Publications by authors named "Kathleen Collins"

188 Publications

Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase.

Proc Natl Acad Sci U S A 2021 Oct;118(42)

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;

Selfish, non-long terminal repeat (non-LTR) retroelements and mobile group II introns encode reverse transcriptases (RTs) that can initiate DNA synthesis without substantial base pairing of primer and template. Biochemical characterization of these enzymes has been limited by recombinant expression challenges, hampering understanding of their properties and the possible exploitation of their properties for research and biotechnology. We investigated the activities of representative RTs using a modified non-LTR RT from and a group II intron RT from Only the non-LTR RT supported robust and serial template jumping, producing one complementary DNA (cDNA) from several templates each copied end to end. We also discovered an unexpected terminal deoxynucleotidyl transferase activity of the RTs that adds nucleotide(s) of choice to 3' ends of single- and/or double-stranded RNA or DNA. Combining these two types of activity with additional insights about nontemplated nucleotide additions to duplexed cDNA product, we developed a streamlined protocol for fusion of next-generation sequencing adaptors to both cDNA ends in a single RT reaction. When benchmarked using a reference pool of microRNAs (miRNAs), library production by Ordered Two-Template Relay (OTTR) using recombinant non-LTR retroelement RT outperformed all commercially available kits and rivaled the low bias of technically demanding home-brew protocols. We applied OTTR to inventory RNAs purified from extracellular vesicles, identifying miRNAs as well as myriad other noncoding RNAs (ncRNAs) and ncRNA fragments. Our results establish the utility of OTTR for automation-friendly, low-bias, end-to-end RNA sequence inventories of complex ncRNA samples.
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http://dx.doi.org/10.1073/pnas.2107900118DOI Listing
October 2021

Establishing the phenotypic spectrum of ZTTK syndrome by analysis of 52 individuals with variants in SON.

Eur J Hum Genet 2021 Sep 15. Epub 2021 Sep 15.

Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands.

Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome, an intellectual disability syndrome first described in 2016, is caused by heterozygous loss-of-function variants in SON. Its encoded protein promotes pre-mRNA splicing of many genes essential for development. Whereas individual phenotypic traits have previously been linked to erroneous splicing of SON target genes, the phenotypic spectrum and the pathogenicity of missense variants have not been further evaluated. We present the phenotypic abnormalities in 52 individuals, including 17 individuals who have not been reported before. In total, loss-of-function variants were detected in 49 individuals (de novo in 47, inheritance unknown in 2), and in 3, a missense variant was observed (2 de novo, 1 inheritance unknown). Phenotypic abnormalities, systematically collected and analyzed in Human Phenotype Ontology, were found in all organ systems. Significant inter-individual phenotypic variability was observed, even in individuals with the same recurrent variant (n = 13). SON haploinsufficiency was previously shown to lead to downregulation of downstream genes, contributing to specific phenotypic features. Similar functional analysis for one missense variant, however, suggests a different mechanism than for heterozygous loss-of-function. Although small in numbers and while pathogenicity of these variants is not certain, these data allow for speculation whether de novo missense variants cause ZTTK syndrome via another mechanism, or a separate overlapping syndrome. In conclusion, heterozygous loss-of-function variants in SON define a recognizable syndrome, ZTTK, associated with a broad, severe phenotypic spectrum, characterized by a large inter-individual variability. These observations provide essential information for affected individuals, parents, and healthcare professionals to ensure appropriate clinical management.
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http://dx.doi.org/10.1038/s41431-021-00960-4DOI Listing
September 2021

The methodological integrity of critical qualitative research: Principles to support design and research review.

J Couns Psychol 2021 Apr;68(3):357-370

University of Massachusetts-Boston.

This article articulates principles and practices that support methodological integrity in relation to critical qualitative research. We begin by describing 2 changes that have occurred in psychological methods over the last 15 years. (a) Building on foundational work advocating for epistemological pluralism, guidelines on how to design, review, and report qualitative and mixed methods have been advanced to support methodological integrity in keeping with a diversity of researchers' aims and approaches. (b) There has been an increased use of critical epistemological perspectives and critical methods. In light of these changes, the current article puts forward principles to support critical qualitative researchers when considering methodological rigor and when formulating rationales to support their methods in the journal article review process. Illustrating the principles with an example of critical research, the article describes common problems and issues in the research design process that can be considered in order to strengthen the returns of critical studies. Recommendations are made for editors and reviewers on how to conduct reviews of critical qualitative research, and pressing concerns for publishing critical qualitative research are detailed. (PsycInfo Database Record (c) 2021 APA, all rights reserved).
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http://dx.doi.org/10.1037/cou0000523DOI Listing
April 2021

Structure of human telomerase holoenzyme with bound telomeric DNA.

