Human follicular mites: Ectoparasites becoming symbionts.

Most humans carry mites in the hair follicles of their skin for their entire lives. Follicular mites are the only metazoans tha continuously live on humans. We propose that Demodex folliculorum (Acari) represents a transitional stage from a host-injuring obligate parasite to an obligate symbiont. Here, we describe the profound impact of this transition on the genome and physiology of the mite. Genome sequencing revealed that the permanent host association of D. folliculorum led to an extensive genome reduction through relaxed selection and genetic drift, resulting in the smallest number of protein-coding genes yet identified among panarthropods. Confocal microscopy revealed that this gene loss coincided with an extreme reduction in the number of cells. Single uninucleate muscle cells are sufficient to operate each of the three segments that form each walking leg. While it has been assumed that the reduction of the cell number in parasites starts early in development, we identified a greater total number of cells in the last developmental stage (nymph) than in the terminal adult stage, suggesting that reduction starts at the adult or ultimate stage of development. This is the first evolutionary step in an arthropod species adopting a reductive, parasitic or endosymbiotic lifestyle. Somatic nuclei show underreplication at the diploid stage. Novel eye structures or photoreceptors as well as a unique human host melatonin-guided day/night rhythm are proposed for the first time. The loss of DNA repair genes coupled with extreme endogamy might have set this mite species on an evolutionary dead-end trajectory.

Induction of phenotypic changes in HER2-postive breast cancer cells in vivo and in vitro.

The influence of breast cancer cells on normal cells of the microenvironment, such as fibroblasts and macrophages, has been heavily studied but the influence of normal epithelial cells on breast cancer cells has not. Here using in vivo and in vitro models we demonstrate the impact epithelial cells and the mammary microenvironment can exert on breast cancer cells. Under specific conditions, signals that originate in epithelial cells can induce phenotypic and genotypic changes in cancer cells. We have termed this phenomenon "cancer cell redirection." Once breast cancer cells are redirected, either in vivo or in vitro, they lose their tumor forming capacity and undergo a genetic expression profile shift away from one that supports a cancer profile towards one that supports a non-tumorigenic epithelial profile. These findings indicate that epithelial cells and the normal microenvironment influence breast cancer cells and that under certain circumstances restrict proliferation of tumorigenic cells.

Sequence conservation of mitochondrial (mt)DNA during expansion of clonal mammary epithelial populations suggests a common mtDNA template in CzechII mice.

One major foundation of cancer etiology is the process of clonal expansion. The mechanisms underlying the complex process of a single cell leading to a clonal dominant tumor, are poorly understood. Our study aims to analyze mitochondrial DNA (mtDNA) for somatic single nucleotide polymorphisms (SNPs) variants, to determine if they are conserved throughout clonal expansion in mammary tissues and tumors. To test this hypothesis, we took advantage of a mouse mammary tumor virus (MMTV)-infected mouse model (CzechII). CzechII mouse mtDNA was extracted, from snap-frozen normal, hyperplastic, and tumor mammary epithelial outgrowth fragments. Next generation deep sequencing was used to determine if mtDNA "de novo" SNP variants are conserved during serial transplantation of both normal and neoplastic mammary clones. Our results support the conclusion that mtDNA "de novo" SNP variants are selected for and maintained during serial passaging of clonal phenotypically heterogeneous normal cellular populations; neoplastic cellular populations; metastatic clonal cellular populations and in individual tumor transplants, grown from the original metastatic tumor. In one case, a mammary tumor arising from a single cell, within a clonal hyperplastic outgrowth, contained only mtDNA copies, harboring a deleterious "de novo" SNP variant, suggesting that only one mtDNA template may act as a template for all mtDNA copies regardless of cell phenotype. This process has been attributed to "heteroplasmic-shifting". A process that is thought to result from selective pressure and may be responsible for pathogenic mutated mtDNA copies becoming homogeneous in clonal dominant oncogenic tissues.

Long-label-retaining mammary epithelial cells are created early in ductal development and distributed throughout the branching ducts.

