Retrospective Analysis of Retroperitoneal-Abdominal-Pelvic Ganglioneuromas: An International Study by the Transatlantic Australasian Retroperitoneal Sarcoma Working Group (TARPSWG).

OBJECTIVE: The Transatlantic Australasian Retroperitoneal Sarcoma Working Group conducted a retrospective study on the disease course and clinical management of ganglioneuromas. BACKGROUND: Ganglioneuromas are rare tumors derived from neural crest cells. Data on these tumors remain limited to case reports and single-institution case series. METHODS: Patients of all ages with pathologically confirmed primary retroperitoneal, intra-abdominal, and pelvic ganglioneuromas between January 1, 2000, and January 1, 2020, were included. We examined demographic, clinicopathologic, and radiologic characteristics, as well as clinical management. RESULTS: Overall, 328 patients from 29 institutions were included. The median age at diagnosis was 37 years with 59.1% of patients being female. Symptomatic presentation comprised 40.9% of cases, and tumors were often located in the extra-adrenal retroperitoneum (67.1%). At baseline, the median maximum tumor diameter was 7.2 cm. One hundred sixteen (35.4%) patients underwent active surveillance, whereas 212 (64.6%) patients underwent resection with 74.5% of operative cases achieving an R0/R1 resection. Serial tumor evaluations showed that malignant transformation to neuroblastoma was rare (0.9%, N=3). Tumors undergoing surveillance had a median follow-up of 1.9 years, with 92.2% of ganglioneuromas stable in size. With a median follow-up of 3.0 years for resected tumors, 84.4% of patients were disease free after resections, whereas recurrences were observed in 4 (1.9%) patients. CONCLUSIONS: Most ganglioneuromas have indolent disease courses and rarely transform to neuroblastoma. Thus, active surveillance may be appropriate for benign and asymptomatic tumors particularly when the risks of surgery outweigh the benefits. For symptomatic or growing tumors, resection may be curative.

Development of the McMaster Embalming Scale (MES) to assess embalming solutions for surgical skills training.

Human cadavers used for surgical training are embalmed using various methods to facilitate tissue storage and longevity while preserving the natural characteristics required to achieve high fidelity functional task alignment. However, there are no standardized means to evaluate the suitability of embalming solutions for this purpose. The McMaster Embalming Scale (MES) was developed to assess the extent to which embalming solutions allow tissues to achieve physical and functional correspondence to clinical contexts. The MES follows a five-point Likert scale format and evaluates the effect of embalming solutions on tissue utility in seven domains. This study aims to determine the reliability and validity of the MES by presenting it to users after performing surgical skills on tissues embalmed using various solutions. A pilot study of the MES was conducted using porcine material. Surgical residents of all levels and faculty were recruited via the Surgical Foundations program at McMaster University. Porcine tissue was unembalmed (fresh- frozen) or embalmed using one of seven solutions identified in the literature. Participants were blinded to the embalming method as they completed four surgical skills on the tissue. After each performance, participants evaluated their experience using the MES. Internal consistency was evaluated using Cronbach's alpha. Domain to total correlations and a g-study were also conducted. Formalin-fixed tissue achieved the lowest average scores, while fresh frozen tissue achieved the highest. Tissues preserved using Surgical Reality Fluid (Trinity Fluids, LLC, Harsens Island, MI) achieved the highest scores among embalmed tissues. The Cronbach's alpha scores varied between 0.85 and 0.92, indicating a random sample of new raters would offer similar ratings using the MES. All domains except odor were positively correlated. The g-study indicated that the MES is able to differentiate between embalming solutions, but an individual rater's preference for certain tissue qualities also contributes to the variance in scores captured. This study evaluated the psychometric characteristics of the MES. Future steps to this investigation include validating the MES on human cadavers.

Factors associated with hypothermia within the first 6 hours of life in infants born at ≥340 weeks' gestation: a multivariable analysis.