Nature 2021 May 21;593(7859):449-453. Epub 2021 Apr 21.

Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.

Telomerase adds telomeric repeats at chromosome ends to compensate for the telomere loss that is caused by incomplete genome end replication. In humans, telomerase is upregulated during embryogenesis and in cancers, and mutations that compromise the function of telomerase result in disease. A previous structure of human telomerase at a resolution of 8 Å revealed a vertebrate-specific composition and architecture, comprising a catalytic core that is flexibly tethered to an H and ACA (hereafter, H/ACA) box ribonucleoprotein (RNP) lobe by telomerase RNA. High-resolution structural information is necessary to develop treatments that can effectively modulate telomerase activity as a therapeutic approach against cancers and disease. Here we used cryo-electron microscopy to determine the structure of human telomerase holoenzyme bound to telomeric DNA at sub-4 Å resolution, which reveals crucial DNA- and RNA-binding interfaces in the active site of telomerase as well as the locations of mutations that alter telomerase activity. We identified a histone H2A-H2B dimer within the holoenzyme that was bound to an essential telomerase RNA motif, which suggests a role for histones in the folding and function of telomerase RNA. Furthermore, this structure of a eukaryotic H/ACA RNP reveals the molecular recognition of conserved RNA and protein motifs, as well as interactions that are crucial for understanding the molecular pathology of many mutations that cause disease. Our findings provide the structural details of the assembly and active site of human telomerase, which paves the way for the development of therapeutic agents that target this enzyme.
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http://dx.doi.org/10.1038/s41586-021-03415-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610991PMC
May 2021

Peeling Back the Layers: How Expressive Writing about Heterosexist Events Benefits Sexual Minority Adults.

J Homosex 2020 Nov 9:1-29. Epub 2020 Nov 9.

Department of Counseling and School Psychology, University of Massachusetts Boston, Boston, MA, USA.

Expressive writing exercises, in which participants self-reflect upon a difficult experience, are widely researched interventions. These exercises have been adapted to address a host of concerns, including trauma, mental distress, physical health, and minority stress. Although several theories exist about the mechanisms through which expressive writing yields psychological benefits, the direct perspectives of those who have completed expressive writing exercise has remained unexplored. This study used grounded theory methods to analyze sexual minority adults' interviews about their experience of engaging in various formats of expressive writing exercises about personal heterosexist events. Participants described both what they found helpful and frustrating about the writing process and reflected on how their perspectives shifted throughout the process. These findings contribute to the literature on how change occurs in expressive writing and provide guidance on how to assist sexual minority adults in healing from heterosexism in their lives.
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http://dx.doi.org/10.1080/00918369.2020.1826834DOI Listing
November 2020

Telomere length set point regulation in human pluripotent stem cells critically depends on the shelterin protein TPP1.

Mol Biol Cell 2020 11 9;31(23):2583-2596. Epub 2020 Sep 9.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.

Telomere maintenance is essential for the long-term proliferation of human pluripotent stem cells, while their telomere length set point determines the proliferative capacity of their differentiated progeny. The shelterin protein TPP1 is required for telomere stability and elongation, but its role in establishing a telomere length set point remains elusive. Here, we characterize the contribution of the shorter isoform of TPP1 (TPP1S) and the amino acid L104 outside the TEL patch, TPP1's telomerase interaction domain, to telomere length control. We demonstrate that cells deficient for TPP1S (TPP1S knockout [KO]), as well as the complete TPP1 KO cell lines, undergo telomere shortening. However, TPP1S KO cells are able to stabilize short telomeres, while TPP1 KO cells die. We compare these phenotypes with those of TPP1 mutant cells, which have short and stable telomeres similar to the TPP1S KO. In contrast to TPP1S KO cells, TPP1 cells respond to increased telomerase levels and maintain protected telomeres. However, TPP1 shows altered sensitivity to expression changes of shelterin proteins suggesting the mutation causes a defect in telomere length feedback regulation. Together this highlights TPP1 as the first shelterin mutant engineered at the endogenous locus of human stem cells with an altered telomere length set point.
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http://dx.doi.org/10.1091/mbc.E19-08-0447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851873PMC
November 2020

Concanamycin A counteracts HIV-1 Nef to enhance immune clearance of infected primary cells by cytotoxic T lymphocytes.

