scholarly journals Molluscum Contagiosum Virus Transcriptome in Abortively Infected Cultured Cells and a Human Skin Lesion

2016 ◽  
Vol 90 (9) ◽  
pp. 4469-4480 ◽  
Author(s):  
Jorge D. Mendez-Rios ◽  
Zhilong Yang ◽  
Karl J. Erlandson ◽  
Jeffrey I. Cohen ◽  
Craig A. Martens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skin Lesion ◽  
Human Skin ◽  
Cultured Cells ◽  
Skin Lesions ◽  
Molluscum Contagiosum ◽  
Rna Seq ◽  
Molluscum Contagiosum Virus ◽  
Human Specific

ABSTRACTMolluscum contagiosum virus (MOCV), the only circulating human-specific poxvirus, has a worldwide distribution and causes benign skin lesions that may persist for months in young children and severe infections in immunosuppressed adults. Studies of MOCV are restricted by the lack of an efficient animal model or a cell culture replication system. We used next-generation sequencing to analyze and compare polyadenylated RNAs from abortive MOCV infections of several cell lines and a human skin lesion. Viral RNAs were detected for 14 days after MOCV infection of cultured cells; however, there was little change in the RNA species during this time and a similar pattern occurred in the presence of an inhibitor of protein synthesis, indicating a block preventing postreplicative gene expression. Moreover, a considerable number of MOCV RNAs mapped to homologs of orthopoxvirus early genes, but few did so to homologs of intermediate or late genes. The RNAs made duringin vitroinfections represent a subset of RNAs detected in human skin lesions which mapped to homologs of numerous postreplicative as well as early orthopoxvirus genes. Transfection experiments using fluorescent protein and luciferase reporters demonstrated that vaccinia virus recognized MOCV intermediate and late promoters, indicating similar gene regulation. The specific recognition of the intermediate promoter in MOCV-infected cells provided evidence for the synthesis of intermediate transcription factors, which are products of early genes, but not for late transcription factors. Transcriptome sequencing (RNA-seq) and reporter gene assays may be useful for testing engineered cell lines and conditions that ultimately could provide anin vitroreplication system.IMPORTANCEThe inability to propagate molluscum contagiosum virus, which causes benign skin lesions in young children and more extensive infections in immunosuppressed adults, has constrained our understanding of the biology of this human-specific virus. In the present study, we characterized the RNAs synthesized in abortively infected cultured cells and a human skin lesion by next-generation sequencing. These studies provided an initial transcription map of the MOCV genome, suggested temporal regulation of gene expression, and indicated that thein vitroreplication block occurs prior to intermediate and late gene expression. RNA-seq and reporter assays, as described here, may help to further evaluate MOCV gene expression and define conditions that could enable MOCV replicationin vitro.

scholarly journals Single cell RNA sequencing of calvarial and long bone endocortical cells

2019 ◽  
Author(s):  
Ugur M. Ayturk ◽  
Joseph P. Scollan ◽  
Alexander Vesprey ◽  
Christina M. Jacobsen ◽  
Paola Divieti Pajevic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Long Bone ◽  
Appendicular Skeleton ◽  
Rna Seq ◽  
Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

scholarly journals A Novel Target and Approach for Identifying Antivirals against Molluscum Contagiosum Virus

2014 ◽  
Vol 58 (12) ◽  
pp. 7383-7389 ◽  
Author(s):  
Hancheng Guan ◽  
Manunya Nuth ◽  
Natalia Zhukovskaya ◽  
Yih Ling Saw ◽  
Edward Bell ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Sexual Transmission ◽  
Skin Lesions ◽  
Molluscum Contagiosum ◽  
Health Concern ◽  
Target System ◽  
Molluscum Contagiosum Virus ◽  
Dermatological Disease ◽  
Novel Target

ABSTRACTThe dermatological disease molluscum contagiosum (MC) presents as lesions restricted solely to the skin. The poxvirus molluscum contagiosum virus (MCV) is responsible for this skin disease that is easily transmitted through casual contact among all populations, with greater frequency in children and immunosuppressed individuals. In addition, sexual transmission of MCV in adolescents and adults is a health concern. Although the skin lesions ultimately resolve in immunocompetent individuals, they can persist for extended periods, be painful, and result in scarring. Treatment is problematic, and there is no drug that specifically targets MCV. The inability of MCV to propagate in cell culture has impeded drug development. To overcome these barriers, we integrated three new developments. First, we identified a new MCV drug target (mD4) that is essential for processive DNA synthesisin vitro. Second, we discovered a small chemical compound that binds to mD4 and prevents DNA synthesisin vitro. Third, and most significant, we engineered a hybrid vaccinia virus (mD4-VV) in which the natural vaccinia D4 (vD4) gene is replaced by the mD4 target gene. This hybrid virus is dependent on mD4 for viral growth in culture and is inhibited by the small compound. This target system provides, for the first time, a platform and approach for the discovery and evaluation of new therapeutics that can be used to treat MC.

