108 results on '"Sullivan CS"'
Search Results
2. Evaluation of blood lactate elevation as an intensity criterion for exercise training.
- Author
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Casaburi R, Storer TW, Sullivan CS, and Wasserman K
- Published
- 1995
- Full Text
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3. Potty Stools, a Pilot Study to Step Up the Management of Functional Constipation in Children.
- Author
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Reeves PT, Meyers T, Howard B, Rogers PL, Jack B, Kolasinski NT, Burklow CS, Min S, and Nylund CM
- Abstract
Functional constipation (FC) comprises 30% of pediatric gastroenterology clinic visits. FC results from withholding behaviors that can be worsened by poor toileting posture. The use of a defecation posture modification device (DPMD)-a potty stool-has demonstrated benefit in adults. The aim of this study was to assess the risks and benefits of incorporating a DPMD as an adjunct to the FC treatment plan for children. Toilet-trained children aged ≥4 years meeting the Rome IV criteria for FC were enrolled and offered a DPMD at no cost and were followed for 4 months. There were no injuries or treatment-related adverse events reported by the 43 subjects who completed the study. Notably, 98% (n = 42) of respondents would recommend the DPMD to assist other children with FC. DPMD users showed a 33% reduction in fecal incontinence compared with 60% non-users. The DPMD represents a safe adjunct to incorporate into the FC management plan of children., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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- 2024
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4. Viral piracy of host RNA phosphatase DUSP11 by avipoxviruses.
- Author
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Szymanik KH, Hancks DC, and Sullivan CS
- Abstract
Proper recognition of viral pathogens is an essential part of the innate immune response. A common viral replicative intermediate and chemical signal that cells use to identify pathogens is the presence of a triphosphorylated 5' end (5'ppp) RNA, which activates the cytosolic RNA sensor RIG-I and initiates downstream antiviral signaling. While 5'pppRNA generated by viral RNA-dependent RNA polymerases (RdRps) can be a potent activator of the immune response, endogenous RNA polymerase III (RNAPIII) transcripts can retain the 5'pppRNA generated during transcription and induce a RIG-I-mediated immune response. We have previously shown that host RNA triphosphatase dual-specificity phosphatase 11 (DUSP11) can act on both host and viral RNAs, altering their levels and reducing their ability to induce RIG-I activation. Our previous work explored how artificially altered DUSP11 can impact immune activation, prompting further exploration into natural contexts of altered DUSP11. Here, we have identified viral DUSP11 homologs (vDUSP11s) present in some avipoxviruses. Consistent with the known functions of endogenous DUSP11, we have shown that expression of vDUSP11s: 1) reduces levels of endogenous RNAPIII transcripts, 2) reduces a cell's sensitivity to 5'pppRNA-mediated immune activation, and 3) restores virus infection defects seen in the absence of DUSP11. Our results identify a virus-relevant context where DUSP11 activity has been co-opted to alter RNA metabolism and influence the outcome of infection.
- Published
- 2024
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5. Inclusion of racial and ethnic groups in clinical trials for COVID-19 and post-acute COVID-19 syndrome: An analysis of studies registered on ClinicalTrials.gov.
- Author
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Chaka A, Pan D, Irshad M, Piranie H, Wells M, Lal Z, Al-Oraibi A, Bird P, Nazareth J, Sze S, Divall P, Sullivan CS, Appleby BE, Teece L, Martin CA, Naidu J, Nellums LB, Gray LJ, Khunti K, and Pareek M
- Subjects
- Humans, Ethnicity, Racial Groups, Clinical Trials as Topic, COVID-19 ethnology, COVID-19 epidemiology, Patient Selection, Post-Acute COVID-19 Syndrome
- Abstract
Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest.
- Published
- 2024
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6. Universal pH electrocatalytic hydrogen evolution with Au-based high entropy alloys.
- Author
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Jeong S, Branco AJ, Bollen SW, Sullivan CS, and Ross MB
- Abstract
The creation of electrocatalysts with reduced concentrations of platinum-group metals remains a critical challenge for electrochemical hydrogen production. High-entropy alloys (HEAs) offer a distinct type of catalyst with tunable compositions and engineered surface activity, significantly enhancing the hydrogen evolution reaction (HER). We present the synthesis of AuPdFeNiCo HEA nanoparticles (NPs) using a wet impregnation method. The composition and structure of the AuPdFeNiCo HEA NPs are characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and high-resolution transmission electron microscopy (HR-TEM). These nanoparticles exhibit robust HER performance quantified over a broad pH range, with higher activity than any of the unary metal counterparts in all pHs. In comparison to a commercial 10%Pt/C electrocatalyst, AuPdFeNiCo HEA NPs exhibit enhanced electrochemical activity in both acidic and alkaline electrolytes at a current density of 10 mA cm
-2 . Additionally, these nanoparticles achieve a current density of 100 mA cm-2 at a voltage of 540 mV in neutral electrolytes, outperforming Pt/C which requires 570 mV. These findings help enable broad use of reduced precious metal electrocatalysts for water electrolysis in a variety of water and pH conditions.- Published
- 2024
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7. PARP14 is pro- and anti-viral host factor that promotes IFN production and affects the replication of multiple viruses.
- Author
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Parthasarathy S, Saenjamsai P, Hao H, Ferkul A, Pfannenstiel JJ, Suder EL, Bejan DS, Chen Y, Schwarting N, Aikawa M, Muhlberger E, Orozco RC, Sullivan CS, Cohen MS, Davido DJ, Hume AJ, and Fehr AR
- Abstract
PARP14 is a 203 kDa multi-domain protein that is primarily known as an ADP-ribosyltransferase, and is involved in a variety of cellular functions including DNA damage, microglial activation, inflammation, and cancer progression. In addition, PARP14 is upregulated by interferon (IFN), indicating a role in the antiviral response. Furthermore, PARP14 has evolved under positive selection, again indicating that it is involved in host-pathogen conflict. We found that PARP14 is required for increased IFN-I production in response to coronavirus infection lacking ADP-ribosylhydrolase (ARH) activity and poly(I:C), however, whether it has direct antiviral function remains unclear. Here we demonstrate that the catalytic activity of PARP14 enhances IFN-I and IFN-III responses and restricts ARH-deficient murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. To determine if PARP14's antiviral functions extended beyond CoVs, we tested the ability of herpes simplex virus 1 (HSV-1) and several negative-sense RNA viruses, including vesicular stomatitis virus (VSV), Ebola virus (EBOV), and Nipah virus (NiV), to infect A549 PARP14 knockout (KO) cells. HSV-1 had increased replication in PARP14 KO cells, indicating that PARP14 restricts HSV-1 replication. In contrast, PARP14 was critical for the efficient infection of VSV, EBOV, and NiV, with EBOV infectivity at less than 1% of WT cells. A PARP14 active site inhibitor had no impact on HSV-1 or EBOV infection, indicating that its effect on these viruses was independent of its catalytic activity. These data demonstrate that PARP14 promotes IFN production and has both pro- and anti-viral functions targeting multiple viruses., Competing Interests: Competing interests: ARF is in the process of submitting a patent application for use of SARS-CoV-2 ΔMac1 virus as a live-attenuated vaccine.
- Published
- 2024
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8. A Pseudomonas aeruginosa small RNA regulates chronic and acute infection.
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Cao P, Fleming D, Moustafa DA, Dolan SK, Szymanik KH, Redman WK, Ramos A, Diggle FL, Sullivan CS, Goldberg JB, Rumbaugh KP, and Whiteley M
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- Animals, Humans, Cystic Fibrosis microbiology, Wounds and Injuries microbiology, Ubiquinone biosynthesis, Anaerobiosis, Sepsis complications, Sepsis microbiology, Oxygen metabolism, Pseudomonas aeruginosa genetics, Pseudomonas aeruginosa pathogenicity, Pseudomonas Infections complications, Pseudomonas Infections microbiology, Pseudomonas Infections pathology, Chronic Disease, Acute Disease, RNA, Bacterial genetics, RNA, Bacterial metabolism, Genes, Bacterial genetics
- Abstract
The ability to switch between different lifestyles allows bacterial pathogens to thrive in diverse ecological niches
1,2 . However, a molecular understanding of their lifestyle changes within the human host is lacking. Here, by directly examining bacterial gene expression in human-derived samples, we discover a gene that orchestrates the transition between chronic and acute infection in the opportunistic pathogen Pseudomonas aeruginosa. The expression level of this gene, here named sicX, is the highest of the P. aeruginosa genes expressed in human chronic wound and cystic fibrosis infections, but it is expressed at extremely low levels during standard laboratory growth. We show that sicX encodes a small RNA that is strongly induced by low-oxygen conditions and post-transcriptionally regulates anaerobic ubiquinone biosynthesis. Deletion of sicX causes P. aeruginosa to switch from a chronic to an acute lifestyle in multiple mammalian models of infection. Notably, sicX is also a biomarker for this chronic-to-acute transition, as it is the most downregulated gene when a chronic infection is dispersed to cause acute septicaemia. This work solves a decades-old question regarding the molecular basis underlying the chronic-to-acute switch in P. aeruginosa and suggests oxygen as a primary environmental driver of acute lethality., (© 2023. The Author(s).)- Published
- 2023
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9. The Uniformed Services Constipation Action Plan: An Effective Tool for the Management of Children with Functional Constipation.