Proc Natl Acad Sci U S A 2020 09 8;117(38):23835-23846. Epub 2020 Sep 8.

Graduate Program in Immunology, University of Michigan, Ann Arbor, MI 48109;

Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.
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http://dx.doi.org/10.1073/pnas.2008615117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519347PMC
September 2020

A year at the helm.

JCI Insight 2020 09 3;5(17). Epub 2020 Sep 3.

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http://dx.doi.org/10.1172/jci.insight.142915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526442PMC
September 2020

A deliberate path toward diversity, equity, and inclusion within the ASCI.

J Clin Invest 2020 10;130(10):5031-5032

Editor, Journal of Clinical Investigation, , 2018-2022; Johns Hopkins University, Baltimore, Maryland, USA.

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http://dx.doi.org/10.1172/JCI142423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524472PMC
October 2020

Vpr Is a VIP: HIV Vpr and Infected Macrophages Promote Viral Pathogenesis.

Viruses 2020 07 27;12(8). Epub 2020 Jul 27.

Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.

HIV infects several cell types in the body, including CD4 T cells and macrophages. Here we review the role of macrophages in HIV infection and describe complex interactions between viral proteins and host defenses in these cells. Macrophages exist in many forms throughout the body, where they play numerous roles in healthy and diseased states. They express pattern-recognition receptors (PRRs) that bind viral, bacterial, fungal, and parasitic pathogens, making them both a key player in innate immunity and a potential target of infection by pathogens, including HIV. Among these PRRs is mannose receptor, a macrophage-specific protein that binds oligosaccharides, restricts HIV replication, and is downregulated by the HIV accessory protein Vpr. Vpr significantly enhances infection in vivo, but the mechanism by which this occurs is controversial. It is well established that Vpr alters the expression of numerous host proteins by using its co-factor DCAF1, a component of the DCAF1-DDB1-CUL4 ubiquitin ligase complex. The host proteins targeted by Vpr and their role in viral replication are described in detail. We also discuss the structure and function of the viral protein Env, which is stabilized by Vpr in macrophages. Overall, this literature review provides an updated understanding of the contributions of macrophages and Vpr to HIV pathogenesis.
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http://dx.doi.org/10.3390/v12080809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472745PMC
July 2020

Truth and transparency in a time of crisis.

JCI Insight 2020 03 17;5(6). Epub 2020 Mar 17.

Lessons from history underline the importance of having direct lines of communication to and from public health officials, who must remain free from policital bias in times of crisis.
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http://dx.doi.org/10.1172/jci.insight.138132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205248PMC
March 2020

Changing the editorial process at JCI and JCI Insight in response to the COVID-19 pandemic.

J Clin Invest 2020 05;130(5):2147

The editors of JCI and JCI Insight are revisiting our editorial processes in light of the strain that the COVID-19 pandemic places on the worldwide scientific community. Here, we discuss adjustments to our decision framework in light of restrictions placed on laboratory working conditions for many of our authors.
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http://dx.doi.org/10.1172/JCI138305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190969PMC
May 2020

Mannose receptor is an HIV restriction factor counteracted by Vpr in macrophages.

Elife 2020 03 2;9. Epub 2020 Mar 2.

Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, United States.

HIV-1 Vpr is necessary for maximal HIV infection and spread in macrophages. Evolutionary conservation of Vpr suggests an important yet poorly understood role for macrophages in HIV pathogenesis. Vpr counteracts a previously unknown macrophage-specific restriction factor that targets and reduces the expression of HIV Env. Here, we report that the macrophage mannose receptor (MR), is a restriction factor targeting Env in primary human monocyte-derived macrophages. Vpr acts synergistically with HIV Nef to target distinct stages of the MR biosynthetic pathway and dramatically reduce MR expression. Silencing MR or deleting mannose residues on Env rescues Env expression in HIV-1-infected macrophages lacking Vpr. However, we also show that disrupting interactions between Env and MR reduces initial infection of macrophages by cell-free virus. Together these results reveal a Vpr-Nef-Env axis that hijacks a host mannose-MR response system to facilitate infection while evading MR's normal role, which is to trap and destroy mannose-expressing pathogens.
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http://dx.doi.org/10.7554/eLife.51035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051176PMC
March 2020

The Impact of Cellular Proliferation on the HIV-1 Reservoir.