Long-label retention has been used by many to prove Cairns' immortal strand hypothesis and to identify potential stem cells. Here, we describe two strategies using 5-ethynl-2'-deoxyuridine (EdU) to identify and understand the distribution of long-label-retaining mammary epithelial cells during formation of the mouse mammary ductal system. First, EdU was given upon two consecutive days per week during weeks 4 through 10 and analyzed for label retention at 13 weeks of age. Alternatively, EdU was given for 14 consecutive days beginning at 28 days of age and ending at 42 days of age. Analyses were conducted at >91 days of age (13 weeks). Many more LREC were detected following the second labeling method and their distribution among the subsequently developed ducts. This finding indicated that the early-labeled cells that retained their label were distributed into portions of the gland that developed after the ending of EdU treatment (i.e. 42->91 days). These observations may have important meaning with respect to the previously demonstrated retention of regenerative capacity throughout the mouse mammary gland despite age or reproductive history. These results suggest LREC may represent long-lived progenitor cells that are responsible for mammary gland homeostasis. Additionally, these cells may act as multipotent stem cells capable of mammary gland regeneration upon random fragment transplantation into epithelium-denuded mammary fat pads.

Microarray and pathway analysis of two COMMA-Dß derived clones reveal important differences relevant to their developmental capacity in-vivo.

Microarray technologies were used to analyze transcriptomes from Comma-Dß and clonal derivatives, SP3 (Lobule-competent) and NSP2 (Lobule-incompetent), during different mouse mammary growth phases: in-vitro, in-vivo 5-weeks, and in-vivo 12-weeks. A differentially expressed gene (DEG) algorithm was used to enrich for genes associated with cellular proliferation, differentiation, cell cycle regulation, and carcinogenesis. A pairwise comparison analysis, of SP3 vs. NSP2 in-vitro, revealed a total of 45 DEGs significantly up-regulated in SP3. Of the 45 DEGs, only Ccnd1 (Cyclin D1), Id2 (Inhibitor of DNA binding 2) and Sox9 (SRY Box 9) were identified to be associated with cellular proliferation, regulation of G1/S mitotic cell cycle, mammary gland and alveolar development in SP3. During the regenerative growth phase, in-vivo 5-weeks, we identified a total of 545 DEGs. 308 DEGs, of the 545 DEGs, were significantly up-regulated and 237 DEGs were significantly down-regulated in SP3 vs. NSP2. In addition, we identified 9 DEGs significantly up-regulated, within SP3's cell cycle pathway and a persistent overexpression of Cyclin D1, Id2, and Sox9, consistent with our in-vitro study. During the maintenance phase, in-vivo 12-weeks, we identified 407 DEGs. Of these, 336 DEGs were up-regulated, and 71 were down-regulated in SP3 vs. NSP2. Our data shows 15 DEGs significantly up-regulated, simultaneously, affecting 8 signal transducing carcinogenic pathways. In conclusion, increased expression of Cyclin D1, Id2 and Sox9 appear to be important for lobular genesis in SP3. Also, in-vivo 12 week displays increase expression of genes and pathways, involved in tumorigenesis.

Assessing prescribing practices of clozapine before and after the implementation of an updated risk evaluation and mitigation strategy.

INTRODUCTION: Clozapine is an atypical antipsychotic medication approved for treatment-resistant schizophrenia and suicidal behavior in schizophrenia or schizoaffective disorders. Despite its therapeutic efficacy, clozapine is associated with several adverse effects, including agranulocytosis. In late 2015, the Food and Drug Administration updated the risk evaluation and mitigation strategy (REMS) for clozapine with new requirements for monitoring, prescribing, and dispensing. The purpose of this study was to evaluate clozapine prescribing practices at a Kentucky state psychiatric hospital before and after the implementation of the updated REMS program. METHODS: The primary outcome of this study was to evaluate clozapine prescribing practices by identifying the number of patients on clozapine therapy in the 6 months pre and post updated REMS implementation. Included in the study were patients at a Kentucky state psychiatric hospital on clozapine therapy for the 24 months before the updated REMS implementation and in the 6-month study period after the implementation. The secondary objective of this study examined psychiatrist comfort level of prescribing clozapine. RESULTS: Since the implementation of the updated REMS program, there has been an increased percentage of patients that were prescribed clozapine at a Kentucky state psychiatric hospital. This increase was not statistically significant (P = .2610). DISCUSSION: The prescribing practices of clozapine within this facility did not differ significantly comparing pre- and post-REMS change in terms of number of patients prescribed clozapine, patient's dose, and therapy duration. Data from this study contributes to the body of knowledge evaluating this new standard of practice under the updated REMS.