- Author
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Reeves PT, Jack BO, Rogers PL, Kolasinski NT, Burklow CS, Min SB, and Nylund CM
- Subjects
- Child, Humans, Male, Female, Ambulatory Care Facilities, Quality of Life, Constipation
- Abstract
Objective: To implement and to evaluate the effectiveness of the Uniformed Services Constipation Action Plan (USCAP) in our gastroenterology clinic for children with functional constipation., Study Design: This implementation science study included toilet-trained subjects aged 4 years and older who met the Rome IV criteria for functional constipation. Children were block randomized to receive either the USCAP or control. All clinic functional constipation plans recommended subjects continue pharmacotherapy for 4 months. Endpoints measured were clinical outcomes (resolution of functional constipation and achievement of a Pediatric Bristol Stool Form Scale [PBSFS] score of 3 or 4), patient-related outcomes (health-related quality of life [HRQoL] total scale score), and health confidence outcomes (Health Confidence Score [HCS])., Results: Fifty-seven treatment group subjects (44%) received a USCAP (52% male; mean age, 10.9 [4.9] years) compared with 73 controls (56%; 48% male; mean age,10.9 [5.3] years). A PBSFS score of 3 or 4 was achieved by 77% of the treatment group compared with 59% of controls (P = .03). Subjects from the treatment group were more likely than the controls to endorse adherence to the 4-month course of pharmacotherapy (P < .001). Subjects who received a USCAP had greater improvements in HRQoL total scale score by the end of the project (P = .04)., Conclusions: The USCAP is a simple, inexpensive tool that has the potential to improve global outcomes for functional constipation in children and should be recommended as standard clinical practice., (Published by Elsevier Inc.)
- Published
- 2023
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10. Evaluation of an Evidence-Based Prenatal Breastfeeding Education Curriculum Adapted for the COVID-19 Pandemic: Ready, Set, Baby Live COVID-19 Edition.
- Author
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Wouk K, Parry KC, Bridgman J, Palmquist AEL, Perkins M, Smetana A, Woods-Barr A, and Sullivan CS
- Abstract
In response to the cessation of in-person prenatal education services during the COVID-19 pandemic, we adapted an evidence-based curriculum to a live virtual format entitled Ready, Set, Baby Live COVID-19 Edition (RSB Live). In a sample of 146 pregnant people, participation in RSB Live was associated with high levels of knowledge about the benefits of breastfeeding, early infant hunger cues, and recommended maternity care practices, as well as high levels of satisfaction with adaptations to the session's content and virtual delivery. Participation was also associated with a significant increase in prenatal breastfeeding intention, a known predictor of breastfeeding outcomes. This study supports live, virtual education with a standardized curriculum as an effective and acceptable means of providing prenatal education., (© Copyright 2023 Lamaze International.)
- Published
- 2023
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11. Interpreting and de-noising genetically engineered barcodes in a DNA virus.
- Author
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Blois S, Goetz BM, Bull JJ, and Sullivan CS
- Subjects
- Mice, Animals, DNA Viruses genetics, Nucleic Acids
- Abstract
The concept of a nucleic acid barcode applied to pathogen genomes is easy to grasp and the many possible uses are straightforward. But implementation may not be easy, especially when growing through multiple generations or assaying the pathogen long-term. The potential problems include: the barcode might alter fitness, the barcode may accumulate mutations, and construction of the marked pathogens may result in unintended barcodes that are not as designed. Here, we generate approximately 5,000 randomized barcodes in the genome of the prototypic small DNA virus murine polyomavirus. We describe the challenges faced with interpreting the barcode sequences obtained from the library. Our Illumina NextSeq sequencing recalled much greater variation in barcode sequencing reads than the expected 5,000 barcodes-necessarily stemming from the Illumina library processing and sequencing error. Using data from defined control virus genomes cloned into plasmid backbones we develop a vetted post-sequencing method to cluster the erroneous reads around the true virus genome barcodes. These findings may foreshadow problems with randomized barcodes in other microbial systems and provide a useful approach for future work utilizing nucleic acid barcoded pathogens., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Blois et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
- Published
- 2022
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12. Development and assessment of a low-health-literacy, pictographic adrenal insufficiency action plan.
- Author
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Reeves PT, Packett AC, Burklow CS, Echelmeyer S, and Larson NS
- Subjects
- Adult, Child, Child, Preschool, Comprehension, Humans, Middle Aged, Adrenal Insufficiency therapy, Health Literacy, Patient Education as Topic
- Abstract
Objectives: Adrenal insufficiency (AI) is an overall rare disorder characterized by the chronic need for pharmacotherapy to prevent threat to life. The Pediatric Endocrine Society has recommended the use of clinical action tools to improve patient education and help guide acute management of AI. We aimed to develop and assess an easy-to-use, patient-friendly, evidence-based, personalized pictogram-based adrenal insufficiency action plan (AIAP) to aid in the management of AI in children., Methods: Patients/caregivers (P/Cs) responded to surveys which measured the concepts of transparency, translucency, and recall in order to assess the pictograms. Readability was assessed using six formulas to generate a composite readability score. Quality was graded by P/Cs using the Consumer Information Rating Form (CIRF) (>80% rating considered acceptable). Understandability and actionability was assessed by medical librarians using the Patient Education Materials Assessment Tool-Printable (PEMAT-P) (>80% rating was acceptable). Suitability was evaluated by clinicians using the Suitability Assessment of Materials (SAM) instrument (>70% rating considered superior)., Results: All pictograms met criteria for inclusion in the AIAP. Composite readability score=5.4 was consistent with a fifth-grade level. P/Cs (n=120) judged the AIAP to be of high quality with CIRF rating=85.2%. Three medical librarians rated the AIAP to have 100% understandability and 100% actionability. Thirty-three clinicians completing the SAM generated a suitability rating of 90.0%., Conclusions: The AIAP visually highlights individualized care plan components to facilitate optimized preventative and acute AI care. Further investigation will determine if AIAP improves clinical outcomes for patients with AI., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)
- Published
- 2021
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13. Impact of Parental Illness and Injury on Pediatric Disorders of Gut-Brain Interaction.
- Author
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Short P, Burklow CS, Nylund CM, Susi A, and Hisle-Gorman E
- Subjects
- Adolescent, Child, Child, Preschool, Female, Humans, Male, Military Family, Odds Ratio, Time Factors, Abdominal Pain epidemiology, Constipation epidemiology, Family Health, Fecal Incontinence epidemiology, Irritable Bowel Syndrome epidemiology, Parents
- Abstract
Objective: To evaluate the relationship between parental injury and illness and disorders of gut-brain interaction (DGBI) in children., Study Design: A self-controlled case series using data from the Military Health System Data Repository compared International Classification of Diseases, Ninth Revision-identified DGBI-related outpatient visits and prescriptions in 442 651 children aged 3-16 years in the 2 years before and the 2 years after the injury and/or illness of their military parent. Negative binomial regression was used to compare visit rates for constipation, fecal incontinence, abdominal pain, irritable bowel syndrome, and a composite of these before and after parental injury and/or illness. Logistic regression, clustered by child, compared the odds of stooling agent and antispasmodic prescription before and after parental injury and/or illness., Results: In the 2 years following parental injury and/or illness, children had increased visits for DGBIs (adjusted incidence rate ratio [aIRR] 1.09; 95% CI 1.07-1.10), constipation (aIRR 1.07; 95% CI 1.04-1.10), abdominal pain (aIRR 1.09; 95% CI 1.07-1.12), and irritable bowel syndrome (aIRR 1.37; 95% CI 1.19-1.58). Following parental injury and/or illness, the odds of stooling agent prescription decreased (aOR 0.95; 95% CI 0.93-0.97) and the odds of antispasmodic prescription increased (aOR 1.26; 95% CI 1.18-1.36)., Conclusions: Parental injury and/or illness is associated with increased healthcare use for DGBIs. Parental health should be considered by clinicians when assessing DGBIs, counseling patients, and formulating treatment plans., (Published by Elsevier Inc.)
- Published
- 2021
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14. Characterization of ALTO-encoding circular RNAs expressed by Merkel cell polyomavirus and trichodysplasia spinulosa polyomavirus.
- Author
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Yang R, Lee EE, Kim J, Choi JH, Kolitz E, Chen Y, Crewe C, Salisbury NJH, Scherer PE, Cockerell C, Smith TR, Rosen L, Verlinden L, Galloway DA, Buck CB, Feltkamp MC, Sullivan CS, and Wang RC
- Subjects
- Exosomes, Gene Expression Regulation, Viral, HEK293 Cells, Humans, MicroRNAs genetics, RNA, Messenger genetics, RNA, Viral genetics, Antigens, Viral, Tumor genetics, Carcinoma, Merkel Cell virology, Merkel cell polyomavirus genetics, Polyomavirus Infections virology, RNA, Circular genetics, Tumor Virus Infections virology
- Abstract
Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving as messenger RNAs that are translated into peptides. Here we describe circular RNAs generated by human polyomaviruses (HPyVs), some of which encode variants of the previously described alternative large T antigen open reading frame (ALTO) protein. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. The translation of MCPyV circALTOs into ALTO protein is negatively regulated by MCPyV-generated miRNAs in cultured cells. MCPyV ALTO expression increases transcription from some recombinant promoters in vitro and upregulates the expression of multiple genes previously implicated in MCPyV pathogenesis. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation in MCPyV-negative cells. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected in infected tissues and produces ALTO protein in cultured cells. Thus, human polyomavirus circRNAs are expressed in human tumors and infected tissues and express proteins that have the potential to modulate the infectious and tumorigenic properties of these viruses., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
- Full Text
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15. Development and Assessment of a Pictographic Pediatric Constipation Action Plan.