Viruses 2020 01 21;12(2). Epub 2020 Jan 21.

Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI 48109, USA.

Human immunodeficiency virus (HIV) is a chronic infection that destroys the immune system in infected individuals. Although antiretroviral therapy is effective at preventing infection of new cells, it is not curative. The inability to clear infection is due to the presence of a rare, but long-lasting latent cellular reservoir. These cells harboring silent integrated proviral genomes have the potential to become activated at any moment, making therapy necessary for life. Latently-infected cells can also proliferate and expand the viral reservoir through several methods including homeostatic proliferation and differentiation. The chromosomal location of HIV proviruses within cells influences the survival and proliferative potential of host cells. Proliferating, latently-infected cells can harbor proviruses that are both replication-competent and defective. Replication-competent proviral genomes contribute to viral rebound in an infected individual. The majority of available techniques can only assess the integration site or the proviral genome, but not both, preventing reliable evaluation of HIV reservoirs.
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http://dx.doi.org/10.3390/v12020127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077244PMC
January 2020

Making intelligible the controversies over femme identities: A functionalist approach to conceptualizing the subversive meanings of femme genders.

J Lesbian Stud 2021 2;25(2):123-140. Epub 2020 Jan 2.

Clinical Psychology, University of Massachusetts Boston, Boston, Massachusetts, USA.

Quite a number of heated arguments have been put forth in the controversy about the meanings and appropriate uses of femme identities. In this article, the authors apply a functionalist theoretical framework, developed to explicate the links between gender and gender identities, to reframe the disputes about femme gender. They position two femme identities as responding to distinctive forms of oppression-one that centralizes the affirmation of gender diversity in the face of cisgenderism, and one that centralizes lesbian, gay, bisexual, transgender, and queer (LGBTQ+) femininity to counter femmephobia. They consider the subversive functions of the two identities in terms of unmet needs across four domains. These needs include the need for authenticity in identity (psychological domain); for the prizing of socially devalued characteristics (cultural domain); for security and affiliation (interpersonal domain); and for aesthetic desirability rather than shame (sexual domain). Instead of seeing the two femme identities as at odds, they see them as serving some shared functions, but also distinctive functions in resisting stigma of varied forms. The framework can be applied to other forms of femme-inity (and other genders) to distinguish the varied meanings inherent in gender identities and facilitate research that advances gender theory.
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http://dx.doi.org/10.1080/10894160.2019.1694788DOI Listing
April 2021

Case 3: Hypoglycemia in an Infant with Cholestasis.

Pediatr Rev 2019 Sep;40(9):488-490

Division of Pediatric Endocrinology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX.

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http://dx.doi.org/10.1542/pir.2017-0209DOI Listing
September 2019

Telomerase structures and regulation: shedding light on the chromosome end.

Curr Opin Struct Biol 2019 04 12;55:185-193. Epub 2019 Jun 12.

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; California Institute for Quantitative Biology (QB3), University of California, Berkeley, CA 94720, USA; Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.

During genome replication, telomerase adds repeats to the ends of chromosomes to balance the loss of telomeric DNA. The regulation of telomerase activity is of medical relevance, as it has been implicated in human diseases such as cancer, as well as in aging. Until recently, structural information on this enzyme that would facilitate its clinical manipulation had been lacking due to telomerase very low abundance in cells. Recent cryo-EM structures of both the human and Tetrahymena thermophila telomerases have provided a picture of both the shared catalytic core of telomerase and its interaction with species-specific factors that play different roles in telomerase RNP assembly and function. We discuss also progress toward an understanding of telomerase RNP biogenesis and telomere recruitment from recent studies.
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http://dx.doi.org/10.1016/j.sbi.2019.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240480PMC
April 2019

Comparison of medical management and genetic counseling options pre- and post-whole exome sequencing for patients with positive and negative results.

J Genet Couns 2019 04 16;28(2):182-193. Epub 2019 Jan 16.

Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.