BACKGROUND: Lack of appropriate temperature management has been associated with significant adverse outcomes in preterm and low birthweight neonates. There is a lack of similar investigations in the late preterm (340-366) and term (≥370) neonate population. Our aim was to identify key risk factors as well as clinical outcomes associated with hypothermia in this population. METHODS: A retrospective chart review was conducted at the Ottawa Hospital including all eligible infants ≥340 weeks' gestation over a one-month period in November 2020. Infant, maternal, and delivery room variables were collected, including prematurity, maternal temperature, delivery mode, birthweight, and premature rupture of membranes, as well as clinical outcomes such as NICU/SCN admission and length of stay. Regression models were generated, adjusted for covariates, and stepwise regression was performed. RESULTS: Four hundred forty infants were included in the analysis; 26.8% (118/440) were hypothermic within 6 hours of delivery. In the multivariable analysis, prematurity, low 5 minute Apgar score (< 7) or need for resuscitation, maternal hypertension, and absence of premature rupture of membranes > 18 hours or suspected maternal infection were significantly associated with hypothermia within 6 hours of delivery (p < 0.05). Multivariable analysis of clinical outcomes demonstrated a significant association between hypothermia within 6 hours of delivery and NICU/SCN admission (OR = 2.87; 95% CI 1.36, 6.04), need for respiratory support or diagnosis of respiratory distress syndrome (OR = 3.94; 95% CI 1.55, 10.50), and length of stay (exp(ß) = 1.20; 95% bootstrap CI 1.04, 1.37). CONCLUSIONS: Our results suggest there are similar factors associated with hypothermia in our study population of infants born at ≥340 weeks' gestation compared to prior studies in preterm and low-birthweight infants. Furthermore, hypothermia is associated with higher risk of adverse outcomes, which highlights the need to prevent hypothermia in all newborns.

Drug-drug interactions with warfarin: A systematic review and meta-analysis.

AIMS: The objective of this paper is to systematically review the literature on drug-drug interactions with warfarin, with a focus on patient-important clinical outcomes. METHODS: MEDLINE, EMBASE and the International Pharmaceutical Abstract (IPA) databases were searched from January 2004 to August 2019. We included studies describing drug-drug interactions between warfarin and other drugs. Screening and data extraction were conducted independently and in duplicate. We synthesized pooled odds ratios (OR) with 95% confidence intervals (CIs), comparing warfarin plus another medication to warfarin alone. We assessed the risk of bias at the study level and evaluated the overall certainty of evidence using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. RESULTS: Of 42 013 citations identified, a total of 72 studies reporting on 3 735 775 patients were considered eligible, including 11 randomized clinical trials and 61 observational studies. Increased risk of clinically relevant bleeding when added to warfarin therapy was observed for antiplatelet (AP) regimens (OR = 1.74; 95% CI 1.56-1.94), many antimicrobials (OR = 1.63; 95% CI 1.45-1.83), NSAIDs including COX-2 NSAIDs (OR = 1.83; 95% CI 1.29-2.59), SSRIs (OR = 1.62; 95% CI 1.42-1.85), mirtazapine (OR = 1.75; 95% CI 1.30-2.36), loop diuretics (OR = 1.92; 95% CI 1.29-2.86) among others. We found a protective effect of proton pump inhibitors (PPIs) against warfarin-related gastrointestinal (GI) bleeding (OR = 0.69; 95% CI 0.64-0.73). No significant effect on thromboembolic events or mortality of any drug group used with warfarin was found, including single or dual AP regimens. CONCLUSIONS: This review found low to moderate certainty evidence supporting the interaction between warfarin and a small group of medications, which result in increased bleeding risk. PPIs are associated with reduced hospitalization for upper GI bleeding for patients taking warfarin. Further studies are required to better understand drug-drug interactions leading to thromboembolic outcomes or death.

Placement of mid-urethral mesh slings at the time of vaginal prolapse repair does not affect post-operative sexual function or orgasm.