- Author
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Reeves PT, Kolasinski NT, Yin HS, Alqurashi W, Echelmeyer S, Chumpitazi BP, Rogers PL, Burklow CS, and Nylund CM
- Subjects
- Adult, Child, Comprehension, Health Literacy, Humans, Middle Aged, Parents education, Sampling Studies, Surveys and Questionnaires, Constipation therapy, Health Communication methods, Patient Education as Topic
- Abstract
Objective: To assess the Uniformed Services Constipation Action Plan (USCAP) as an evidence-based, personalized, clinical action tool with pictograms to aid clinicians and families in the management of functional constipation., Study Design: The USCAP facilitates the management functional constipation by using a health literacy-informed approach to provide instructions for pharmacotherapies and lifestyle modifications. This study included part 1 (pictogram validation) and part 2 (assessment). For part 1, pictogram transparency, translucency, and recall were assessed by parent survey (transparency ≥85%, mean translucency score ≥5, recall ≥85% required for validation). For part 2, the USCAP was assessed by parents, clinical librarians, and clinicians. Parental perceptions (n = 65) were assessed using the Consumer Information Rating Form (17 questions) to gauge comprehensibility, design quality and usefulness. Readability was assessed by 5 formulas and a Readability Composite Score was calculated. Clinical librarians (n = 3) used the Patient Education Materials Assessment Tool to measure understandability (19 questions) and actionability (7 questions) (>80% rating was acceptable). Suitability was assessed by clinicians (n = 34) using Doak's Suitability Assessment of Materials (superior ≥70% rating)., Results: All 12 pictograms demonstrated appropriate transparency, translucency, and recall. Parental perceptions reflected appropriate comprehensibility, design quality, and usefulness. The Readability Composite Score was consistent with a fifth-grade level. Clinical librarians reported acceptable understandability and actionability. Clinicians reported superior suitability., Conclusions: The USCAP met all criteria for clinical implementation and future study of USCAP implementation for treating children with chronic functional constipation., (Published by Elsevier Inc.)
- Published
- 2021
- Full Text
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16. DUSP11 and triphosphate RNA balance during virus infection.
- Author
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Choi JH and Sullivan CS
- Subjects
- Dual-Specificity Phosphatases genetics, Humans, RNA, Viral chemistry, Virus Diseases genetics, Dual-Specificity Phosphatases metabolism, Host-Pathogen Interactions, Polyphosphates chemistry, RNA, Viral genetics, Virus Diseases virology, Viruses pathogenicity
- Abstract
Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
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17. DUSP11-mediated control of 5'-triphosphate RNA regulates RIG-I sensitivity.
- Author
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Choi JH, Burke JM, Szymanik KH, Nepal U, Battenhouse A, Lau JT, Stark A, Lam V, and Sullivan CS
- Subjects
- Animals, Cell Line, HEK293 Cells, Host-Pathogen Interactions, Humans, Immunity, Innate genetics, Immunity, Innate immunology, Interferons metabolism, Liposomes immunology, Mice, Mice, Inbred C57BL, Polyphosphates, RNA Viruses physiology, RNA, Viral metabolism, Virus Replication genetics, DEAD Box Protein 58 immunology, Dual-Specificity Phosphatases immunology, Dual-Specificity Phosphatases metabolism, RNA immunology, Virus Diseases immunology
- Abstract
Deciphering the mechanisms that regulate the sensitivity of pathogen recognition receptors is imperative to understanding infection and inflammation. Here we demonstrate that the RNA triphosphatase dual-specificity phosphatase 11 (DUSP11) acts on both host and virus-derived 5'-triphosphate RNAs rendering them less active in inducing a RIG-I-mediated immune response. Reducing DUSP11 levels alters host triphosphate RNA packaged in extracellular vesicles and induces enhanced RIG-I activation in cells exposed to extracellular vesicles. Virus infection of cells lacking DUSP11 results in a higher proportion of triphosphorylated viral transcripts and attenuated virus replication, which is rescued by reducing RIG-I expression. Consistent with the activity of DUSP11 in the cellular RIG-I response, mice lacking DUSP11 display lower viral loads, greater sensitivity to triphosphorylated RNA, and a signature of enhanced interferon activity in select tissues. Our results reveal the importance of controlling 5'-triphosphate RNA levels to prevent aberrant RIG-I signaling and demonstrate DUSP11 as a key effector of this mechanism., (© 2020 Choi et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2020
- Full Text
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18. Ready, Set, BABY Live Virtual Prenatal Breastfeeding Education for COVID-19.
- Author
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Palmquist AEL, Parry KC, Wouk K, Lawless GC, Smith JL, Smetana AR, Bourg JF, Hendricks MJ, and Sullivan CS
- Subjects
- Curriculum, Female, Humans, Internet, Pregnancy, United States, Breast Feeding, COVID-19 prevention & control, Prenatal Education methods, Telemedicine methods
- Published
- 2020
- Full Text
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19. Comparison of media and standards for SARS-CoV-2 RT-qPCR without prior RNA preparation.
- Author
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Ragan KB, Bhadra S, Choi JH, Towers D, Sullivan CS, and Ellington AD
- Abstract
Since the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there have been demands on the testing infrastructure that have strained testing capacity. As a simplification of method, we confirm the efficacy of RNA extraction-free RT-qPCR and saline as an alternative patient sample storage buffer. In addition, amongst potential reagent shortages, it has sometimes been difficult to obtain inactivated viral particles. We have therefore also characterized armored SARS-CoV-2 RNA from Asuragen as an alternative diagnostic standard to ATCC genomic SARS-CoV-2 RNA and heat inactivated virions and provide guidelines for its use in RT-qPCR.
- Published
- 2020
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20. Correction for Kincaid et al., "Noncanonical MicroRNA (miRNA) Biogenesis Gives Rise to Retroviral Mimics of Lymphoproliferative and Immunosuppressive Host miRNAs".
- Author
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Kincaid RP, Chen Y, Cox JE, Rethwilm A, and Sullivan CS
- Published
- 2020
- Full Text
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21. Developmental Regulation of Basket Interneuron Synapses and Behavior through NCAM in Mouse Prefrontal Cortex.
- Author
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Sullivan CS, Mohan V, Manis PB, Moy SS, Truong Y, Duncan BW, and Maness PF
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- Animals, Behavior, Animal, Female, Male, Memory, Short-Term physiology, Mice, Mice, Inbred C57BL, Prefrontal Cortex growth & development, Interneurons metabolism, Neural Cell Adhesion Molecules metabolism, Neurogenesis physiology, Prefrontal Cortex metabolism, Synapses physiology
- Abstract
Parvalbumin (PV)-expressing basket interneurons in the prefrontal cortex (PFC) regulate pyramidal cell firing, synchrony, and network oscillations. Yet, it is unclear how their perisomatic inputs to pyramidal neurons are integrated into neural circuitry and adjusted postnatally. Neural cell adhesion molecule NCAM is expressed in a variety of cells in the PFC and cooperates with EphrinA/EphAs to regulate inhibitory synapse density. Here, analysis of a novel parvalbumin (PV)-Cre: NCAM F/F mouse mutant revealed that NCAM functions presynaptically in PV+ basket interneurons to regulate postnatal elimination of perisomatic synapses. Mutant mice exhibited an increased density of PV+ perisomatic puncta in PFC layer 2/3, while live imaging in mutant brain slices revealed fewer puncta that were dynamically eliminated. Furthermore, EphrinA5-induced growth cone collapse in PV+ interneurons in culture depended on NCAM expression. Electrophysiological recording from layer 2/3 pyramidal cells in mutant PFC slices showed a slower rise time of inhibitory synaptic currents. PV-Cre: NCAM F/F mice exhibited impairments in working memory and social behavior that may be impacted by altered PFC circuitry. These findings suggest that the density of perisomatic synapses of PV+ basket interneurons is regulated postnatally by NCAM, likely through EphrinA-dependent elimination, which is important for appropriate PFC network function and behavior., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2020
- Full Text
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22. Close Homolog of L1 Regulates Dendritic Spine Density in the Mouse Cerebral Cortex Through Semaphorin 3B.