Whole exome sequencing (WES) is expected to impact patient management, but data surrounding the types of downstream effects and how frequently these effects are observed depending on the type of WES results received is limited. This study investigated changes to medical management and genetic counseling (GC) options following WES for individuals with positive and negative results. Electronic medical records of patients who had positive (n = 37) or negative (n = 41) WES results from Cincinnati Children's Hospital were retrospectively reviewed. Pre- and post-WES management and GC options were analyzed as were differences between positive and negative results. Almost all participants (97%) were observed to have at least one difference in medical management and/or GC options following WES. Comparing pre- and post-WES detected significant differences (p ≤ 0.05) in genetic testing, imaging, and metabolic testing regardless of WES results. Participants with positive results also had significant differences in recurrence risk, reproductive options, testing for family members, and support groups. Pre- to post-WES differences were significantly different between participants with positive and negative results in specialist referrals, lifestyle recommendations, recurrence risk, and all GC options (p ≤ 0.05); specifically, participants with positive results were more likely to have differences in these categories. Overall, differences in medical management and/or GC options were observed for participants with both types of WES results (positive and negative). Results from this study may contribute to the understanding of how WES impacts patients and their care and thus improve its utilization.
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http://dx.doi.org/10.1002/jgc4.1054DOI Listing
April 2019

Hematopoietic Stem and Progenitor Cells Are a Distinct HIV Reservoir that Contributes to Persistent Viremia in Suppressed Patients.

Cell Rep 2018 12;25(13):3759-3773.e9

Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA; Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA; Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI, USA; Medical Scientist Training Program, University of Michigan, Ann Arbor, MI, USA; Division of Infectious Disease, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA; Graduate Program in Immunology, University of Michigan, Ann Arbor, MI, USA. Electronic address:

Long-lived reservoirs of persistent HIV are a major barrier to a cure. CD4 hematopoietic stem and progenitor cells (HSPCs) have the capacity for lifelong survival, self-renewal, and the generation of daughter cells. Recent evidence shows that they are also susceptible to HIV infection in vitro and in vivo. Whether HSPCs harbor infectious virus or contribute to plasma virus (PV) is unknown. Here, we provide strong evidence that clusters of identical proviruses from HSPCs and their likely progeny often match residual PV. A higher proportion of these sequences match residual PV than proviral genomes from bone marrow and peripheral blood mononuclear cells that are observed only once. Furthermore, an analysis of near-full-length genomes isolated from HSPCs provides evidence that HSPCs harbor functional HIV proviral genomes that often match residual PV. These results support the conclusion that HIV-infected HSPCs form a distinct and functionally significant reservoir of persistent HIV in infected people.
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http://dx.doi.org/10.1016/j.celrep.2018.11.104DOI Listing
December 2018

Predictors of mortality after extracorporeal cardiopulmonary resuscitation.

Crit Care Resusc 2018 Sep;20(3):223-230

Department of Intensive Care and Hyperbaric Medicine, Alfred Hospital, Melbourne, VIC, Australia.

Objective: Extracorporeal membrane oxygenation (ECMO) is a promising adjunct to cardiopulmonary resuscitation (CPR) in refractory cardiac arrest (CA). Factors associated with outcome are incompletely characterised. The aim of our study was to identify pre-ECMO factors associated with in-hospital mortality after extracorporeal CPR (ECPR).

Design: Retrospective analysis of a prospective cohort of patients.

Setting: Academic quaternary referral hospital.

Participants: All patients who underwent ECPR from January 2012 through April 2017.

Interventions: A retrospective chart review was performed for CPR and ECMO. A multivariable logistic regression was performed to identify factors associated with mortality after ECPR.

Main Outcome Measures: Primary outcome was in-hospital mortality. Secondary outcomes included survival with favourable neurologic outcome, days on ECMO, and intensive care unit (ICU) length of stay.

Results: During the study period, 75 patients received ECPR. Median age was 59 years, 81% were male, 51% had out-of-hospital CA, and 57% had an initial shockable rhythm. Median time from arrest to ECMO was 91 minutes (IQR, 56-129) for non-survivors and 51 minutes (IQR, 37-84) for survivors ( =0.02). Twenty-six patients (39%) were successfully separated from ECMO, with 31% surviving to hospital discharge and 29% with a cerebral performance category score of 1 or 2. In multivariable analysis, significant predictors of in-hospital mortality were ongoing CPR at the time of ECMO initiation ( < 0.01) and arrest to ECMO cannulation time ( =0.02).