scholarly journals Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM

2019 ◽  
Author(s):  
Marcus Alvarez ◽  
Elior Rahmani ◽  
Brandon Jew ◽  
Kristina M. Garske ◽  
Zong Miao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Types ◽  
Supervised Machine Learning ◽  
Data Sets ◽  
Rna Seq ◽  
Novel Approach ◽  
Single Nucleus ◽  
Downstream Analysis

AbstractSingle-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. Contrary to single-cell RNA seq (scRNA-seq), we observe that snRNA-seq is commonly subject to contamination by high amounts of extranuclear background RNA, which can lead to identification of spurious cell types in downstream clustering analyses if overlooked. We present a novel approach to remove debris-contaminated droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: 1) human differentiating preadipocytes in vitro, 2) fresh mouse brain tissue, and 3) human frozen adipose tissue (AT) from six individuals. All three data sets showed various degrees of extranuclear RNA contamination. We observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq data, we also successfully applied DIEM to single-cell data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

scholarly journals In vitro integration of an ichnovirus genome segment into the genomic DNA of lepidopteran cells

2007 ◽  
Vol 88 (1) ◽  
pp. 105-113 ◽  
Author(s):  
Daniel Doucet ◽  
Anic Levasseur ◽  
Catherine Béliveau ◽  
Renée Lapointe ◽  
Don Stoltz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virus Replication ◽  
Genomic Dna ◽  
Cultured Cells ◽  
Germ Line ◽  
Choristoneura Fumiferana ◽  
Genome Segment ◽  
Host Cells ◽  
Dsdna Viruses

Polydnaviruses (PDVs) are dsDNA viruses transmitted by ichneumonid and braconid endoparasitoids to their lepidopteran hosts during oviposition. Wasp carriers are asymptomatic and transmit the virus to their progeny through the germ line; replication is confined to the calyx region of the wasp ovary, where the virus accumulates in the fluid bathing the eggs. In the lepidopteran host, however, no virus replication takes place, but PDV gene expression is essential for successful parasitism. Sustained gene expression in the absence of virus replication thus requires that the circular PDV genome segments persist for days within host cells. Available evidence suggests that most genome segments persist as episomes, but recent studies have indicated that some genome segments may undergo integration within lepidopteran genomic DNA, at least in vitro. In the present study, an integrated form of a Tranosema rostrale ichnovirus (TrIV) genome segment was cloned from genomic DNA extracted from infected Choristoneura fumiferana CF-124T cells and junction regions on either side of the viral DNA sequence were sequenced. This is the first proven example of integration of an ichnovirus genome segment in infected lepidopteran cells. Interestingly, circular forms of this genome segment do not appear to persist in these cells; none the less, a gene (TrFrep1) carried by this genome segment displays long-term transcription in infected cultured cells.

Effect of SMARCB1 deficiency in renal medullary carcinoma (RMC) on genes associated with nucleosome assembly and telomere organization.

2018 ◽  
Vol 36 (6_suppl) ◽  
pp. 614-614 ◽  
Author(s):  
Pavlos Msaouel ◽  
Gabriel G. Malouf ◽  
Xiaoping Su ◽  
Hui Yao ◽  
Durga N Tripathi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Rate ◽  
Sickle Cell Trait ◽  
Regulation Of Gene Expression ◽  
Kidney Tissue ◽  
Rna Seq ◽  
Nucleosome Assembly ◽  
P Values ◽  
Go Analysis