INTRODUCTION AND HYPOTHESIS: Because of the relationship between the clitoral neurovascular supply and the urethra, the dissection for placement of mid-urethral slings (MUS) may negatively impact orgasmic function. We aimed to analyze the role of MUS in orgasmic and overall sexual function in patients undergoing prolapse surgery. METHODS: A single institution retrospective review was performed on 157 patients undergoing prolapse surgery with and without MUS from 2008 to 2014. Pelvic Organ Prolapse Incontinence Sexual Questionnaires (PISQ-12) scores at baseline, 6, and 12 months post-operatively were compared. The difference in overall mean post-operative PISQ-12 scores at 6 and 12 months in those undergoing POP with or without MUS placement was assessed using Wilcoxon rank tests. RESULTS: Of 157 women who underwent prolapse surgery, 81 (52%) had concomitant MUS. Mean baseline PISQ-12 scores were 32 in both groups (p = 0.98). Post-operative PISQ-12 scores between the two groups did not differ at 6 (p = 0.96) or 12 months (p = 0.65). Within the MUS group, mean overall PISQ-12 scores improved at 6 (p = 0.05) and 12 months (p < 0.01). Mean overall PISQ-12 scores did not improve in patients who did not have slings placed at 6 (p = 0.10) or 12 months (p = 0.15). Orgasm frequency and intensity did not differ between the two groups at 6 (p = 0.39, p = 0.91, respectively) or 12 months (p = 0.11, p = 0.44, respectively). CONCLUSION: MUS at the time of prolapse repair did not affect orgasmic or overall sexual function. PISQ-12 scores improved after prolapse surgery with concomitant MUS placement. Our findings may help counsel patients regarding the risk of MUS placement affecting sexual function.

Strategies to Mitigate Anxiety and Pain in Intrauterine Device Insertion: A Systematic Review.

One barrier to the use of intrauterine devices (IUDs) as a contraceptive method is the experience of anxiety and pain during the insertion procedure. Previous reviews have focused on pharmacological methods used to relieve pain during IUD insertion; however, few similar reviews have examined non-pharmacological methods to relieve pain or strategies to reduce anxiety. The objectives of this study were to identify and categorize strategies for reducing anxiety and pain with respect to IUD insertion and the ways in which anxiety and pain were assessed. In particular, the study aimed to identify non-pharmacological interventions and studies that included anxiety as a research outcome. A literature search was conducted of all English-language studies between inception and the week of July 29, 2018 from the following online databases: Medline, Embase, Cochrane Library, and PubMed. The search revealed 426 studies after removal of duplicates, 35 of which fulfilled the inclusion criteria. A total of 29 studies were identified as assessing pharmacological interventions for the management of pain, and six studies assessed non-pharmacological interventions. Only one study included a measurement of patient anxiety during the procedure as an outcome measure. Research on non-pharmacological interventions for the management of anxiety and pain during IUD insertion is lacking. This review found that evidence for the studied pharmacological interventions is conflicting, and there is very little evidence on understanding the effectiveness of strategies to manage anxiety during the IUD insertion procedure. Further high-quality research on non-pharmacological pain and anxiety management strategies is warranted.

Crystal structure of Pistol, a class of self-cleaving ribozyme.

Small self-cleaving ribozymes have been discovered in all evolutionary domains of life. They can catalyze site-specific RNA cleavage, and as a result, they have relevance in gene regulation. Comparative genomic analysis has led to the discovery of a new class of small self-cleaving ribozymes named Pistol. We report the crystal structure of Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine base in the active site pocket to carry out the phosphoester transfer reaction. The guanine G40 is in close proximity to serve as the general base for activating the nucleophile by deprotonating the 2'-hydroxyl to initiate the reaction (phosphoester transfer). Furthermore, G40 can also establish hydrogen bonding interactions with the nonbridging oxygen of the scissile phosphate. The proximity of G32 to the O5' leaving group suggests that G32 may putatively serve as the general acid. The RNA structure of Pistol also contains A-minor interactions, which seem to be important to maintain its tertiary structure and compact fold. Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutionary mechanism used by ribozymes for catalysis.

Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells.

Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2-like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.

Evaluation of online clinical pharmacology curriculum resources for medical students.

AIMS: To identify and evaluate clinical pharmacology (CP) online curricular (e-Learning) resources that are internationally available for medical students. METHODS: Literature searches of Medline, EMBASE and ERIC databases and an online survey of faculty members of international English language medical schools, were used to identify CP e-Learning resources. Resources that were accessible online in English and aimed to improve the quality of prescribing specific medications were then evaluated using a summary percentage score for comprehensiveness, usability and quality, and for content suitability. RESULTS: Our literature searches and survey of 252 faculty (40.7% response rate) in 219 medical schools identified 22 and 59 resources respectively. After screening and removing duplicates, 8 eligible resources remained for evaluation. Mean total score was 53% (standard deviation = 13). The Australian National Prescribing Curriculum, ranked highest with a score of 77%, based primarily on very good ratings for usability, quality and suitable content. CONCLUSION: Using a novel method and evaluation metric to identify, classify, and rate English language CP e-Learning resources, the National Prescribing Curriculum was the highest ranked open access resource. Future work is required to implement and evaluate its effectiveness on prescribing competence.

