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1.
Int J Geriatr Psychiatry ; 38(6): e5943, 2023 Jun.
Article in English | MEDLINE | ID: mdl-37294207

ABSTRACT

OBJECTIVES: Assessing for recovery in delirium is essential in guiding ongoing investigation and treatment. Yet, there is little scrutiny and no research or clinical consensus on how recovery should be measured. We reviewed studies which used tests of neuropsychological domains and functional ability to track recovery of delirium longitudinally in acute hospital settings. METHODS/DESIGN: We systematically searched databases (MEDLINE, PsycInfo, CINAHL, Embase, ClinicalTrials.gov, Cochrane Central Register of Controlled Trials), from inception to October 14th , 2022. Inclusion criteria were: adult acute hospital patients (≥18 years) diagnosed with delirium by a validated tool; 1+ repeat assessment using an assessment tool measuring domains of delirium/functional recovery ≤7 days from baseline. Two reviewers independently screened articles, performed data extraction, and assessed risk of bias. A narrative data synthesis was completed. RESULTS: From 6533 screened citations, we included 39 papers (reporting 32 studies), with 2370 participants with delirium. Studies reported 21 tools with an average of four repeat assessments including baseline (range 2-10 assessments within ≤7 days), measuring 15 specific domains. General cognition, functional ability, arousal, attention and psychotic features were most commonly assessed for longitudinal change. Risk of bias was moderate to high for most studies. CONCLUSIONS: There was no standard approach for tracking change in specific domains of delirium. The methodological heterogeneity of studies was too high to draw firm conclusions on the effectiveness of assessment tools to measure delirium recovery. This highlights the need for standardised methods for assessing recovery from delirium.


Subject(s)
Activities of Daily Living , Delirium , Humans , Delirium/diagnosis , Delirium/drug therapy , Hospitals
2.
Delirium Commun ; 1: 56675, 2022 Dec 19.
Article in English | MEDLINE | ID: mdl-36968519

ABSTRACT

Background: A crucial part of delirium care is determining if the delirium episode has resolved. Yet, there is no clear evidence or consensus on which assessments clinicians should use to assess for delirium recovery. Objective: To evaluate current opinions from delirium specialists on assessment of delirium recovery. Design: Online questionnaire-based survey distributed internationally to healthcare professionals involved in delirium care. Methods: The survey covered methods for assessing recovery, the importance of different symptom domains for capturing recovery, and local guidance or pathways that recommend monitoring for delirium recovery. Results: Responses from 199 clinicians were collected. Respondents were from the UK (51%), US (13%), Australia (9%), Canada (7%), Ireland (7%) and 16 other countries. Most respondents were doctors (52%) and nurses (27%). Clinicians worked mostly in geriatrics (52%), ICUs (21%) and acute assessment units (17%). Ninety-four percent of respondents indicated that they conduct repeat delirium assessments (i.e., on ≥2 occasions) to monitor delirium recovery. The symptom domains considered most important for capturing recovery were: arousal (92%), inattention (84%), motor disturbance (84%), and hallucinations and delusions (83%). The most used tool for assessing recovery was the 4 'A's Test (4AT, 51%), followed by the Confusion Assessment Method (CAM, 26%), the CAM for the ICU (CAM-ICU, 17%) and the Single Question in Delirium (SQiD, 11%). Twenty-eight percent used clinical features only. Less than half (45%) of clinicians reported having local guidance that recommends monitoring for delirium recovery. Conclusions: The survey results suggest a lack of standardisation regarding tools and methods used for repeat delirium assessment, despite consensus surrounding the key domains for capturing delirium recovery. These findings emphasise the need for further research to establish best practice for assessing delirium recovery.

3.
Viruses ; 7(6): 3186-200, 2015 Jun 19.
Article in English | MEDLINE | ID: mdl-26102578

ABSTRACT

The T4 bacteriophage encodes eight proteins, which are sufficient to carry out coordinated leading and lagging strand DNA synthesis. These purified proteins have been used to reconstitute DNA synthesis in vitro and are a well-characterized model system. Recent work on the T4 replisome has yielded more detailed insight into the dynamics and coordination of proteins at the replication fork. Since the leading and lagging strands are synthesized in opposite directions, coordination of DNA synthesis as well as priming and unwinding is accomplished by several protein complexes. These protein complexes serve to link catalytic activities and physically tether proteins to the replication fork. Essential to both leading and lagging strand synthesis is the formation of a holoenzyme complex composed of the polymerase and a processivity clamp. The two holoenzymes form a dimer allowing the lagging strand polymerase to be retained within the replisome after completion of each Okazaki fragment. The helicase and primase also form a complex known as the primosome, which unwinds the duplex DNA while also synthesizing primers on the lagging strand. Future studies will likely focus on defining the orientations and architecture of protein complexes at the replication fork.