Conclusion: Following ECPR, the factors most strongly associated with mortality were ongoing CPR at the time of ECMO initiation and arrest to ECMO cannulation time. Interventions aimed at reducing time to ECMO initiation may lead to improved outcomes.
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September 2018

Mechanisms of template handling and pseudoknot folding in human telomerase and their manipulation to expand the sequence repertoire of processive repeat synthesis.

Nucleic Acids Res 2018 09;46(15):7886-7901

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

Telomerase adds telomeric repeats to chromosome ends by processive copying of a template within the telomerase RNA bound to telomerase reverse transcriptase. Telomerase RNAs have single-stranded regions that separate the template from a 5' stem and 3' pseudoknot, and mammals gained additional stem P2a.1 separating the template from the pseudoknot. Using human telomerase, we show that the length of template 3'-flanking single-stranded RNA is a determinant of repeat addition processivity whereas template 5'-flanking single-stranded RNA and P2a.1 are critical for activity but not processivity. In comparison, requirements for the template sequence itself are confounding: different substitutions of the same position have strikingly different consequences, from improved processivity and activity to complete inactivation. We discovered that some altered-template sequences stabilize an alternative RNA conformation that precludes the pseudoknot by base-pairing of one pseudoknot strand to the template 3' end. Using mutations to reduce over-stability of the alternative conformation, we restore high activity and processivity to otherwise inactive altered-template telomerase ribonucleoproteins. In cells, over-stabilization or destabilization of the alternative state severely inhibited biogenesis of active telomerase. Our findings delineate roles for human telomerase RNA template-flanking regions, establish a biologically relevant pseudoknot-alternative RNA conformation, and expand the repertoire of human telomerase repeat synthesis.
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http://dx.doi.org/10.1093/nar/gky601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125678PMC
September 2018

The National Health and Nutrition Examination Survey's Food Insecurity Questionnaire Completed by Children: Effects of Assessment Mode (Classroom versus Interview).

J Hunger Environ Nutr 2018 19;13(2):205-227. Epub 2017 Jun 19.

Analyst, Institute for Families in Society, College of Social Work, University of South Carolina, Columbia, SC, USA, 29208; Manager, South Carolina Revenue and Fiscal Affairs Office, Health and Demographics, Columbia, SC, USA, 29201.

The National Health and Nutrition Examination Survey's food insecurity questionnaire was administered to 155 children (77 African American, 65 White, 13 "Other" [7 Hispanic; 6 mixed races]) in grade 4 twice, 28-32 days apart. Test-retest reliabilities were modest and somewhat similar for assessment mode (classroom, interview) and subgroup variables (gender, race, socioeconomic status, academic achievement, body mass index percentile, social desirability). As academic achievement increased, White and Other children reported less food insecurity, and African-American children reported slightly less. As social desirability increased, White and African-American children reported slightly more food insecurity, and Other children reported substantially more. Although the questionnaire may be acceptable for use with diverse groups of children in grade 4, validation is needed.
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http://dx.doi.org/10.1080/19320248.2017.1315325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995461PMC
June 2017

Cryo-EM structure of substrate-bound human telomerase holoenzyme.

Nature 2018 05 25;557(7704):190-195. Epub 2018 Apr 25.

Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.

The enzyme telomerase adds telomeric repeats to chromosome ends to balance the loss of telomeres during genome replication. Telomerase regulation has been implicated in cancer, other human diseases, and ageing, but progress towards clinical manipulation of telomerase has been hampered by the lack of structural data. Here we present the cryo-electron microscopy structure of the substrate-bound human telomerase holoenzyme at subnanometre resolution, showing two flexibly RNA-tethered lobes: the catalytic core with telomerase reverse transcriptase (TERT) and conserved motifs of telomerase RNA (hTR), and an H/ACA ribonucleoprotein (RNP). In the catalytic core, RNA encircles TERT, adopting a well-ordered tertiary structure with surprisingly limited protein-RNA interactions. The H/ACA RNP lobe comprises two sets of heterotetrameric H/ACA proteins and one Cajal body protein, TCAB1, representing a pioneering structure of a large eukaryotic family of ribosome and spliceosome biogenesis factors. Our findings provide a structural framework for understanding human telomerase disease mutations and represent an important step towards telomerase-related clinical therapeutics.
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http://dx.doi.org/10.1038/s41586-018-0062-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223129PMC
May 2018

Cannabis Use in Individuals With Spinal Cord Injury or Moderate to Severe Traumatic Brain Injury in Colorado.