- Author
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Mohan V, Wade SD, Sullivan CS, Kasten MR, Sweetman C, Stewart R, Truong Y, Schachner M, Manis PB, and Maness PF
- Subjects
- Aging physiology, Animals, Cell Adhesion Molecules deficiency, Cells, Cultured, Female, GABA Agonists pharmacology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Nerve Tissue Proteins physiology, Neuropilin-2 physiology, Patch-Clamp Techniques, Prefrontal Cortex cytology, Prefrontal Cortex growth & development, Protein Interaction Mapping, Pyramidal Cells drug effects, Pyramidal Cells physiology, Pyramidal Cells ultrastructure, Pyridazines pharmacology, Receptors, Cell Surface physiology, Synaptic Transmission, Visual Cortex cytology, Visual Cortex growth & development, Cell Adhesion Molecules physiology, Dendritic Spines physiology, Prefrontal Cortex physiology, Semaphorins physiology, Visual Cortex physiology
- Abstract
Dendritic spines in the developing mammalian neocortex are initially overproduced and then eliminated during adolescence to achieve appropriate levels of excitation in mature networks. We show here that the L1 family cell adhesion molecule Close Homolog of L1 (CHL1) and secreted repellent ligand Semaphorin 3B (Sema3B) function together to induce dendritic spine pruning in developing cortical pyramidal neurons. Loss of CHL1 in null mutant mice in both genders resulted in increased spine density and a greater proportion of immature spines on apical dendrites in the prefrontal and visual cortex. Electron microscopy showed that excitatory spine synapses with postsynaptic densities were increased in the CHL1-null cortex, and electrophysiological recording in prefrontal slices from mutant mice revealed deficiencies in excitatory synaptic transmission. Mechanistically, Sema3B protein induced elimination of spines on apical dendrites of cortical neurons cultured from wild-type but not CHL1-null embryos. Sema3B was secreted by the cortical neuron cultures, and its levels increased when cells were treated with the GABA antagonist gabazine. In vivo CHL1 was coexpressed with Sema3B in pyramidal neuron subpopulations and formed a complex with Sema3B receptor subunits Neuropilin-2 and PlexinA4. CHL1 and NrCAM, a closely related L1 adhesion molecule, localized primarily to distinct spines and promoted spine elimination to Sema3B or Sema3F, respectively. These results support a new concept in which selective spine elimination is achieved through different secreted semaphorins and L1 family adhesion molecules to sculpt functional neural circuits during postnatal maturation. SIGNIFICANCE STATEMENT Dendritic spines in the mammalian neocortex are initially overproduced and then pruned in adolescent life through unclear mechanisms to sculpt maturing cortical circuits. Here, we show that spine and excitatory synapse density of pyramidal neurons in the developing neocortex is regulated by the L1 adhesion molecule, Close Homolog of L1 (CHL1). CHL1 mediated spine pruning in response to the secreted repellent ligand Semaphorin 3B and associated with receptor subunits Neuropilin-2 and PlexinA4. CHL1 and related L1 adhesion molecule NrCAM localized to distinct spines, and promoted spine elimination to Semaphorin 3B and -3F, respectively. These results support a new concept in which selective elimination of individual spines and nascent synapses can be achieved through the action of distinct secreted semaphorins and L1 adhesion molecules., (Copyright © 2019 the authors.)
- Published
- 2019
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23. The coronavirus macrodomain is required to prevent PARP-mediated inhibition of virus replication and enhancement of IFN expression.
- Author
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Grunewald ME, Chen Y, Kuny C, Maejima T, Lease R, Ferraris D, Aikawa M, Sullivan CS, Perlman S, and Fehr AR
- Subjects
- ADP-Ribosylation, Animals, Coronavirus drug effects, Coronavirus Infections drug therapy, Coronavirus Infections immunology, Coronavirus Infections metabolism, Humans, Immunity, Innate drug effects, Mice, Poly(ADP-ribose) Polymerases chemistry, Poly(ADP-ribose) Polymerases genetics, Protein Domains, Receptor, Interferon alpha-beta genetics, Receptor, Interferon alpha-beta metabolism, Virulence, Coronavirus immunology, Coronavirus Infections virology, Immunity, Innate immunology, Interferons metabolism, Poly(ADP-ribose) Polymerase Inhibitors pharmacology, Poly(ADP-ribose) Polymerases metabolism, Virus Replication
- Abstract
ADP-ribosylation is a ubiquitous post-translational addition of either monomers or polymers of ADP-ribose to target proteins by ADP-ribosyltransferases, usually by interferon-inducible diphtheria toxin-like enzymes known as PARPs. While several PARPs have known antiviral activities, these activities are mostly independent of ADP-ribosylation. Consequently, less is known about the antiviral effects of ADP-ribosylation. Several viral families, including Coronaviridae, Togaviridae, and Hepeviridae, encode for macrodomain proteins that bind to and hydrolyze ADP-ribose from proteins and are critical for optimal replication and virulence. These results suggest that macrodomains counter cellular ADP-ribosylation, but whether PARPs or, alternatively, other ADP-ribosyltransferases cause this modification is not clear. Here we show that pan-PARP inhibition enhanced replication and inhibited interferon production in primary macrophages infected with macrodomain-mutant but not wild-type coronavirus. Specifically, knockdown of two abundantly expressed PARPs, PARP12 and PARP14, led to increased replication of mutant but did not significantly affect wild-type virus. PARP14 was also important for the induction of interferon in mouse and human cells, indicating a critical role for this PARP in the regulation of innate immunity. In summary, these data demonstrate that the macrodomain is required to prevent PARP-mediated inhibition of coronavirus replication and enhancement of interferon production., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
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24. Temporal Regulation of Dendritic Spines Through NrCAM-Semaphorin3F Receptor Signaling in Developing Cortical Pyramidal Neurons.
- Author
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Mohan V, Sullivan CS, Guo J, Wade SD, Majumder S, Agarwal A, Anton ES, Temple BS, and Maness PF
- Subjects
- Animals, Guanylate Kinases physiology, Mice, Transgenic, Models, Molecular, Signal Transduction, Cell Adhesion Molecules physiology, Dendritic Spines physiology, Frontal Lobe growth & development, Membrane Proteins physiology, Nerve Tissue Proteins physiology, Pyramidal Cells physiology, Visual Cortex growth & development
- Abstract
Neuron-glial related cell adhesion molecule NrCAM is a newly identified negative regulator of spine density that genetically interacts with Semaphorin3F (Sema3F), and is implicated in autism spectrum disorders (ASD). To investigate a role for NrCAM in spine pruning during the critical adolescent period when networks are established, we generated novel conditional, inducible NrCAM mutant mice (Nex1Cre-ERT2: NrCAMflox/flox). We demonstrate that NrCAM functions cell autonomously during adolescence in pyramidal neurons to restrict spine density in the visual (V1) and medial frontal cortex (MFC). Guided by molecular modeling, we found that NrCAM promoted clustering of the Sema3F holoreceptor complex by interfacing with Neuropilin-2 (Npn2) and PDZ scaffold protein SAP102. NrCAM-induced receptor clustering stimulated the Rap-GAP activity of PlexinA3 (PlexA3) within the holoreceptor complex, which in turn, inhibited Rap1-GTPase and inactivated adhesive β1 integrins, essential for Sema3F-induced spine pruning. These results define a developmental function for NrCAM in Sema3F receptor signaling that limits dendritic spine density on cortical pyramidal neurons during adolescence.
- Published
- 2019
- Full Text
- View/download PDF
25. RNA triphosphatase DUSP11 enables exonuclease XRN-mediated restriction of hepatitis C virus.
- Author
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Kincaid RP, Lam VL, Chirayil RP, Randall G, and Sullivan CS
- Subjects
- Acid Anhydride Hydrolases genetics, Carcinoma, Hepatocellular genetics, Carcinoma, Hepatocellular virology, Cell Line, Tumor, Dual-Specificity Phosphatases genetics, Exoribonucleases genetics, Gene Knockout Techniques, Genome, Viral, Hepacivirus physiology, Hepatitis C, Chronic genetics, Hepatitis C, Chronic virology, Hepatocytes virology, Humans, Liver Neoplasms genetics, Liver Neoplasms virology, RNA, Small Interfering metabolism, RNA, Viral metabolism, Virus Replication genetics, Acid Anhydride Hydrolases metabolism, Dual-Specificity Phosphatases metabolism, Exoribonucleases metabolism, Hepacivirus pathogenicity, Host-Pathogen Interactions physiology, MicroRNAs metabolism
- Abstract
Seventy percent of people infected with hepatitis C virus (HCV) will suffer chronic infection, putting them at risk for liver disease, including hepatocellular carcinoma. The full range of mechanisms that render some people more susceptible to chronic infection and liver disease is still being elucidated. XRN exonucleases can restrict HCV replication and may help to resolve HCV infections. However, it is unknown how 5' triphosphorylated HCV transcripts, primary products of the viral polymerase, become susceptible to attack by 5' monophosphate-specific XRNs. Here, we show that the 5' RNA triphosphatase DUSP11 acts on HCV transcripts, rendering them susceptible to XRN-mediated attack. Cells lacking DUSP11 show substantially enhanced HCV replication, and this effect is diminished when XRN expression is reduced. MicroRNA-122 (miR-122), a target of current phase II anti-HCV drugs, is known to protect HCV transcripts against XRNs. We show that HCV replication is less dependent on miR-122 in cells lacking DUSP11. Combined, these results implicate DUSP11 as an important component of XRN-mediated restriction of HCV., Competing Interests: The authors declare no conflict of interest.
- Published
- 2018
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- View/download PDF
26. The Murine Polyomavirus MicroRNA Locus Is Required To Promote Viruria during the Acute Phase of Infection.
- Author
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Burke JM, Bass CR, Kincaid RP, Ulug ET, and Sullivan CS
- Subjects
- Animals, Mice, MicroRNAs genetics, Polyomavirus genetics, RNA, Viral genetics, Virus Shedding, MicroRNAs metabolism, Polyomavirus pathogenicity, Polyomavirus Infections pathology, Polyomavirus Infections virology, RNA, Viral metabolism, Urine virology
- Abstract
Polyomaviruses (PyVs) can cause serious disease in immunosuppressed hosts. Several pathogenic PyVs encode microRNAs (miRNAs), small RNAs that regulate gene expression via RNA silencing. Despite recent advances in understanding the activities of PyV miRNAs, the biological functions of PyV miRNAs during in vivo infections are mostly unknown. The studies presented here used murine polyomavirus (MuPyV) as a model to assess the roles of the PyV miRNAs in a natural host. This analysis revealed that a MuPyV mutant that is unable to express miRNAs has enhanced viral DNA loads in select tissues at late times after infection. This is consistent with the PyV miRNAs functioning to reduce viral replication during the persistent phase of infection in a natural host. Additionally, the MuPyV miRNA locus promotes viruria during the acute phase of infection as evidenced by a defect in shedding during infection with the miRNA mutant virus. The viruria defect of the miRNA mutant virus could be rescued by infecting Rag2
-/- mice. These findings implicate the miRNA locus as functioning in both the persistent and acute phases of infection and suggest a role for MuPyV miRNA in evading the adaptive immune response. IMPORTANCE MicroRNAs are expressed by diverse viruses, but for only a few is there any understanding of their in vivo function. PyVs can cause serious disease in immunocompromised hosts. Therefore, increased knowledge of how these viruses interact with the immune response is of clinical relevance. Here we show a novel activity for a viral miRNA locus in promoting virus shedding. This work indicates that in addition to any role for the PyV miRNA locus in long-term persistence, it also has biological activity during the acute phase. As this mutant phenotype is alleviated by infection of mice lacking an adaptive immune response, our work also connects the in vivo activity of the PyV miRNA locus to the immune response. Given that PyV-associated disease is associated with alterations in the immune response, our findings help to better understand how the balance between PyVs and the immune response becomes altered in pathogenic states., (Copyright © 2018 American Society for Microbiology.)- Published
- 2018
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27. Identification of virus-encoded microRNAs in divergent Papillomaviruses.
