated IDO-dependent T-cell suppression by DCs, even though cells with phenotypic attributes matching IDO-competent cells developed normally and expressed IDO in response to interferon gamma.

T cell regulatory plasmacytoid dendritic cells expressing indoleamine 2,3 dioxygenase.

Mature dendritic cells (DCs) are potent stimulators of T cells that recognize antigens presented by the DCs. In this chapter we describe mature DCs that suppress T cell responses to antigens they present due to expression of the intracellular enzyme indoleamine 2,3 dioxygenase (IDO). IDO-competent DCs are a subset of plasmacytoid DCs that can be induced to express IDO under certain inflammatory conditions in humans and mice.

Chronic inflammation that facilitates tumor progression creates local immune suppression by inducing indoleamine 2,3 dioxygenase.

Topical application of phorbol myristate acetate (PMA) elicits intense local inflammation that facilitates outgrowth of premalignant lesions in skin after carcinogen exposure. The inflammatory response to PMA treatment activates immune stimulatory mechanisms. However, we show here that PMA exposure also induces plasmacytoid dendritic cells (pDCs) in local draining lymph nodes (dLNs) to express indoleamine 2,3 dioxygenase (IDO), which confers T cell suppressor activity on pDCs.

Role of CD28 in fatal autoimmune disorder in scurfy mice.

Scurfy mice develop CD4 T-cell-mediated lymphoproliferative disease leading to death within 4 weeks of age. The scurfy mutation causes loss of function of the foxp3 gene (foxp3(sf)), which is essential for development and maintenance of naturally occurring regulatory CD4 T cells (nTregs). In humans, mutations of the foxp3 gene cause immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX).

Cell-autonomous control of interferon type I expression by indoleamine 2,3-dioxygenase in regulatory CD19+ dendritic cells.

Following CD80/86 (B7) and TLR9 ligation, small subsets of splenic dendritic cells expressing CD19 (CD19(+) DC) acquire potent T cell regulatory functions due to induced expression of the intracellular enzyme indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan. In CD19(+) DC, IFN type I (IFN-alpha) is the obligate inducer of IDO. We now report that IFN-alpha production needed to stimulate high-level expression of IDO following B7 ligation is itself dependent on basal levels of IDO activity.

Cutting edge: CpG oligonucleotides induce splenic CD19+ dendritic cells to acquire potent indoleamine 2,3-dioxygenase-dependent T cell regulatory functions via IFN Type 1 signaling.

CpG oligodeoxynucleotides (CpG-ODNs) stimulate innate and adaptive immunity by binding to TLR9 molecules. Paradoxically, expression of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) is induced following i.v.

A minor population of splenic dendritic cells expressing CD19 mediates IDO-dependent T cell suppression via type I IFN signaling following B7 ligation.

By ligating CD80/CD86 (B7) molecules, the synthetic immunomodulatory reagent CTLA4-Ig (soluble synthetic CTLA4 fusion protein) induces expression of the enzyme indoleamine 2,3-dioxygenase (IDO) in some dendritic cells (DCs), which acquire potent T cell regulatory functions as a consequence. Here we show that this response occurred exclusively in a population of splenic DCs co-expressing the marker CD19. B7 ligation induced activation of the transcription factor signal transducer and activator of transcription (STAT1) in sorted CD19+, but not CD19(NEG), DCs.