Arch Phys Med Rehabil 2018 08 7;99(8):1584-1590. Epub 2018 Mar 7.

Research Department, Craig Hospital, Englewood, Colorado.

Objectives: To describe the prevalence of cannabis use in an adult sample with spinal cord injury (SCI) or traumatic brain injury (TBI) in Colorado, and to describe the self-reported reasons and side effects of cannabis use in this sample.

Design: Mixed-methods observational study, using focus group data and telephone survey.

Setting: Community.

Participants: Colorado adults who sustained SCI or moderate to severe TBI and received services through Craig Hospital.

Interventions: None.

Main Outcome Measures: Survey.

Results: Focus group participants identified issues that were then included in the survey development. Seventy percent of the 116 participants surveyed reported cannabis use before their injury (67% SCI, 74% TBI) and 48% reported use after their injury (53% SCI, 45% TBI). Overall, the most common reason for use was recreational (67%), followed by reducing stress/anxiety (62.5%) and improving sleep (59%). Among the respondents with SCI, the most common reasons for use were to reduce spasticity (70%), recreation (63%), and to improve sleep (63%). Among those with TBI, reasons endorsed were recreational (72%), reducing stress/anxiety (62%), and improving sleep (55%). Smoking was the most common method of use.

Conclusions: A majority of this sample reported using cannabis before injury, and approximately half reported using cannabis after injury. Both groups reported recreational use, whereas the group with SCI also highly endorsed using cannabis to address chronic medical conditions. Clinicians should be aware of the high prevalence of cannabis use in these populations and the impact such use may have on the individual's medical management. Further research in this area is needed.
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http://dx.doi.org/10.1016/j.apmr.2018.02.003DOI Listing
August 2018

Self-report depressive symptoms are dissociated from tremor severity in essential tremor.

Parkinsonism Relat Disord 2018 05 20;50:87-93. Epub 2018 Feb 20.

Division of Movement Disorders, Department of Neurology, Yale School of Medicine, Yale University, New Haven, CT, USA; Department of Chronic Disease Epidemiology, Yale School of Public Health, Yale University, New Haven, CT, USA; Center for Neuroepidemiology and Clinical Neurological Research, Yale School of Medicine, Yale University, New Haven, CT, USA.

Background: Depressive symptoms are associated with essential tremor (ET). However, the relationship between cognitive, functional, and motor measures with depressive symptoms in ET is not yet understood.

Methods: The following measures were cross-sectionally assessed in a group of 223 subjects with ET: the Montreal Cognitive Assessment (MoCA) Scale, the Lawton Independent Activities of Daily Living (IADL) Scale, a neurologist assessment of tremor severity, and the Geriatric Depression Scale (GDS).

Results: 20% (44) of the subjects met GDS criteria for depression (GDS ≥ 10). 43% (94) of the subjects showed at least some cognitive impairment (≤24 on the MoCA), and 15.3% (34) reported significant functional impairment (IADL score < 7). There was no significant association between GDS score and tremor scale score. The total GDS was negatively associated with the total MoCA score (Spearman's r = -0.15, p = 0.03). The total GDS was also negatively associated with the IADL score (Spearman's r = -0.19, p = 0.02), (logistic model odds ratio, OR = 4.91, p < 0.01). Over 60% of subjects who were depressed, per GDS cut-off score (≥10), were not receiving medical treatment for depression.

Conclusions: There was a high point prevalence of depressive symptoms in subjects with ET. Self-report depressive symptoms are dissociated from tremor severity. Hence, these data do not support the hypothesis that depression in ET represents a psychological reaction to the tremor. There appears to be a clustering of cognitive, functional, and depressive symptoms in ET. Screening of depression in ET can improve our understanding and treatment of this disorder.
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http://dx.doi.org/10.1016/j.parkreldis.2018.02.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943134PMC
May 2018

Class 1-Selective Histone Deacetylase (HDAC) Inhibitors Enhance HIV Latency Reversal while Preserving the Activity of HDAC Isoforms Necessary for Maximal HIV Gene Expression.