- Author
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Chirayil R, Kincaid RP, Dahlke C, Kuny CV, Dälken N, Spohn M, Lawson B, Grundhoff A, and Sullivan CS
- Subjects
- HEK293 Cells, Humans, Papillomavirus Infections genetics, RNA, Viral genetics, Transcriptome, Gene Expression Profiling methods, Gene Expression Regulation, Viral genetics, MicroRNAs genetics, Papillomaviridae genetics, RNA, Viral analysis
- Abstract
MicroRNAs (miRNAs) are small RNAs that regulate diverse biological processes including multiple aspects of the host-pathogen interface. Consequently, miRNAs are commonly encoded by viruses that undergo long-term persistent infection. Papillomaviruses (PVs) are capable of undergoing persistent infection, but as yet, no widely-accepted PV-encoded miRNAs have been described. The incomplete understanding of PV-encoded miRNAs is due in part to lack of tractable laboratory models for most PV types. To overcome this, we have developed miRNA Discovery by forced Genome Expression (miDGE), a new wet bench approach to miRNA identification that screens numerous pathogen genomes in parallel. Using miDGE, we screened over 73 different PV genomes for the ability to code for miRNAs. Our results show that most PVs are unlikely to code for miRNAs and we conclusively demonstrate a lack of PV miRNA expression in cancers associated with infections of several high risk HPVs. However, we identified five different high-confidence or highly probable miRNAs encoded by four different PVs (Human PVs 17, 37, 41 and a Fringilla coelebs PV (FcPV1)). Extensive in vitro assays confirm the validity of these miRNAs in cell culture and two FcPV1 miRNAs are further confirmed to be expressed in vivo in a natural host. We show that miRNAs from two PVs (HPV41 & FcPV1) are able to regulate viral transcripts corresponding to the early region of the PV genome. Combined, these findings identify the first canonical PV miRNAs and support that miRNAs of either host or viral origin are important regulators of the PV life cycle., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2018
- Full Text
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28. Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons.
- Author
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Sullivan CS, Gotthard I, Wyatt EV, Bongu S, Mohan V, Weinberg RJ, and Maness PF
- Subjects
- Animals, Cerebral Cortex cytology, Cerebral Cortex metabolism, Humans, Mice, Neural Cell Adhesion Molecules chemistry, Protein Binding, Protein Interaction Domains and Motifs, Receptor Protein-Tyrosine Kinases chemistry, Receptor, EphA3, GABAergic Neurons metabolism, Interneurons metabolism, Neural Cell Adhesion Molecules metabolism, Neurocan metabolism, Receptor Protein-Tyrosine Kinases metabolism, Signal Transduction
- Abstract
Perineuronal nets (PNNs) are implicated in closure of critical periods of synaptic plasticity in the brain, but the molecular mechanisms by which PNNs regulate synapse development are obscure. A receptor complex of NCAM and EphA3 mediates postnatal remodeling of inhibitory perisomatic synapses of GABAergic interneurons onto pyramidal cells in the mouse frontal cortex necessary for excitatory/inhibitory balance. Here it is shown that enzymatic removal of PNN glycosaminoglycan chains decreased the density of GABAergic perisomatic synapses in mouse organotypic cortical slice cultures. Neurocan, a key component of PNNs, was expressed in postnatal frontal cortex in apposition to perisomatic synapses of parvalbumin-positive interneurons. Polysialylated NCAM (PSA-NCAM), which is required for ephrin-dependent synapse remodeling, bound less efficiently to neurocan than mature, non-PSA-NCAM. Neurocan bound the non-polysialylated form of NCAM at the EphA3 binding site within the immunoglobulin-2 domain. Neurocan inhibited NCAM/EphA3 association, membrane clustering of NCAM/EphA3 in cortical interneuron axons, EphA3 kinase activation, and ephrin-A5-induced growth cone collapse. These studies delineate a novel mechanism wherein neurocan inhibits NCAM/EphA3 signaling and axonal repulsion, which may terminate postnatal remodeling of interneuron axons to stabilize perisomatic synapses in vivo.
- Published
- 2018
- Full Text
- View/download PDF
29. MMTV does not encode viral microRNAs but alters the levels of cancer-associated host microRNAs.
- Author
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Kincaid RP, Panicker NG, Lozano MM, Sullivan CS, Dudley JP, and Mustafa F
- Subjects
- Animals, High-Throughput Nucleotide Sequencing, Mice, Gene Expression Regulation, Host-Pathogen Interactions, Mammary Neoplasms, Experimental virology, Mammary Tumor Virus, Mouse physiology, MicroRNAs analysis
- Abstract
Mouse mammary tumor virus (MMTV) induces breast cancer in mice in the absence of known virally-encoded oncogenes. Tumorigenesis by MMTV is thought to occur primarily through insertional mutagenesis, leading to the activation of cellular proto-oncogenes and outgrowth of selected cells. Here we investigated whether MMTV encodes microRNAs (miRNAs) and/or modulates host miRNAs that could contribute to tumorigenesis. High throughput small RNA sequencing analysis of MMTV-infected cells and MMTV-induced mammary tumors demonstrates that MMTV does not encode miRNAs. However, infected tissues have altered levels of several host miRNAs, including increased expression of members of the oncogenic miRNA cluster, miR-17-92. Notably, similar changes in miRNA levels have been previously reported in human breast cancers. Combined, our results demonstrate that virally encoded miRNAs do not contribute to MMTV-mediated tumorigenesis, but that changes in specific host miRNAs in infected cells may contribute to virus replication and tumor biology., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
30. Parent-infant room-sharing is complex and important for breastfeeding.
- Author
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Tully KP and Sullivan CS
- Subjects
- Beds, Humans, Infant, Parents, Breast Feeding, Infant Care
- Abstract
Competing Interests: Competing interests: None declared.
- Published
- 2018
- Full Text
- View/download PDF
31. DUSP11 - An RNA phosphatase that regulates host and viral non-coding RNAs in mammalian cells.
- Author
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Burke JM and Sullivan CS
- Subjects
- Animals, Argonaute Proteins genetics, Argonaute Proteins metabolism, Caenorhabditis elegans, DEAD-box RNA Helicases genetics, DEAD-box RNA Helicases metabolism, Dual-Specificity Phosphatases metabolism, Gene Expression Regulation, Humans, MicroRNAs metabolism, RNA Polymerase II genetics, RNA Polymerase II metabolism, RNA Polymerase III genetics, RNA Polymerase III metabolism, RNA, Small Interfering metabolism, RNA, Viral metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, RNA-Induced Silencing Complex genetics, RNA-Induced Silencing Complex metabolism, Ribonuclease III genetics, Ribonuclease III metabolism, Signal Transduction, Viruses genetics, Viruses metabolism, Dual-Specificity Phosphatases genetics, Host-Pathogen Interactions genetics, MicroRNAs genetics, RNA, Small Interfering genetics, RNA, Viral genetics
- Abstract
Dual-specificity phosphatase 11 (DUSP11) is a conserved protein tyrosine phosphatase (PTP) in metazoans. The cellular substrates and physiologic activities of DUSP11 remain largely unknown. In nematodes, DUSP11 is required for normal development and RNA interference against endogenous RNAs (endo-RNAi) via molecular mechanisms that are not well understood. However, mammals lack analogous endo-RNAi pathways and consequently, a role for DUSP11 in mammalian RNA silencing was unanticipated. Recent work from our laboratory demonstrated that DUSP11 activity alters the silencing potential of noncanonical viral miRNAs in mammalian cells. Our studies further uncovered direct cellular substrates of DUSP11 and suggest that DUSP11 is part of regulatory pathway that controls the abundance of select triphosphorylated noncoding RNAs. Here, we highlight recent findings and present new data that advance understanding of mammalian DUSP11 during gene silencing and discuss the emerging biological activities of DUSP11 in mammalian cells.