614 Background: RMC is a highly aggressive tumor with close to universal fatality despite therapy. It is almost exclusively found in young African-Americans with sickle cell trait, and is characterized by complete loss of expression of SMARCB1, a major chromatin remodeler involved in regulation of gene expression. We investigated the effects of SMARCB1 loss on mutation frequency, gene expression, and cell growth in RMC. Methods: Whole exome sequencing (WES) and RNA sequencing (RNA-seq) were performed in RMC tissues from 15 and 11 patients respectively, each with matched adjacent normal kidney tissue controls. In vitro experiments were performed in a cell line (RMC2C) we established from a patient with RMC. SMARCB1 was conditionally re-expressed using a tetracycline-inducible lentivector. Gene ontology (GO) analysis was performed using DAVID. Results: WES showed that RMC harbors a low number (median of < 25/tumor sample) of non-synonymous exomic single nucleotide variants (SNVs) or small indels. GO analysis revealed that the most significant pathways upregulated in RMC compared with normal tissue were those associated with nucleosome assembly and telomere organization (p values < 0.0001). Re-expression of SMARCB1 at near-endogenous levels suppressed the growth rate of RMC2C cells. Subsequent silencing of SMARCB1 expression restored the growth rate of these cells. RNA-seq of RMC2C cells expressing SMARCB1 demonstrated that the most significant downregulated pathways compared with SMARCB1-negative RMC2C cells were those associated with nucleosome assembly and telomere organization (p values < 0.0001). Conclusions: RMC harbors a remarkably simple genome, as evidenced by our WES analysis. Therefore, consistently detected alterations, such as SMARCB1 loss, are likely to serve as drivers for this disease. Indeed, in vitro restoration of SMARCB1 expression suppressed the growth of RMC cells and repressed genes associated with nucleosome assembly and telomere organization, identifying for the first time a causal link between loss of SMARCB1 and dysregulation of these genes. These results provide the basis for future therapeutic strategies targeting SMARCB1 loss in RMC.

scholarly journals Molluscum Contagiosum Virus MC159 Abrogates cIAP1-NEMO Interactions and Inhibits NEMO Polyubiquitination

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Sunetra Biswas ◽  
Joanna L. Shisler
Keyword(s):  
Host Cell ◽  
Skin Neoplasms ◽  
Skin Lesions ◽  
Molluscum Contagiosum ◽  
Host Cells ◽  
Molluscum Contagiosum Virus ◽  
Antiviral Responses ◽  
Iκb Kinase ◽  
Apoptosis Protein ◽  
Ikk Complex

ABSTRACT Molluscum contagiosum virus (MCV) is a dermatotropic poxvirus that causes benign skin lesions. MCV lesions persist because of virally encoded immune evasion molecules that inhibit antiviral responses. The MCV MC159 protein suppresses NF-κB activation, a powerful antiviral response, via interactions with the NF-κB essential modulator (NEMO) subunit of the IκB kinase (IKK) complex. Binding of MC159 to NEMO does not disrupt the IKK complex, implying that MC159 prevents IKK activation via an as-yet-unidentified strategy. Here, we demonstrated that MC159 inhibited NEMO polyubiquitination, a posttranslational modification required for IKK and downstream NF-κB activation. Because MCV cannot be propagated in cell culture, MC159 was expressed independent of infection or during a surrogate vaccinia virus infection to identify how MC159 prevented polyubiquitination. Cellular inhibitor of apoptosis protein 1 (cIAP1) is a cellular E3 ligase that ubiquitinates NEMO. Mutational analyses revealed that MC159 and cIAP1 each bind to the same NEMO region, suggesting that MC159 may competitively inhibit cIAP1-NEMO interactions. Indeed, MC159 prevented cIAP1-NEMO interactions. MC159 also diminished cIAP1-mediated NEMO polyubiquitination and cIAP1-induced NF-κB activation. These data suggest that MC159 competitively binds to NEMO to prevent cIAP1-induced NEMO polyubiquitination. To our knowledge, this is the first report of a viral protein disrupting NEMO-cIAP1 interactions to strategically suppress IKK activation. All viruses must antagonize antiviral signaling events for survival. We hypothesize that MC159 inhibits NEMO polyubiquitination as a clever strategy to manipulate the host cell environment to the benefit of the virus. IMPORTANCE Molluscum contagiosum virus (MCV) is a human-specific poxvirus that causes persistent skin neoplasms. The persistence of MCV has been attributed to viral downregulation of host cell immune responses such as NF-κB activation. We show here that the MCV MC159 protein interacts with the NEMO subunit of the IKK complex to prevent NEMO interactions with the cIAP1 E3 ubiquitin ligase. This interaction correlates with a dampening of cIAP1 to polyubiquitinate NEMO and to activate NF-κB. This inhibition of cIAP1-NEMO interactions is a new viral strategy to minimize IKK activation and to control NEMO polyubiquitination. This research provides new insights into mechanisms that persistent viruses may use to cause long-term infection of host cells.