J Virol 2018 03 26;92(6). Epub 2018 Feb 26.

Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA

Combinations of drugs that affect distinct mechanisms of HIV latency aim to induce robust latency reversal leading to cytopathicity and elimination of the persistent HIV reservoir. Thus far, attempts have focused on combinations of protein kinase C (PKC) agonists and pan-histone deacetylase inhibitors (HDIs) despite the knowledge that HIV gene expression is regulated by class 1 histone deacetylases. We hypothesized that class 1-selective HDIs would promote more robust HIV latency reversal in combination with a PKC agonist than pan-HDIs because they preserve the activity of proviral factors regulated by non-class 1 histone deacetylases. Here, we show that class 1-selective agents used alone or with the PKC agonist bryostatin-1 induced more HIV protein expression per infected cell. In addition, the combination of entinostat and bryostatin-1 induced viral outgrowth, whereas bryostatin-1 combinations with pan-HDIs did not. When class 1-selective HDIs were used in combination with pan-HDIs, the amount of viral protein expression and virus outgrowth resembled that of pan-HDIs alone, suggesting that pan-HDIs inhibit robust gene expression induced by class 1-selective HDIs. Consistent with this, pan-HDI-containing combinations reduced the activity of NF-κB and Hsp90, two cellular factors necessary for potent HIV protein expression, but did not significantly reduce overall cell viability. An assessment of viral clearance from cultures indicated that maximal protein expression induced by class 1-selective HDI treatment was crucial for reservoir clearance. These findings elucidate the limitations of current approaches and provide a path toward more effective strategies to eliminate the HIV reservoir. Despite effective antiretroviral therapy, HIV evades eradication in a latent form that is not affected by currently available drug regimens. Pharmacologic latency reversal that leads to death of cellular reservoirs has been proposed as a strategy for reservoir elimination. Because histone deacetylases (HDACs) promote HIV latency, HDAC inhibitors have been a focus of HIV cure research. However, many of these inhibitors broadly affect multiple classes of HDACs, including those that promote HIV gene expression (class 1 HDACs). Here, we demonstrate that targeted treatment with class 1-selective HDAC inhibitors induced more potent HIV latency reversal than broadly acting agents. Additionally, we provide evidence that broadly acting HDIs are limited by inhibitory effects on non-class 1 HDACs that support the activity of proviral factors. Thus, our work demonstrates that the use of targeted approaches to induce maximum latency reversal affords the greatest likelihood of reservoir elimination.
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http://dx.doi.org/10.1128/JVI.02110-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827401PMC
March 2018

Psychological Suffering in Essential Tremor: A Study of Patients and Those Who Are Close to Them.

Tremor Other Hyperkinet Mov (N Y) 2017 18;7:526. Epub 2017 Dec 18.

Division of Movement Disorders, Department of Neurology, Yale School of Medicine, Yale University, New Haven, CT, USA.

Background: Although the motor and non-motor features of essential tremor (ET) have been characterized in detail, it is not known whether ET patients suffer psychologically and whether those who are close to them consider them to be suffering in this way.

Methods: Fifty ET patients and 50 "close others" (COs), identified by patients "as someone who knows you well and sees you often" and who can "provide a different perspective on your well-being", reported their own depressive symptoms, daily stress, and perceptions of patient psychological suffering and patient overall suffering with validated scales. ET patients' tremor severity, duration, disability, cognition, and number of medications were also assessed.

Results: ET patients reported levels of psychological suffering within the range documented in arthritis and dementia patients from previous studies, and COs perceived significantly more psychological suffering in patients than patients reported themselves. Regression models, controlling for tremor severity, duration, and disability revealed that patients' greater psychological suffering was associated with greater patient depression. The greater perceptions of COs of patient psychological and overall suffering were associated with greater CO depression and daily stress. Sensitivity analysis showed that patients' cognitive status or number of medications did not affect the results.

Discussion: Multidisciplinary teams caring for ET patients should look beyond simple clinical ET indicators. They should be aware of patient experiences and perceptions of COs of psychological and overall suffering. This will help guide the development of evidence-based, supportive interventions that improve communication about the needs of ET patients and those who are close to them.
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http://dx.doi.org/10.7916/D8Q53WF0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740226PMC
September 2018
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