The Risks and Benefits of Cavernous Neurovascular Bundle Sparing during Radical Prostatectomy: A Systematic Review and Meta-Analysis.

PURPOSE: We summarize published data on associations between cavernous neurovascular bundle preservation (nerve sparing) during prostatectomy and positive surgical margins, erectile function, urinary function and other patient reported outcomes. MATERIALS AND METHODS: A systematic literature search of MEDLINE®, Embase® and Cochrane Reviews databases was performed for interventional or observational studies published between 2000 and 2014. English language articles that compared clinical outcomes of patients undergoing nerve sparing and nonnerve sparing radical prostatectomy were included. Meta-analyses were performed to calculate pooled relative risk estimates for positive surgical margins, erectile dysfunction and urinary incontinence in nerve sparing and nonnerve sparing groups. Sensitivity analyses compared outcomes among unilateral and bilateral nerve sparing vs nonnerve sparing groups. RESULTS: Of the 1,883 articles identified, 124 studies (73,448 patients) were included in the analysis. Nerve sparing did not increase the risk of positive surgical margins in patients with pT2 (RR 0.92, 95% CI 0.75-1.13) or pT3 disease (RR 0.83, 95% CI 0.71-0.96), potentially due to appropriate patient selection. The risk of incontinence was lower in nerve sparing cases (RR 0.75, 95% CI 0.65-0.85 and RR 0.61, 95% CI 0.44-0.84) at 3 and 12 months, respectively. The relative risk of erectile dysfunction with nerve sparing was 0.77 (95% CI 0.70-0.85) at 3 months and 0.53 (95% CI 0.39-0.71) at 12 months. Subgroup analyses of unilateral and bilateral nerve sparing approaches demonstrated similar results. CONCLUSIONS: Among cohort studies nerve sparing was not associated with worse cancer outcomes. Nerve sparing is associated with better urinary and erectile function. These results should be interpreted with caution given the potential for selection bias and unadjusted confounding factors.

Host SAMHD1 protein promotes HIV-1 recombination in macrophages.

Template switching can occur during the reverse transcription of HIV-1. Deoxynucleotide triphosphate (dNTP) concentrations have been biochemically shown to impact HIV-1 reverse transcriptase (RT)-mediated strand transfer. Lowering the dNTP concentrations promotes RT pausing and RNA template degradation by RNase H activity of the RT, subsequently leading to strand transfer. Terminally differentiated/nondividing macrophages, which serve as a key HIV-1 reservoir, contain extremely low dNTP concentrations (20-50 nm), which results from the cellular dNTP hydrolyzing sterile α motif and histidine aspartic domain containing protein 1 (SAMHD1) protein, when compared with activated CD4(+) T cells (2-5 µm). In this study, we first observed that HIV-1 template switching efficiency was nearly doubled in human primary macrophages when compared with activated CD4(+) T cells. Second, SAMHD1 degradation by viral protein X (Vpx), which elevates cellular dNTP concentrations, decreased HIV-1 template switching efficiency in macrophages to the levels comparable with CD4(+) T cells. Third, differentiated SAMHD1 shRNA THP-1 cells have a 2-fold increase in HIV-1 template switching efficiency. Fourth, SAMHD1 degradation by Vpx did not alter HIV-1 template switching efficiency in activated CD4(+) T cells. Finally, the HIV-1 V148I RT mutant that is defective in dNTP binding and has DNA synthesis delay promoted RT stand transfer when compared with wild type RT, particularly at low dNTP concentrations. Here, we report that SAMHD1 regulation of the dNTP concentrations influences HIV-1 template switching efficiency, particularly in macrophages.

SAMHD1 prevents autoimmunity by maintaining genome stability.