scholarly journals α-Melanocyte-Stimulating Hormone Counteracts the Suppressive Effect of UVB on Nrf2 and Nrf-Dependent Gene Expression in Human Skin

Endocrinology ◽  
2009 ◽  
Vol 150 (7) ◽  
pp. 3197-3206 ◽  
Author(s):  
Agatha Kokot ◽  
Dieter Metze ◽  
Nicolas Mouchet ◽  
Marie-Dominique Galibert ◽  
Meinhard Schiller ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Human Skin ◽  
Uv Light ◽  
Ex Vivo ◽  
Suppressive Effect ◽  
Heme Oxygenase 1 ◽  
Organ Cultures ◽  
Normal Keratinocytes

Human skin is constantly exposed to UV light, the most ubiquitous environmental stressor. Here, we investigated the expression and regulation of Nrf1-3, transcription factors crucially involved in protection against oxidative stress in human skin cells in vitro, ex vivo, and in situ. In particular, we examined whether α-MSH, a UV-induced peptide, is capable of modulating Nrf2 and Nrf-dependent gene expression. Nrf1, -2, and -3 were found to be expressed in various cutaneous cell types in vitro. Surprisingly, UVB irradiation at physiological doses (10 mJ/cm2) reduced Nrf2 and Nrf-dependent gene expression in normal keratinocytes and melanocytes in vitro as well as ex vivo in skin organ cultures. α-MSH alone significantly increased Nrf2 as well as Nrf-dependent heme oxygenase-1, γ-glutamylcysteine-synthetase, and glutathione-S-transferase Pi gene expression in both keratinocytes and melanocytes. This effect of α-MSH occurred at physiological doses and was due to transcriptional induction, mimicked by the artificial cAMP inducer forskolin, and blocked by protein kinase A pathway inhibition. In silico promoter analysis of Nrf2 further identified several putative binding sites for activator protein 1 and cAMP response element-binding protein, transcription factors typically activated by α-MSH. Importantly, α-MSH prevented or even overcompensated the UVB-induced suppression of Nrf2 and Nrf-dependent genes not only in normal keratinocytes and melanocytes in vitro but also in skin organ cultures. These findings, for the first time, show regulation of Nrf2 and Nrf-dependent genes by α-MSH. Our data also highlight a novel facet in the cytoprotective and antioxidative effector mechanisms of α-MSH and perhaps of related melanocortin peptides.

scholarly journals Establishment of long-term monolayer cultures of somatic cells from human fetal testes and expansion of peritubular myoid cells in the presence of androgen

Reproduction ◽  
2010 ◽  
Vol 139 (4) ◽  
pp. 749-757 ◽  
Author(s):  
Gillian Cowan ◽  
Andrew J Childs ◽  
Richard A Anderson ◽  
Philippa T K Saunders
Keyword(s):  
Gene Expression ◽  
Cultured Cells ◽  
Smooth Muscle Actin ◽  
Cell Monolayer ◽  
Somatic Cells ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Monolayer Cultures ◽  
The Impact

The somatic (Sertoli cell (SC), Leydig cell (LC), and peritubular myoid (PTM) cell) cells play key roles in development of the fetal testis. We established monolayer cultures from second trimester human testes and investigated the pattern of expression of cell-lineage characteristic mRNAs. Expression of some SC-associated genes (SRY, SOX9, WT1, GATA4, and SF1) was detectable up to and including passage 3 (P3), while others (anti-Müllerian hormone; desert hedgehog) present prior to dissociation were not expressed in the cultured cells. Transcripts encoding the androgen receptor were expressed but addition of dihydrotestosterone (DHT) had no impact on expression of mRNAs expressed in SC or LC. Total concentrations of mRNAs encoding smooth muscle actin (ACTA2) and desmin increased from P1 to P3; an increasing proportion of the cells in the cultures were immunopositive for ACTA2 consistent with proliferation/differentiation of PTM cells. In conclusion, somatic cell monolayer cultures were established from human fetal testes; these cultures could form the basis for future studies based on isolation of purified populations of somatic cells and manipulation of gene expression that is difficult to achieve with organ culture systems. Our results suggest that fetal SC do not maintain a fully differentiated phenotype in vitro, yet PTM (ACTA2 positive) cells readily adapt to monolayer culture conditions in the presence of DHT. This culture system provides an opportunity to study the impact of regulatory factors on gene expression in PTM cells, a population thought to play a key role in mediating androgen action within the developing testis.

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