Does the Mouse Mammary Gland Arise from Unipotent or Multipotent Mammary Stem/Progenitor Cells?

The presence of long-lived lineage restricted progenitor and multipotent progenitor cells in adult mouse mammary gland for cancer development is compelling. Mammary cancers are phenotypically diverse This might be explained by transformation of long-lived, lineage-limited progenitor subpopulations. Mammary multipotent epithelial stem cells and their environmental niches must be considered, since their niche(s), once empty might be occupied by lineage-limited progenitors that are proximal. The existence of premalignant mammary populationst that manifest characteristics of lineage limitation argues strongly for this proposition.

Sexual Dimorphism and Retinal Mosaic Diversification following the Evolution of a Violet Receptor in Butterflies.

Numerous animal lineages have expanded and diversified the opsin-based photoreceptors in their eyes underlying color vision behavior. However, the selective pressures giving rise to new photoreceptors and their spectral tuning remain mostly obscure. Previously, we identified a violet receptor (UV2) that is the result of a UV opsin gene duplication specific to Heliconius butterflies. At the same time the violet receptor evolved, Heliconius evolved UV-yellow coloration on their wings, due to the pigment 3-hydroxykynurenine (3-OHK) and the nanostructure architecture of the scale cells. In order to better understand the selective pressures giving rise to the violet receptor, we characterized opsin expression patterns using immunostaining (14 species) and RNA-Seq (18 species), and reconstructed evolutionary histories of visual traits in five major lineages within Heliconius and one species from the genus Eueides. Opsin expression patterns are hyperdiverse within Heliconius. We identified six unique retinal mosaics and three distinct forms of sexual dimorphism based on ommatidial types within the genus Heliconius. Additionally, phylogenetic analysis revealed independent losses of opsin expression, pseudogenization events, and relaxation of selection on UVRh2 in one lineage. Despite this diversity, the newly evolved violet receptor is retained across most species and sexes surveyed. Discriminability modeling of behaviorally preferred 3-OHK yellow wing coloration suggests that the violet receptor may facilitate Heliconius color vision in the context of conspecific recognition. Our observations give insights into the selective pressures underlying the origins of new visual receptors.

In vivo reprogramming of non-mammary cells to an epithelial cell fate is independent of amphiregulin signaling.

Amphiregulin (AREG)-/- mice demonstrate impaired mammary development and form only rudimentary ductal epithelial trees; however, AREG-/- glands are still capable of undergoing alveologenesis and lactogenesis during pregnancy. Transplantation of AREG-/- mammary epithelial cells into cleared mouse mammary fat pads results in a diminished capacity for epithelial growth (∼15%) as compared to that of wild-type mammary epithelial cells. To determine whether estrogen receptor α (ERα, also known as ESR1) and/or AREG signaling were necessary for non-mammary cell redirection, we inoculated either ERα-/- or AREG-/- mammary cells with non-mammary progenitor cells (WAP-Cre/Rosa26LacZ+ male testicular cells or GFP-positive embryonic neuronal stem cells). ERα-/- cells possessed a limited ability to grow or reprogram non-mammary cells in transplanted mammary fat pads. AREG-/- mammary cells were capable of redirecting both types of non-mammary cell populations to mammary phenotypes in regenerating mammary outgrowths. Transplantation of fragments from AREG-reprogrammed chimeric outgrowths resulted in secondary outgrowths in six out of ten fat pads, demonstrating the self-renewing capacity of the redirected non-mammary cells to contribute new progeny to chimeric outgrowths. Nestin was detected at the leading edges of developing alveoli, suggesting that its expression may be essential for lobular expansion.

Mammary extracellular matrix directs differentiation of testicular and embryonic stem cells to form functional mammary glands in vivo.