- Published
- 2017
- Full Text
- View/download PDF
32. Expression of short hairpin RNAs using the compact architecture of retroviral microRNA genes.
- Author
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Burke JM, Kincaid RP, Aloisio F, Welch N, and Sullivan CS
- Subjects
- Algorithms, Base Pairing, Base Sequence, Gene Expression Regulation, Gene Silencing, Genes, Reporter, Genetic Vectors, HEK293 Cells, Humans, Leukemia Virus, Bovine metabolism, Luciferases genetics, Luciferases metabolism, MicroRNAs metabolism, Promoter Regions, Genetic, RNA Polymerase III metabolism, RNA, Small Interfering metabolism, RNA, Viral metabolism, RNA-Induced Silencing Complex genetics, RNA-Induced Silencing Complex metabolism, Sequence Analysis, RNA, User-Computer Interface, Gene Targeting methods, Leukemia Virus, Bovine genetics, MicroRNAs genetics, RNA Polymerase III genetics, RNA, Small Interfering genetics, RNA, Viral genetics
- Abstract
Short hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, typical U6/H1 promoters encompass the majority of the ∼270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by >2-fold as compared to the typical U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2017
- Full Text
- View/download PDF
33. Innovative Prenatal Breastfeeding Education Curriculum: Ready, Set, BABY.
- Author
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Parry KC, Tully KP, Moss SL, and Sullivan CS
- Subjects
- Curriculum, Female, Food Services, Health Promotion methods, Humans, Infant, Infant, Newborn, North Carolina, Organizational Innovation, Pamphlets, Pilot Projects, Pregnancy, Breast Feeding psychology, Health Knowledge, Attitudes, Practice, Prenatal Education methods
- Published
- 2017
- Full Text
- View/download PDF
34. NCAM Regulates Inhibition and Excitability in Layer 2/3 Pyramidal Cells of Anterior Cingulate Cortex.
- Author
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Zhang X, Sullivan CS, Kratz MB, Kasten MR, Maness PF, and Manis PB
- Subjects
- Animals, Gyrus Cinguli cytology, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Mice, Transgenic, Microscopy, Confocal, Neural Inhibition physiology, Patch-Clamp Techniques, Pyramidal Cells cytology, Electrophysiological Phenomena physiology, Gyrus Cinguli physiology, Neural Cell Adhesion Molecules physiology, Pyramidal Cells physiology
- Abstract
The neural cell adhesion molecule (NCAM), has been shown to be an obligate regulator of synaptic stability and pruning during critical periods of cortical maturation. However, the functional consequences of NCAM deletion on the organization of inhibitory circuits in cortex are not known. In vesicular gamma-amino butyric acid (GABA) transporter (VGAT)-channelrhodopsin2 (ChR2)-enhanced yellow fluorescent protein (EYFP) transgenic mice, NCAM is expressed postnatally at perisomatic synaptic puncta of EYFP-labeled parvalbumin, somatostatin and calretinin-positive interneurons, and in the neuropil in the anterior cingulate cortex (ACC). To investigate how NCAM deletion affects the spatial organization of inhibitory inputs to pyramidal cells, we used laser scanning photostimulation in brain slices of VGAT-ChR2-EYFP transgenic mice crossed to either NCAM-null or wild type (WT) mice. Laser scanning photostimulation revealed that NCAM deletion increased the strength of close-in inhibitory connections to layer 2/3 pyramidal cells of the ACC. In addition, in NCAM-null mice, the intrinsic excitability of pyramidal cells increased, whereas the intrinsic excitability of GABAergic interneurons did not change. The increase in inhibitory tone onto pyramidal cells, and the increased pyramidal cell excitability in NCAM-null mice will alter the delicate coordination of excitation and inhibition (E/I coordination) in the ACC, and may be a factor contributing to circuit dysfunction in diseases such as schizophrenia and bipolar disorder, in which NCAM has been implicated.
- Published
- 2017
- Full Text
- View/download PDF
35. The Neural Cell Adhesion Molecule (NCAM) Promotes Clustering and Activation of EphA3 Receptors in GABAergic Interneurons to Induce Ras Homolog Gene Family, Member A (RhoA)/Rho-associated protein kinase (ROCK)-mediated Growth Cone Collapse.
- Author
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Sullivan CS, Kümper M, Temple BS, and Maness PF
- Subjects
- Animals, Ephrin-A5 genetics, Ephrin-A5 metabolism, Mice, Mice, Mutant Strains, Neural Cell Adhesion Molecules genetics, Phosphorylation physiology, rho GTP-Binding Proteins genetics, rho-Associated Kinases genetics, rhoA GTP-Binding Protein, GABAergic Neurons metabolism, Growth Cones metabolism, Neural Cell Adhesion Molecules metabolism, Receptor, EphA3 metabolism, Signal Transduction physiology, rho GTP-Binding Proteins metabolism, rho-Associated Kinases metabolism
- Abstract
Establishment of a proper balance of excitatory and inhibitory connectivity is achieved during development of cortical networks and adjusted through synaptic plasticity. The neural cell adhesion molecule (NCAM) and the receptor tyrosine kinase EphA3 regulate the perisomatic synapse density of inhibitory GABAergic interneurons in the mouse frontal cortex through ephrin-A5-induced growth cone collapse. In this study, it was demonstrated that binding of NCAM and EphA3 occurred between the NCAM Ig2 domain and EphA3 cysteine-rich domain (CRD). The binding interface was further refined through molecular modeling and mutagenesis and shown to be comprised of complementary charged residues in the NCAM Ig2 domain (Arg-156 and Lys-162) and the EphA3 CRD (Glu-248 and Glu-264). Ephrin-A5 induced co-clustering of surface-bound NCAM and EphA3 in GABAergic cortical interneurons in culture. Receptor clustering was impaired by a charge reversal mutation that disrupted NCAM/EphA3 association, emphasizing the importance of the NCAM/EphA3 binding interface for cluster formation. NCAM enhanced ephrin-A5-induced EphA3 autophosphorylation and activation of RhoA GTPase, indicating a role for NCAM in activating EphA3 signaling through clustering. NCAM-mediated clustering of EphA3 was essential for ephrin-A5-induced growth cone collapse in cortical GABAergic interneurons, and RhoA and a principal effector, Rho-associated protein kinase, mediated the collapse response. This study delineates a mechanism in which NCAM promotes ephrin-A5-dependent clustering of EphA3 through interaction of the NCAM Ig2 domain and the EphA3 CRD, stimulating EphA3 autophosphorylation and RhoA signaling necessary for growth cone repulsion in GABAergic interneurons in vitro, which may extend to remodeling of axonal terminals of interneurons in vivo., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
- Full Text
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36. DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs.
- Author
-
Burke JM, Kincaid RP, Nottingham RM, Lambowitz AM, and Sullivan CS
- Subjects
- Acid Anhydride Hydrolases metabolism, Adenoviridae genetics, Gene Knockout Techniques, HEK293 Cells, Humans, Leukemia Virus, Bovine genetics, Phosphorylation, RNA Polymerase III metabolism, RNA, Viral metabolism, Argonaute Proteins metabolism, Dual-Specificity Phosphatases genetics, Dual-Specificity Phosphatases metabolism, MicroRNAs metabolism, RNA, Untranslated metabolism
- Abstract
RNA silencing is a conserved eukaryotic gene expression regulatory mechanism mediated by small RNAs. In Caenorhabditis elegans, the accumulation of a distinct class of siRNAs synthesized by an RNA-dependent RNA polymerase (RdRP) requires the PIR-1 phosphatase. However, the function of PIR-1 in RNAi has remained unclear. Since mammals lack an analogous siRNA biogenesis pathway, an RNA silencing role for the mammalian PIR-1 homolog (dual specificity phosphatase 11 [DUSP11]) was unexpected. Here, we show that the RNA triphosphatase activity of DUSP11 promotes the RNA silencing activity of viral microRNAs (miRNAs) derived from RNA polymerase III (RNAP III) transcribed precursors. Our results demonstrate that DUSP11 converts the 5' triphosphate of miRNA precursors to a 5' monophosphate, promoting loading of derivative 5p miRNAs into Argonaute proteins via a Dicer-coupled 5' monophosphate-dependent strand selection mechanism. This mechanistic insight supports a likely shared function for PIR-1 in C. elegans Furthermore, we show that DUSP11 modulates the 5' end phosphate group and/or steady-state level of several host RNAP III transcripts, including vault RNAs and Alu transcripts. This study shows that steady-state levels of select noncoding RNAs are regulated by DUSP11 and defines a previously unknown portal for small RNA-mediated silencing in mammals, revealing that DUSP11-dependent RNA silencing activities are shared among diverse metazoans., (© 2016 Burke et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2016
- Full Text
- View/download PDF
37. Uncovering Barriers to Teaching Assistants (TAs) Implementing Inquiry Teaching: Inconsistent Facilitation Techniques, Student Resistance, and Reluctance to Share Control over Learning with Students.
- Author
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Gormally C, Sullivan CS, and Szeinbaum N
- Abstract
Inquiry-based teaching approaches are increasingly being adopted in biology laboratories. Yet teaching assistants (TAs), often novice teachers, teach the majority of laboratory courses in US research universities. This study analyzed the perspectives of TAs and their students and used classroom observations to uncover challenges faced by TAs during their first year of inquiry-based teaching. Our study revealed three insights about barriers to effective inquiry teaching practices: 1) TAs lack sufficient facilitation skills; 2) TAs struggle to share control over learning with students as they reconcile long-standing teaching beliefs with newly learned approaches, consequently undermining their fledgling ability to use inquiry approaches; and 3) student evaluations reinforce teacher-centered behaviors as TAs receive positive feedback conflicting with inquiry approaches. We make recommendations, including changing instructional feedback to focus on learner-centered teaching practices. We urge TA mentors to engage TAs in discussions to uncover teaching beliefs underlying teaching choices and support TAs through targeted feedback and practice.