Subject(s)
Bacteriophage T4/physiology , DNA Replication , DNA, Viral/metabolism , Multienzyme Complexes/metabolism , Viral Proteins/metabolism
4.
J Biol Chem ; 288(28): 20683-91, 2013 Jul 12.
Article in English | MEDLINE | ID: mdl-23744077

ABSTRACT

Newly identified anti-HIV host factor, SAMHD1, restricts replication of lentiviruses such as HIV-1, HIV-2, and simian immunodeficiency virus in macrophages by enzymatically hydrolyzing and depleting cellular dNTPs, which are the substrates of viral DNA polymerases. HIV-2 and some simian immunodeficiency viruses express viral protein X (VPX), which counteracts SAMHD1 and elevates cellular dNTPs, enhancing viral replication in macrophages. Because nucleoside reverse transcriptase inhibitors (NRTIs), the most commonly used anti-HIV drugs, compete against cellular dNTPs for incorporation into proviral DNA, we tested whether SAMHD1 directly affects the efficacy of NRTIs in inhibiting HIV-1. We found that reduction of SAMHD1 levels with the use of virus-like particles expressing Vpx- and SAMHD1-specific shRNA subsequently elevates cellular dNTPs and significantly decreases HIV-1 sensitivity to various NRTIs in macrophages. However, virus-like particles +Vpx treatment of activated CD4(+) T cells only minimally reduced NRTI efficacy. Furthermore, with the use of HPLC, we could not detect SAMHD1-mediated hydrolysis of NRTI-triphosphates, verifying that the reduced sensitivity of HIV-1 to NRTIs upon SAMHD1 degradation is most likely caused by the elevation in cellular dNTPs.


Subject(s)
Deoxyribonucleosides/metabolism , HIV-1/drug effects , Monomeric GTP-Binding Proteins/metabolism , Reverse Transcriptase Inhibitors/pharmacology , Blotting, Western , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Cell Line , Cells, Cultured , Dose-Response Relationship, Drug , HIV-1/genetics , HIV-1/physiology , Host-Pathogen Interactions , Humans , Macrophages/drug effects , Macrophages/metabolism , Macrophages/virology , Monomeric GTP-Binding Proteins/genetics , Nevirapine/pharmacology , RNA Interference , SAM Domain and HD Domain-Containing Protein 1 , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/physiology , Virion/drug effects , Virion/genetics , Virion/physiology , Virus Replication/drug effects , Zidovudine/pharmacology
5.
J Biol Chem ; 288(18): 12522-32, 2013 May 03.
Article in English | MEDLINE | ID: mdl-23479739

ABSTRACT

HIV-1 reverse transcriptase (RT) frequently incorporates ribonucleoside triphosphates (rNTPs) during proviral DNA synthesis, particularly under the limited dNTP conditions found in macrophages. We investigated the mechanistic impacts of an rNMP embedded in DNA templates on HIV-1 RT-mediated DNA synthesis. We observed that the template-embedded rNMP induced pausing of RT and delayed DNA synthesis kinetics at low macrophage dNTP concentrations but not at high T cell dNTP concentrations. Although the binding affinity of RT to the rNMP-containing template-primer was not altered, the dNTP incorporation kinetics of RT were significantly reduced at one nucleotide upstream and downstream of the rNMP site, leading to pause sites. Finally, HIV-1 RT becomes more error-prone at rNMP sites with an elevated mismatch extension capability but not enhanced misinsertion capability. Together these data suggest that rNMPs embedded in DNA templates may influence reverse transcription kinetics and impact viral mutagenesis in macrophages.


Subject(s)
DNA, Viral/biosynthesis , Deoxyribonucleotides/chemistry , HIV Reverse Transcriptase/chemistry , HIV-1/enzymology , Proviruses/enzymology , Ribonucleotides/chemistry , Cell-Free System , DNA, Viral/chemistry , DNA, Viral/genetics , Deoxyribonucleotides/genetics , Deoxyribonucleotides/metabolism , HIV Reverse Transcriptase/genetics , HIV Reverse Transcriptase/metabolism , HIV-1/genetics , Kinetics , Mutation , Proviruses/genetics , Ribonucleotides/genetics , Ribonucleotides/metabolism
6.
Virology ; 436(2): 247-54, 2013 Feb 20.
Article in English | MEDLINE | ID: mdl-23260109