Previously, we demonstrated the ability of the normal mammary microenvironment (niche) to direct non-mammary cells including testicular and embryonic stem cells (ESCs) to adopt a mammary epithelial cell (MEC) fate. These studies relied upon the interaction of transplanted normal MECs with non-mammary cells within the mammary fat-pads of recipient mice that had their endogenous epithelium removed. Here, we tested whether acellular mammary extracellular matrix (mECM) preparations are sufficient to direct differentiation of testicular-derived cells and ESCs to form functional mammary epithelial trees in vivo. We found that mECMs isolated from adult mice and rats were sufficient to redirect testicular derived cells to produce normal mammary epithelial trees within epithelial divested mouse mammary fat-pads. Conversely, ECMs isolated from omental fat and lung did not redirect testicular cells to a MEC fate, indicating the necessity of tissue specific components of the mECM. mECM preparations also completely inhibited teratoma formation from ESC inoculations. Further, a phenotypically normal ductal outgrowth resulted from a single inoculation of ESCs and mECM. To the best of our knowledge, this is the first demonstration of a tissue specific ECM driving differentiation of cells to form a functional tissue in vivo.

Techniques for the Reprogramming of Exogenous Stem/Progenitor Cell Populations Towards a Mammary Epithelial Cell Fate.

This chapter considers the techniques necessary and required for the reprogramming of exogenous stem/progenitor cell populations towards a mammary epithelial cell fate. The protocols describe how to isolate cells from alternate mouse organs such as testicles of male mice and mix them with mammary cells to generate chimeric glands comprised of male and female epithelial cells that are fully competent. During the reformation of mammary stem cell niches by dispersed epithelial cells, in the context of the intact epithelium-free mammary stroma, non-mammary cells are sequestered and reprogrammed to perform mammary epithelial cell functions including those ascribed to mammary stem/progenitor cells. This therefore is a powerful technique for the redirection of cells from other organs/cancer cells to a normal mammary phenotype.

Gene Duplication and Gene Expression Changes Play a Role in the Evolution of Candidate Pollen Feeding Genes in Heliconius Butterflies.

Heliconius possess a unique ability among butterflies to feed on pollen. Pollen feeding significantly extends their lifespan, and is thought to have been important to the diversification of the genus. We used RNA sequencing to examine feeding-related gene expression in the mouthparts of four species of Heliconius and one nonpollen feeding species, Eueides isabella We hypothesized that genes involved in morphology and protein metabolism might be upregulated in Heliconius because they have longer proboscides than Eueides, and because pollen contains more protein than nectar. Using de novo transcriptome assemblies, we tested these hypotheses by comparing gene expression in mouthparts against antennae and legs. We first looked for genes upregulated in mouthparts across all five species and discovered several hundred genes, many of which had functional annotations involving metabolism of proteins (cocoonase), lipids, and carbohydrates. We then looked specifically within Heliconius where we found eleven common upregulated genes with roles in morphology (CPR cuticle proteins), behavior (takeout-like), and metabolism (luciferase-like). Closer examination of these candidates revealed that cocoonase underwent several duplications along the lineage leading to heliconiine butterflies, including two Heliconius-specific duplications. Luciferase-like genes also underwent duplication within lepidopterans, and upregulation in Heliconius mouthparts. Reverse-transcription PCR confirmed that three cocoonases, a peptidase, and one luciferase-like gene are expressed in the proboscis with little to no expression in labial palps and salivary glands. Our results suggest pollen feeding, like other dietary specializations, was likely facilitated by adaptive expansions of preexisting genes-and that the butterfly proboscis is involved in digestive enzyme production.

Mammary Epithelial Cell Lineage Analysis via the Lyon's Hypothesis.

Implants of mammary glands from a single mammary fat pad in a H253 transgenic female mouse heterozygous for a lacZ-labeled X chromosome were analyzed at various time points following transplantation into the epithelium-cleared mammary fat pads of immune-compromised mice. The results show that the lacZ-marked X chromosome, demonstrated by nuclear-associated X-gal staining, was confined to a single epithelial clone that gave rise to the cap cells of all growing terminal end buds (TEB) in the expanding mammary outgrowths and also the basal cells of the elongated ducts. The nuclei of luminal cells in these ducts were uniformly negative for lacZ expression indicating that they were derived from cellular precursors that contained a silenced lac-Z marked X chromosome. This observation confirms the earlier work of Williams and Daniel, who concluded that cap cells were the precursors of the basal (myoepithelial cells) of the subtending mammary ducts.