- Published
- 2016
- Full Text
- View/download PDF
38. Virus-Host Interactions and the ARTD/PARP Family of Enzymes.
- Author
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Kuny CV and Sullivan CS
- Subjects
- Humans, Poly(ADP-ribose) Polymerases classification, Poly(ADP-ribose) Polymerases genetics, Stress, Physiological, Virus Physiological Phenomena, Viruses genetics, Host-Pathogen Interactions, Poly(ADP-ribose) Polymerases metabolism, Viruses immunology
- Published
- 2016
- Full Text
- View/download PDF
39. Lessons Learned from In Vivo Studies of a Viral Noncoding RNA.
- Author
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Kincaid RP and Sullivan CS
- Abstract
Despite increasing interest in the biology of noncoding RNAs (ncRNAs), few functions have been uncovered for viral ncRNAs in vivo. In their recent article in mSphere, Feldman and colleagues [E. R. Feldman et al., mSphere 1(2):e00105-15, 2016, doi:10.1128/mSphere.00105-15] demonstrate a highly specific activity of a gammaherpesviral ncRNA in viral dissemination and establishment of latent infection. Their work highlights several interesting features that should be informative to future studies of viral ncRNA.
- Published
- 2016
- Full Text
- View/download PDF
40. Correction for Chen et al., Divergent MicroRNA Targetomes of Closely Related Circulating Strains of a Polyomavirus.
- Author
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Chen CJ, Cox JE, Kincaid RP, Martinez A, and Sullivan CS
- Published
- 2016
- Full Text
- View/download PDF
41. Identification, validation, and characterization of noncanonical miRNAs.
- Author
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Burke JM, Kuny CV, Kincaid RP, and Sullivan CS
- Subjects
- Eukaryota metabolism, Herpesviridae genetics, RNA Polymerase III metabolism, Retroviridae genetics, Transcription, Genetic, Algorithms, Herpesviridae metabolism, MicroRNAs biosynthesis, RNA, Viral biosynthesis, Retroviridae metabolism
- Abstract
Many eukaryotes and some viruses encode microRNAs (miRNAs), small RNAs that post-transcriptionally regulate gene expression. While most miRNAs are generated through the activity of RNA Polymerase II (RNAP II) and subsequent processing by Drosha and Dicer, some viral miRNAs utilize alternative pathways of biogenesis. Some members of the herpesvirus and retrovirus families can direct synthesis of miRNAs through RNAP III transcription rather than RNAP II and can utilize atypical enzymes to generate miRNAs. Though the advantages of alternative miRNA biogenesis remain unclear for herpesviruses, the retroviral miRNA biogenesis routes allow the RNAP II transcribed retroviral genome to escape Drosha cleavage while still expressing abundant, biologically-active miRNAs. These RNAP III-derived miRNAs have unique characteristics that allow for their identification and characterization. In this article, we describe procedures to predict, validate, and characterize RNAP III-transcribed miRNAs and other small RNAs, while providing resources that are also useful for canonical miRNAs., (Copyright © 2015. Published by Elsevier Inc.)
- Published
- 2015
- Full Text
- View/download PDF
42. Pan-viral-microRNA screening identifies interferon inhibition as a common function of diverse viruses.
- Author
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Cox JE, McClure LV, Goga A, and Sullivan CS
- Subjects
- Blotting, Northern, Blotting, Western, Cell Line, Tumor, DNA Primers genetics, Gene Library, HEK293 Cells, Herpesviridae metabolism, Humans, MicroRNAs metabolism, Oligonucleotides genetics, Gene Expression Regulation, Viral genetics, Herpesviridae genetics, Interferons antagonists & inhibitors, MicroRNAs genetics, Signal Transduction genetics
- Abstract
Diverse viruses encode regulatory RNAs called microRNAs (miRNAs). Despite much progress, the functions of the majority of viral miRNAs remain unknown. Most previous studies have used biochemical methods to uncover targets of viral miRNAs, but it is unclear what fraction of these targets is functionally important. Here, we apply an alternative strategy based on the premise that assorted viral miRNAs will share functionality. Screening a library of >70 human viral miRNAs showed that three unrelated miRNAs from distantly related herpesviruses significantly inhibited IFN signaling. Strikingly, each of these miRNAs directly reduced expression of the cyclic AMP-responsive element-binding protein (CBP), which as part of the p300-CBP complex, mediates IFN signaling. We show that both 5' and 3' derivatives from Epstein-Barr virus (EBV) encoded miR-BART-18 precursor miRNA (pre-miRNA) and the orthologous pre-miRNA from Rhesus lymphocryptovirus contribute to reducing IFN signaling. Thus, through both convergent and divergent evolutionary mechanisms, varied herpesviral miRNAs share the ability to decrease IFN signaling. Restoring miR-BART-18 to cells infected with an EBV miRNA mutant conveyed a cellular growth advantage upon IFN treatment, and relevant miRNAs from other herpesviruses were able to complement this activity. Blocking miR-BART-18 function in an EBV(+) tumor cell line renders cells more susceptible to IFN-mediated effects. These findings provide a mechanism that can at least partially explain the resistance of some EBV-associated tumors to IFN therapy. Our work suggests that similar pan-viral-miRNA functional-based screening strategies are warranted for determining relevant activities of other viral miRNAs.
- Published
- 2015
- Full Text
- View/download PDF
43. Identification of a polyomavirus microRNA highly expressed in tumors.
- Author
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Chen CJ, Cox JE, Azarm KD, Wylie KN, Woolard KD, Pesavento PA, and Sullivan CS
- Subjects
- Animals, Base Sequence, Gene Expression Regulation, Viral, Humans, MicroRNAs metabolism, Molecular Sequence Data, Phylogeny, Polyomavirus classification, Polyomavirus isolation & purification, Polyomavirus metabolism, RNA, Viral metabolism, Raccoons, MicroRNAs genetics, Neoplasms virology, Polyomavirus genetics, RNA, Viral genetics
- Abstract
Polyomaviruses (PyVs) are associated with tumors including Merkel cell carcinoma (MCC). Several PyVs encode microRNAs (miRNAs) but to date no abundant PyV miRNAs have been reported in tumors. To better understand the function of the Merkel cell PyV (MCPyV) miRNA, we examined phylogenetically-related viruses for miRNA expression. We show that two primate PyVs and the more distantly-related raccoon PyV (RacPyV) encode miRNAs that share genomic position and partial sequence identity with MCPyV miRNAs. Unlike MCPyV miRNA in MCC, RacPyV miRNA is highly abundant in raccoon tumors. RacPyV miRNA negatively regulates reporters of early viral (T antigen) transcripts, yet robust viral miRNA expression is tolerated in tumors. We also identify raccoon miRNAs expressed in RacPyV-associated neuroglial brain tumors, including several likely oncogenic miRNAs (oncomiRs). This work describes the first PyV miRNA abundantly expressed in tumors and is consistent with a possible role for both host and viral miRNAs in RacPyV-associated tumors., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
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44. Identification of tri-phosphatase activity in the biogenesis of retroviral microRNAs and RNAP III-generated shRNAs.
- Author
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Burke JM, Bass CR, Kincaid RP, and Sullivan CS
- Subjects
- HEK293 Cells, Humans, MicroRNAs biosynthesis, MicroRNAs chemistry, MicroRNAs metabolism, Phosphates analysis, RNA Precursors biosynthesis, RNA, Small Interfering metabolism, RNA, Small Untranslated chemistry, RNA, Small Untranslated metabolism, Ribonuclease III metabolism, Acid Anhydride Hydrolases metabolism, Leukemia Virus, Bovine genetics, RNA Polymerase III metabolism, RNA, Small Interfering biosynthesis
- Abstract
Transcripts possessing a 5'-triphosphate are a hallmark of viral transcription and can trigger the host antiviral response. 5'-triphosphates are also found on common host transcripts transcribed by RNA polymerase III (RNAP III), yet how these transcripts remain non-immunostimulatory is incompletely understood. Most microRNAs (miRNAs) are 5'-monophosphorylated as a result of sequential endonucleolytic processing by Drosha and Dicer from longer RNA polymerase II (RNAP II)-transcribed primary transcripts. In contrast, bovine leukemia virus (BLV) expresses subgenomic RNAP III transcripts that give rise to miRNAs independent of Drosha processing. Here, we demonstrate that each BLV pre-miRNA is directly transcribed by RNAP III from individual, compact RNAP III type II genes. Thus, similar to manmade RNAP III-generated short hairpin RNAs (shRNAs), the BLV pre-miRNAs are initially 5'-triphosphorylated. Nonetheless, the derivative 5p miRNAs and shRNA-generated 5p small RNAs (sRNAs) possess a 5'-monophosphate. Our enzymatic characterization and small RNA sequencing data demonstrate that BLV 5p miRNAs are co-terminal with 5'-triphosphorylated miRNA precursors (pre-miRNAs). Thus, these results identify a 5'-tri-phosphatase activity that is involved in the biogenesis of BLV miRNAs and shRNA-generated sRNAs. This work advances our understanding of retroviral miRNA and shRNA biogenesis and may have implications regarding the immunostimulatory capacity of RNAP III transcripts., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2014
- Full Text
- View/download PDF
45. Balance and Stealth: The Role of Noncoding RNAs in the Regulation of Virus Gene Expression.
- Author
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Cox JE and Sullivan CS
- Abstract
In the past two decades, our knowledge of gene regulation has been greatly expanded by the discovery of microRNAs (miRNAs). miRNAs are small (19-24 nt) noncoding RNAs (ncRNAs) found in metazoans, plants, and some viruses. They have been shown to regulate many cellular processes, including differentiation, maintenance of homeostasis, apoptosis, and the immune response. At present, there are over 300 known viral miRNAs encoded by diverse virus families. One well-characterized function of some viral miRNAs is the regulation of viral transcripts. Host miRNAs can also regulate viral gene expression. We propose that viruses take advantage of both host and viral ncRNA regulation to balance replication and infectious state (for example, latent versus lytic infection). As miRNA regulation can be reversed upon certain cellular stresses, we hypothesize that ncRNAs can serve viruses as barometers for cellular stress.