OBJECTIVES: The HIV restriction factor, SAMHD1 (SAM domain and HD domain-containing protein 1), is a triphosphohydrolase that degrades deoxyribonucleoside triphosphates (dNTPs). Mutations in SAMHD1 cause Aicardi-Goutières syndrome (AGS), an inflammatory disorder that shares phenotypic similarity with systemic lupus erythematosus, including activation of antiviral type 1 interferon (IFN). To further define the pathomechanisms underlying autoimmunity in AGS due to SAMHD1 mutations, we investigated the physiological properties of SAMHD1. METHODS: Primary patient fibroblasts were examined for dNTP levels, proliferation, senescence, cell cycle progression and DNA damage. Genome-wide transcriptional profiles were generated by RNA sequencing. Interaction of SAMHD1 with cyclin A was assessed by coimmunoprecipitation and fluorescence cross-correlation spectroscopy. Cell cycle-dependent phosphorylation of SAMHD1 was examined in synchronised HeLa cells and using recombinant SAMHD1. SAMHD1 was knocked down by RNA interference. RESULTS: We show that increased dNTP pools due to SAMHD1 deficiency cause genome instability in fibroblasts of patients with AGS. Constitutive DNA damage signalling is associated with cell cycle delay, cellular senescence, and upregulation of IFN-stimulated genes. SAMHD1 is phosphorylated by cyclin A/cyclin-dependent kinase 1 in a cell cycle-dependent manner, and its level fluctuates during the cell cycle, with the lowest levels observed in G1/S phase. Knockdown of SAMHD1 by RNA interference recapitulates activation of DNA damage signalling and type 1 IFN activation. CONCLUSIONS: SAMHD1 is required for genome integrity by maintaining balanced dNTP pools. dNTP imbalances due to SAMHD1 deficiency cause DNA damage, leading to intrinsic activation of IFN signalling. These findings establish a novel link between DNA damage signalling and innate immune activation in the pathogenesis of autoimmunity.

Effect of ribonucleotides embedded in a DNA template on HIV-1 reverse transcription kinetics and fidelity.

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.

Improving Admission Temperature in Infants ≥34 Weeks' Gestation: A Quality Improvement Initiative.

BACKGROUND: NICU admission for hypothermia is a problem worldwide, with associated morbidity, mortality, and financial costs. Many interventions have been studied for smaller infants, but there has been little focus on infants born ≥34 weeks' gestational age (GA), though most deliveries occur at this gestation. Our primary aim was to improve the proportion of infants ≥34 weeks' GA with normal NICU admission temperature. Secondary outcomes included improvement of the proportion of normal first temperature in all infants ≥34 weeks' GA, independent of NICU admission, and improvement of predefined outcome measures. METHODS: We completed a root cause analysis, using fishbone and process mapping to determine what factors were contributing to hypothermia. A series of changes were trialed using plan-do-study-act cycles to develop a standard operating procedure, covering both vaginal and cesarean section births. Outcome measures were analyzed using a P-chart as well as traditional statistical tests. RESULTS: We successfully increased the proportion of infants ≥34 weeks' GA with normothermia on NICU admission from 62% to 80% without increasing hyperthermia. In addition, the interventions improved the proportion of delivery room normothermia in all infants born ≥34 weeks' GA and were associated with a decreased need for intravenous therapy for hypoglycemia and the incidence of metabolic acidosis. CONCLUSIONS: This quality improvement initiative was successful at improving our institution's rates of normal infant temperature. The methodology used can be applied to other similar centers to improve this common problem.

Tight interplay among SAMHD1 protein level, cellular dNTP levels, and HIV-1 proviral DNA synthesis kinetics in human primary monocyte-derived macrophages.

Recently, SAMHD1 has come under intense focus as a host anti-HIV factor. SAMHD1 is a dNTP triphosphohydrolase, which leads to the regulation of DNA metabolism in host cells. HIV-2/SIV (simian immunodeficiency virus) viral protein x (Vpx) has been shown to promote the degradation of SAMHD1. In this study, we examine the kinetics of SAMHD1 degradation, the increase in the dNTP pool level, and the efficiency of proviral DNA synthesis in Vpx+ virus-like particle (VLP)-treated monocyte-derived macrophages (MDMs). Our results indicate a very close temporal link with a reduction in SAMHD1 detected within the first few hours of Vpx+ VLP treatment. This loss of SAMHD1 is followed by a significant increase in cellular dNTP levels by 8 h after Vpx+ VLP addition, ultimately leading to the enhancement of the HIV proviral DNA synthesis rate and HIV infection in MDMs. Finally, the pretreatment of MDMs with the Vpx+ VLPs, which is a widely used protocol, displayed identical proviral DNA synthesis as compared with MDMs co-treated with Vpx+ VLP and HIV vector. These findings further indicate that Vpx degradation of SAMHD1 is sufficiently rapid to enable appropriate progression of reverse transcription in MDMs, even when present at the time of infection. Overall, this study demonstrates a tight interplay between SAMHD1 level, dNTP levels, and HIV proviral DNA synthesis kinetics in MDMs.