Binuclear Cells in the Lactating Mammary Gland: New Insights on an Old Concept?

In a recent paper (Rios et al. Nat Commun. 7:11400, 2016), it was reported that polyploid cells are frequent in lactating mammary tissues. This phenomenon was observed in mammary tissue sampled from five separate mammalian species. According to that report, these binucleated cells occur late in pregnancy and early in lactation. Unfortunately, this paper did not mention a number of earlier observations and findings that remain pertinent to this day (Banerjee et al. Life sciences Pt 2: Biochemistry, general and molecular biology. 10(15):867-77, 1971; Banerjee MR, Wagner JE. Biochem. Biophys. Res. Commun. 49(2):480-7, 1972). In these classical experiments, the authors demonstrated in vivo that DNA synthesis continued without commensurate cell division during late pregnancy and lactation, and that this DNA synthesis was imperative for functional differentiation of the mammary epithelium. Later studies showed that DNA synthesis was indispensable to the induction of milk protein production in explant cultures of mammary tissue from unprimed, nulliparous mice. This dependence on DNA synthesis in mammary explant cultures stimulated by lactogenic hormones was found to be dispensable following a single pregnancy. The absolute requirement for DNA synthesis in nulliparous mouse mammary explants stimulated to synthesize milk protein in vitro has remained unexplained, as has the need for DNA synthesis prior to the onset of lactation. From a historical perspective, it is more likely that binuclear secretory cells in the lactating mammary gland are a consequence of the DNA synthesis requirement for lactation, rather than an essential element.

Telomerase and estrogen-sensing activities are essential for continued mammary growth in vivo but dispensable for "reprogramming" neural stem cells.

It has been proposed that the erosion of telomere length is a limiting factor in replicative capacity and important in cell senescence. To determine if this activity was essential in the mouse mammary gland in vivo, we serially transplanted mammary fragments from wild type (TER+/+), heterozygous (TER+/-), and homozygous (TER-/-) mammary tissues into the cleared mammary fat pads of immune-compromised nude mice. Individual implants from both homozygous and heterozygous TER null outgrowths showed growth senescence beginning at transplant generation two, earlier than implants from TER+/+ mammary glands which continued to show growth. This result suggests that either mammary epithelial stem cells maintain their telomere length in order to self renew, or that the absence or reduction of telomerase template results in more frequent death/extinction of stem cells during symmetric divisions. A third possibility is the inability of signaling cells in the niche to replicate resulting in reduction of the maintenance signals necessary for stem cell renewal. Consistent with this, examination of senescent outgrowths revealed the absence of estrogen receptor alpha (ERα+) epithelium although progesterone receptor (PR+) cells were abundant. Despite their inability to establish mammary growth in vivo, TER+/- cells were able to direct neural stem cells to mammary cell fates.

Transcriptome-Wide Differential Gene Expression in Bicyclus anynana Butterflies: Female Vision-Related Genes Are More Plastic.

Vision is energetically costly to maintain. Consequently, over time many cave-adapted species downregulate the expression of vision genes or even lose their eyes and associated eye genes entirely. Alternatively, organisms that live in fluctuating environments, with different requirements for vision at different times, may evolve phenotypic plasticity for expression of vision genes. Here, we use a global transcriptomic and candidate gene approach to compare gene expression in the heads of a polyphenic butterfly. Bicyclus anynana have two seasonal forms that display sexual dimorphism and plasticity in eye morphology, and female-specific plasticity in opsin gene expression. Nonchoosy dry season females downregulate opsin expression, consistent with the high physiological cost of vision. To identify other genes associated with sexually dimorphic and seasonally plastic differences in vision, we analyzed RNA-sequencing data from whole head tissues. We identified two eye development genes (klarsicht and warts homologs) and an eye pigment biosynthesis gene (henna) differentially expressed between seasonal forms. By comparing sex-specific expression across seasonal forms, we found that klarsicht, warts, henna, and another eye development gene (domeless) were plastic in a female-specific manner. In a male-only analysis, white (w) was differentially expressed between seasonal forms. Reverse transcription polymerase chain reaction confirmed that warts and white are expressed in eyes only, whereas klarsicht, henna and domeless are expressed in both eyes and brain. We find that differential expression of eye development and eye pigment genes is associated with divergent eye phenotypes in B. anynana seasonal forms, and that there is a larger effect of season on female vision-related genes.