- Published
- 2014
- Full Text
- View/download PDF
46. Naturally arising strains of polyomaviruses with severely attenuated microRNA expression.
- Author
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Chen CJ, Burke JM, Kincaid RP, Azarm KD, Mireles N, Butel JS, and Sullivan CS
- Subjects
- Animals, Cell Line, Humans, Immunocompromised Host, JC Virus isolation & purification, JC Virus physiology, Macaca mulatta, MicroRNAs genetics, Mutagenesis, Insertional, Sequence Deletion, Simian virus 40 isolation & purification, Simian virus 40 physiology, Virus Replication, Gene Expression Regulation, Viral, JC Virus genetics, MicroRNAs biosynthesis, Polyomavirus Infections veterinary, Polyomavirus Infections virology, Simian virus 40 genetics
- Abstract
Unlabelled: Several different polyomaviruses (PyVs) encode microRNAs (miRNAs) that regulate viral as well as host gene expression. However, the functions of polyomaviral miRNAs, particularly during in vivo infection, remain poorly understood. Here we identify rare naturally arising PyVs that are severely attenuated or null for miRNA expression. We identify hypomorphic or null strains for miRNA expression from rhesus macaque simian virus 40 (SV40) and human JC virus. These strains were isolated from immunocompromised hosts and derive from insertions or deletions in the viral DNA that preserve the amino acid reading frame of opposing-strand large T antigen gene. Characterization of the SV40 miRNA hypomorph, K661, shows that it is inhibited at the early miRNA biogenesis step of Drosha-mediated processing. Despite having a nonrearranged enhancer, which a previous study has shown renders some PyVs more susceptible to the autoregulatory activities of the miRNA, restoring miRNA expression to K661 has little effect on virus growth in either immortalized or primary monkey kidney cells. Thus, in addition to any effect of accompanying genomic elements, these results suggest that the cellular context also determines susceptibility to PyV miRNA-mediated effects. Combined, these results demonstrate that polyomaviruses lacking miRNAs can arise infrequently and that the functional importance of polyomaviral miRNAs is context dependent, consistent with an activity connected to the immune status of the host., Importance: Diverse virus families encode miRNAs, yet much remains unknown about viral miRNA function and contribution to the infectious cycle. Polyomaviruses (PyVs) are small DNA viruses, long known to be important as etiological agents of rare diseases and valuable models of DNA virus infection. Here, in immunosuppressed hosts, we uncover rare naturally arising variants of different PyVs that have lost the ability to express miRNAs. This represents some of the only known natural viruses to have lost miRNA expression. By probing the biogenesis pathways of these variants, we uncover that miRNA expression is lost via small insertions or deletions that render the transcripts resistant to early steps of miRNA biogenesis while preserving the reading frame of the opposing T antigen transcripts. Overall, our study informs how miRNA genes evolve/devolve in viruses and suggests that miRNA function is exquisitely dependent not only on viral genomic context but also on the cellular and host environment., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
- Full Text
- View/download PDF
47. A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA.
- Author
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Burke JM, Kelenis DP, Kincaid RP, and Sullivan CS
- Subjects
- Base Sequence, Binding Sites genetics, HEK293 Cells, Humans, MicroRNAs chemistry, Molecular Sequence Data, Proteins metabolism, RNA Cleavage genetics, RNA Precursors chemistry, RNA Precursors metabolism, RNA-Binding Proteins, Ribonuclease III genetics, Simian virus 40 genetics, MicroRNAs metabolism, Nucleic Acid Conformation, RNA Processing, Post-Transcriptional genetics, RNA, Viral chemistry, RNA, Viral metabolism, Ribonuclease III metabolism
- Abstract
Processing of primary microRNA (pri-miRNA) stem-loops by the Drosha-DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha-DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha-DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design., (© 2014 Burke et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2014
- Full Text
- View/download PDF
48. The adaptor protein GULP promotes Jedi-1-mediated phagocytosis through a clathrin-dependent mechanism.
- Author
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Sullivan CS, Scheib JL, Ma Z, Dang RP, Schafer JM, Hickman FE, Brodsky FM, Ravichandran KS, and Carter BD
- Subjects
- 3T3 Cells, Actins metabolism, Amino Acid Motifs, Animals, HeLa Cells, Humans, Membrane Proteins chemistry, Mice, Protein Interaction Domains and Motifs, Protein Transport, Adaptor Proteins, Signal Transducing physiology, Clathrin metabolism, Membrane Proteins metabolism, Phagocytosis
- Abstract
During the development of the peripheral nervous system, the large number of apoptotic neurons generated are phagocytosed by glial precursor cells. This clearance is mediated, in part, through the mammalian engulfment receptor Jedi-1. However, the mechanisms by which Jedi-1 mediates phagocytosis are poorly understood. Here we demonstrate that Jedi-1 associates with GULP, the mammalian homologue of CED-6, an adaptor protein required for phagocytosis mediated by the nematode engulfment receptor CED-1. Silencing GULP or mutating the NPXY motif in Jedi-1, which is required for GULP binding, prevents Jedi-1-mediated phagocytosis. How GULP promotes engulfment is not known. Of interest, we find that Jedi-1-induced phagocytosis requires GULP binding to clathrin heavy chain (CHC). During engulfment, CHC is tyrosine phosphorylated, which is required for Jedi-mediated engulfment. Both phosphoclathrin and actin accumulate around engulfed microspheres. Furthermore, knockdown of CHC in HeLa cells prevents Jedi-1-mediated engulfment of microspheres, and knockdown in glial precursors prevents the engulfment of apoptotic neurons. Taken together, these results reveal that Jedi-1 signals through recruitment of GULP, which promotes phagocytosis through a noncanonical phosphoclathrin-dependent mechanism., (© 2014 Sullivan et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)
- Published
- 2014
- Full Text
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49. A host MicroRNA brokers truce with HSV-1.
- Author
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Pare JM and Sullivan CS
- Subjects
- Animals, Humans, Male, Herpesvirus 1, Human genetics, Immediate-Early Proteins biosynthesis, Immediate-Early Proteins genetics, MicroRNAs genetics, Trigeminal Ganglion virology, Ubiquitin-Protein Ligases biosynthesis, Ubiquitin-Protein Ligases genetics
- Abstract
Establishing lifelong infection and periodically shedding infectious progeny is a successful strategy employed by several persistent pathogens. In this issue of Cell Host & Microbe, Pan et al. (2014) demonstrate that a cell-type-specific host microRNA can restrict gene expression and pathogenicity of herpes simplex virus 1, thereby promoting long-term infection., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
50. Noncanonical microRNA (miRNA) biogenesis gives rise to retroviral mimics of lymphoproliferative and immunosuppressive host miRNAs.
- Author
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Kincaid RP, Chen Y, Cox JE, Rethwilm A, and Sullivan CS
- Subjects
- Animals, Host-Pathogen Interactions, RNA Polymerase III metabolism, Sequence Homology, Simian foamy virus genetics, MicroRNAs biosynthesis, Primates virology, Simian foamy virus physiology, Transcription, Genetic
- Abstract
MicroRNAs (miRNAs) play regulatory roles in diverse processes in both eukaryotic hosts and their viruses, yet fundamental questions remain about which viruses code for miRNAs and the functions that they serve. Simian foamy viruses (SFVs) of Old World monkeys and apes can zoonotically infect humans and, by ill-defined mechanisms, take up lifelong infections in their hosts. Here, we report that SFVs encode multiple miRNAs via a noncanonical mode of biogenesis. The primary SFV miRNA transcripts (pri-miRNAs) are transcribed by RNA polymerase III (RNAP III) and take multiple forms, including some that are cleaved by Drosha. However, these miRNAs are generated in a context-dependent fashion, as longer RNAP II transcripts spanning this region are resistant to Drosha cleavage. This suggests that the virus may avoid any fitness penalty that could be associated with viral genome/transcript cleavage. Two SFV miRNAs share sequence similarity and functionality with notable host miRNAs, the lymphoproliferative miRNA miR-155 and the innate immunity suppressor miR-132. These results have important implications regarding foamy virus biology, viral miRNAs, and the development of retroviral-based vectors. IMPORTANCE Fundamental questions remain about which viruses encode miRNAs and their associated functions. Currently, few natural viruses with RNA genomes have been reported to encode miRNAs. Simian foamy viruses are retroviruses that are prevalent in nonhuman host populations, and some can zoonotically infect humans who hunt primates or work as animal caretakers. We identify a cluster of miRNAs encoded by SFV. Characterization of these miRNAs reveals evolutionarily conserved, unconventional mechanisms to generate small RNAs. Several SFV miRNAs share sequence similarity and functionality with host miRNAs, including the oncogenic miRNA miR-155 and innate immunity suppressor miR-132. Strikingly, unrelated herpesviruses also tap into one or both of these same regulatory pathways, implying relevance to a broad range of viruses. These findings provide new insights with respect to foamy virus biology and vectorology.
- Published
- 2014
- Full Text
- View/download PDF
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