ABSTRACT

Retroviruses consume cellular deoxynucleoside triphosphates (dNTPs) to convert their RNA genomes into proviral DNA through reverse transcription. While all retroviruses replicate in dividing cells, lentiviruses uniquely replicate in nondividing cells such as macrophages. Importantly, dNTP levels in nondividing cells are extremely low, compared to dividing cells. Indeed, a recently discovered anti-HIV/SIV restriction factor, SAMHD1, which is a dNTP triphosphohydrolase, is responsible for the limited dNTP pool of nondividing cells. Lentiviral reverse transcriptases (RT) uniquely stay functional even at the low dNTP concentrations in nondividing cells. Interestingly, Vpx of HIV-2/SIVsm proteosomally degrades SAMHD1, which elevates cellular dNTP pools and accelerates lentiviral replication in nondividing cells. These Vpx-encoding lentiviruses rapidly replicate in nondividing cells by encoding both highly functional RTs and Vpx. Here, we discuss a series of mechanistic and virological studies that have contributed to conceptually linking cellular dNTP levels and the adaptation of lentiviral replication in nondividing cells.


Subject(s)
Cytoplasm/chemistry , Lentivirus/physiology , Nucleotides/metabolism , Retroviridae/physiology , Reverse Transcription , Virus Replication , Animals , Humans , Monomeric GTP-Binding Proteins/metabolism , Nucleotides/analysis , SAM Domain and HD Domain-Containing Protein 1 , Viral Regulatory and Accessory Proteins/metabolism
7.
Virology ; 433(1): 27-34, 2012 Nov 10.
Article in English | MEDLINE | ID: mdl-22841552

ABSTRACT

The influenza A polymerase is a heterotrimer which transcribes viral mRNAs and replicates the viral genome. To initiate synthesis of mRNA, the polymerase binds a host pre-mRNA and cleaves a short primer downstream of the 5' end cap structure. The N-terminal domain of PA has been demonstrated to have endonuclease activity in vitro. Here we sought to better understand the biochemical nature of the PA endonuclease by developing an improved assay using full-length PA protein. This full-length protein is active against both RNA and DNA in a cap-independent manner and can use several different divalent cations as cofactors, which affects the secondary structure of the full-length PA. Our in vitro assay was also able to demonstrate the minimal substrate size and sequence selectivity of the PA protein, which is crucial information for inhibitor design. Finally, we confirmed the observed endonuclease activity of the full-length PA with a FRET-based assay.


Subject(s)
Endonucleases/metabolism , Influenza A virus/enzymology , RNA Caps/chemistry , RNA, Viral/chemistry , RNA-Dependent RNA Polymerase/metabolism , Viral Proteins/metabolism , Biological Assay , Cations, Divalent , Coenzymes/metabolism , Endonucleases/chemistry , Endonucleases/genetics , Fluorescence Resonance Energy Transfer , Humans , Influenza A virus/genetics , Magnesium/metabolism , Manganese/metabolism , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , RNA Caps/genetics , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Viral Proteins/chemistry , Viral Proteins/genetics
8.
J Biol Chem ; 286(26): 22965-70, 2011 Jul 01.
Article in English | MEDLINE | ID: mdl-21555520

ABSTRACT

Each segment of the influenza A virus (IAV) genome contains conserved sequences at the 5'- and 3'-terminal ends, which form the promoter region necessary for polymerase binding and initiation of RNA synthesis. Although several models of interaction have been proposed it remains unclear if these two short, partially complementary, and highly conserved sequences can form a stable RNA duplex at physiological temperatures. First, our time-resolved FRET analysis revealed that a 14-mer 3'-RNA and a 15-mer 5'-RNA associate in solution, even at 42 °C. We also found that a nonfunctional RNA promoter containing the 3'-G3U mutation, as well as a promoter containing the compensatory 3'-G3U/C8A mutations, was able to form a duplex as efficiently as wild type. Second, UV melting analysis demonstrated that the wild-type and mutant RNA duplexes have similar stabilities in solution. We also observed an increase in thermostability for a looped promoter structure. The absence of differences in the stability and binding kinetics between wild type and a nonfunctional sequence suggests that the IAV promoter can be functionally inactivated without losing the capability to form a stable RNA duplex. Finally, using uridine specific chemical probing combined with mass spectrometry, we confirmed that the 5' and 3' sequences form a duplex which protects both RNAs from chemical modification, consistent with the previously published panhandle structure. These data support that these short, conserved promoter sequences form a stable complex at physiological temperatures, and this complex likely is important for polymerase recognition and viral replication.


Subject(s)
Influenza A virus/chemistry , Promoter Regions, Genetic , RNA, Double-Stranded/chemistry , RNA, Viral/chemistry , Hot Temperature , Influenza A virus/genetics , Influenza A virus/metabolism , Mutation , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Ultraviolet Rays , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication/physiology , Virus Replication/radiation effects
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