Hormone signaling requirements for the conversion of non-mammary mouse cells to mammary cell fate(s) in vivo.

Mammotropic hormones and growth factors play a very important role in mammary growth and differentiation. Here, hormones including Estrogen, Progesterone, Prolactin, their cognate receptors, and the growth factor Amphiregulin, are tested with respect to their roles in signaling non-mammary cells from the mouse to redirect to mammary epithelial cell fate(s). This was done in the context of glandular regeneration in pubertal athymic female mice. Our previous studies demonstrated that mammary stem cell niches are recapitulated during gland regeneration in vivo. During this process, cells of exogenous origin cooperate with mammary epithelial cells to form mammary stem cell niches and thus respond to normal developmental signals. In all cases tested with the possible exception of estrogen receptor alpha (ER-α), hormone signaling is dispensable for non-mammary cells to undertake mammary epithelial cell fate(s), proliferate, and contribute progeny to chimeric mammary outgrowths. Importantly, redirected non-mammary cell progeny, regardless of their source, have the ability to self-renew and contribute offspring to secondary mammary outgrowths derived from transplanted chimeric mammary fragments; thus suggesting that some of these cells are capable of mammary stem cell/progenitor functions.

Molecular evolution and expression of the CRAL_TRIO protein family in insects.

CRAL_TRIO domain proteins are known to bind small lipophilic molecules such as retinal, inositol and Vitamin E and include such gene family members as PINTA, α-tocopherol transfer (ATT) proteins, retinoid binding proteins, and clavesins. In insects, very little is known about either the molecular evolution of this family of proteins or their ligand specificity. Here we characterize insect CRAL_TRIO domain proteins and present the first insect CRAL_TRIO protein phylogeny constructed by performing reciprocal BLAST searches of the reference genomes of Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum, Bombyx mori, Manduca sexta and Danaus plexippus. We find several highly conserved amino acid residues in the CRAL_TRIO domain-containing genes across insects and a gene expansion resulting in more than twice as many gene family members in lepidopterans than in other surveyed insect species, but no lepidopteran homolog of the PINTA gene in Drosophila. In addition, we examined the expression pattern of CRAL_TRIO domain genes in Manduca sexta heads using RNA-Seq data. Of the 42 gene family members found in the M. sexta reference genome, we found 30 expressed in the head tissue with similar expression profiles between males and females. Our results suggest this gene family underwent a large expansion in lepidopteran, making the lepidopteran CRAL_TRIO domain family distinct from other holometabolous insect lineages.

Genome-wide DNA methylation patterns in wild samples of two morphotypes of threespine stickleback (Gasterosteus aculeatus).

Epigenetic marks such as DNA methylation play important biological roles in gene expression regulation and cellular differentiation during development. To examine whether DNA methylation patterns are potentially associated with naturally occurring phenotypic differences, we examined genome-wide DNA methylation within Gasterosteus aculeatus, using reduced representation bisulfite sequencing. First, we identified highly methylated regions of the stickleback genome, finding such regions to be located predominantly within genes, and associated with genes functioning in metabolism and biosynthetic processes, cell adhesion, signaling pathways, and blood vessel development. Next, we identified putative differentially methylated regions (DMRs) of the genome between complete and low lateral plate morphs of G. aculeatus. We detected 77 DMRs that were mainly located in intergenic regions. Annotations of genes associated with these DMRs revealed potential functions in a number of known divergent adaptive phenotypes between G. aculeatus ecotypes, including cardiovascular development, growth, and neuromuscular development.