Interferon block to HIV-1 transduction in macrophages despite SAMHD1 degradation and high deoxynucleoside triphosphates supply.

BACKGROUND: Interferon-α (IFN-α) is an essential mediator of the antiviral response, which potently inhibits both early and late phases of HIV replication. The SAMHD1 deoxynucleoside triphosphate (dNTP) hydrolase represents the prototype of a new antiviral strategy we referred to as "nucleotide depletion". SAMHD1 depletes dNTP levels in myeloid cells below those required for optimal synthesis of HIV viral DNA. HIV-2 and its SIVsm and SIVmac close relatives encode a protein termed Vpx, which counteracts SAMHD1. The potentiality of IFN-α to cooperate with nucleotide depletion has been poorly investigated so far. Here we wondered whether IFN-α affects SAMHD1 expression, Vpx-induced SAMHD1 degradation, Vpx-mediated rescue of HIV-1 transduction and the dNTP supply in monocyte-derived macrophages (MDMs). RESULTS: IFN-α inhibited HIV-1 transduction in monocytes and in MDMs while SAMHD1 expression was not up-regulated. Vpx triggered SAMHD1 degradation in IFN-α treated cells, and weakly restored HIV-1 transduction from the IFN-α block. Vpx helper effect towards HIV-1 transduction was gradually inhibited with increasing doses of IFN-α. dNTP levels were not significantly affected in MDMs and CD4+ primary activated T lymphocytes by IFN-α and, in correlation with SAMHD1 degradation, restoration of dNTP levels by Vpx was efficient in MDMs treated with the cytokine. In contrast, IFN-α inhibited Vpx-mediated SAMHD1 degradation in THP-1 cells, where, accordingly, Vpx could not rescue HIV-1 transduction. CONCLUSION: Our results suggest that the early antiviral effect of IFN-α results from a mechanism independent of nucleotide depletion in MDMs. In addition, they indicate that the macrophage-like THP-1 cell line may provide a system to characterize an IFN-α-induced cell response that inhibits Vpx-mediated SAMHD1 degradation.

Contribution of oligomerization to the anti-HIV-1 properties of SAMHD1.

BACKGROUND: SAMHD1 is a restriction factor that potently blocks infection by HIV-1 and other retroviruses. We have previously demonstrated that SAMHD1 oligomerizes in mammalian cells by immunoprecipitation. Here we investigated the contribution of SAMHD1 oligomerization to retroviral restriction. RESULTS: Structural analysis of SAMHD1 and homologous HD domain proteins revealed that key hydrophobic residues Y146, Y154, L428 and Y432 stabilize the extensive dimer interface observed in the SAMHD1 crystal structure. Full-length SAMHD1 variants Y146S/Y154S and L428S/Y432S lost their ability to oligomerize tested by immunoprecipitation in mammalian cells. In agreement with these observations, the Y146S/Y154S variant of a bacterial construct expressing the HD domain of human SAMHD1 (residues 109-626) disrupted the dGTP-dependent tetramerization of SAMHD1 in vitro. Tetramerization-defective variants of the full-length SAMHD1 immunoprecipitated from mammalian cells and of the bacterially-expressed HD domain construct lost their dNTPase activity. The nuclease activity of the HD domain construct was not perturbed by the Y146S/Y154S mutations. Remarkably, oligomerization-deficient SAMHD1 variants potently restricted HIV-1 infection. CONCLUSIONS: These results suggested that SAMHD1 oligomerization is not required for the ability of the protein to block HIV-1 infection.

A bivalent Epstein-Barr virus vaccine induces neutralizing antibodies that block infection and confer immunity in humanized mice.

Epstein-Barr virus (EBV) is the major cause of infectious mononucleosis and is associated with several human cancers and, more recently, multiple sclerosis. Despite its prevalence and health impact, there are currently no vaccines or treatments. Four viral glycoproteins (gp), gp350 and gH/gL/gp42, mediate entry into the major sites of viral replication, B cells, and epithelial cells. Here, we designed a nanoparticle vaccine displaying these proteins and showed that it elicits potent neutralizing antibodies that protect against infection in vivo. We designed single-chain gH/gL and gH/gL/gp42 proteins that were each fused to bacterial ferritin to form a self-assembling nanoparticle. Structural analysis revealed that single-chain gH/gL and gH/gL/gp42 adopted a similar conformation to the wild-type proteins, and the protein spikes were observed by electron microscopy. Single-chain gH/gL or gH/gL/gp42 nanoparticle vaccines were constructed to ensure product homogeneity needed for clinical development. These vaccines elicited neutralizing antibodies in mice, ferrets, and nonhuman primates that inhibited EBV entry into both B cells and epithelial cells. When mixed with a previously reported gp350 nanoparticle vaccine, gp350D123, no immune competition was observed. To confirm its efficacy in vivo, humanized mice were challenged with EBV after passive transfer of IgG from mice vaccinated with control, gH/gL/gp42+gp350D123, or gH/gL+gp350D123 nanoparticles. Although all control animals were infected, only one mouse in each vaccine group that received immune IgG had detectable transient viremia. Furthermore, no EBV lymphomas were detected in immune animals. This bivalent EBV nanoparticle vaccine represents a promising candidate to prevent EBV infection and EBV-related malignancies in humans.

Ribonucleoside triphosphates as substrate of human immunodeficiency virus type 1 reverse transcriptase in human macrophages.

We biochemically simulated HIV-1 DNA polymerization in physiological nucleotide pools found in two HIV-1 target cell types: terminally differentiated/non-dividing macrophages and activated/dividing CD4(+) T cells. Quantitative tandem mass spectrometry shows that macrophages harbor 22-320-fold lower dNTP concentrations and a greater disparity between ribonucleoside triphosphate (rNTP) and dNTP concentrations than dividing target cells. A biochemical simulation of HIV-1 reverse transcription revealed that rNTPs are efficiently incorporated into DNA in the macrophage but not in the T cell environment. This implies that HIV-1 incorporates rNTPs during viral replication in macrophages and also predicts that rNTP chain terminators lacking a 3'-OH should inhibit HIV-1 reverse transcription in macrophages. Indeed, 3'-deoxyadenosine inhibits HIV-1 proviral DNA synthesis in human macrophages more efficiently than in CD4(+) T cells. This study reveals that the biochemical landscape of HIV-1 replication in macrophages is unique and that ribonucleoside chain terminators may be a new class of anti-HIV-1 agents specifically targeting viral macrophage infection.

Cost-related medication nonadherence in Canada: a systematic review of prevalence, predictors, and clinical impact.

BACKGROUND: Cost-related nonadherence to medications (CRNA) is common in many countries and thought to be associated with adverse outcomes. The characteristics of CRNA in Canada, with its patchwork coverage of increasingly expensive medications, are unclear. OBJECTIVES: Our objective in this systematic review was to summarize the literature evaluating CRNA in Canada in three domains: prevalence, predictors, and effect on clinical outcomes. METHODS: We searched MEDLINE, Embase, Google Scholar, and the Cochrane Library from 1992 to December 2019 using search terms covering medication adherence, costs, and Canada. Eligible studies, without restriction on design, had to have original data on at least one of the three domains specifically for Canadian participants. Articles were identified and reviewed in duplicate. Risk of bias was assessed using design-specific tools. RESULTS: Twenty-six studies of varying quality (n = 483,065 Canadians) were eligible for inclusion. Sixteen studies reported on the overall prevalence of CRNA, with population-based estimates ranging from 5.1 to 10.2%. Factors predicting CRNA included high out-of-pocket spending, low income or financial flexibility, lack of drug insurance, younger age, and poorer health. A single randomized trial of free essential medications with free delivery in Ontario improved adherence but did not find any change in clinical outcomes at 1 year. CONCLUSION: CRNA affects many Canadians. The estimated percentage depends on the sampling frame, the main predictors tend to be financial, and its association with clinical outcomes in Canada remains unproven.