Interleukin(IL)-1/IL-6-inhibitor-associated drug reaction with eosinophilia and systemic symptoms (DReSS) in systemic inflammatory illnesses.

BACKGROUND: After introducing interleukin(IL)-1/IL-6 inhibitors, some Still and Still-like patients developed unusual often fatal pulmonary disease. This complication was associated with scoring as DReSS (drug reaction with eosinophilia and systemic symptoms) implicating these inhibitors, although DReSS can be difficult to recognize in the setting of systemic inflammatory disease. OBJECTIVE: We sought to facilitate recognition of IL-1/IL-6 inhibitor-DReSS in systemic inflammatory illnesses (Still/Still-like) by looking at timing and reaction-associated features. We evaluated outcomes of stopping or not-stopping IL-1/IL-6-inhibitors after DReSS reaction began. METHODS: In an international study collaborating primarily with pediatric specialists, we characterized features of 89 drug-reaction cases versus 773 drug-exposed controls and compared outcomes of 52 cases stopping IL-1/IL-6-inhibitors to 37 cases not-stopping these drugs. RESULTS: Before reaction began, drug-reaction cases and controls were clinically comparable, except for younger disease onset age for reaction cases with pre-existing cardiothoracic comorbidities. After reaction began, increased rates of pulmonary complications and macrophage activation syndrome (MAS), differentiated drug-reaction cases from drug-tolerant controls (p=4.7x10-35; p=1.1x10-24, respectively). Initial DReSS feature was typically reported 2-8 weeks after initiating IL-1/IL-6-inhibition. In drug-reaction cases stopping versus not-stopping IL-1/IL-6-inhibitor treatment, reaction related features were indistinguishable, including pulmonary complication rates [75%(39/52] versus [76%(28/37)]. Those stopping subsequently required fewer medications for treatment of systemic inflammation, had decreased rates of MAS, and improved survival (p=0.005, multivariate regression). Resolution of pulmonary complications occurred in 67%(26/39) of drug-reaction cases who stopped and in none who continued inhibitors. CONCLUSIONS: In systemic inflammatory illnesses, recognition of IL-1/IL-6-inhibitor-associated reactions followed by avoidance of IL-1/IL-6-inhibitors significantly improved outcomes.

HIV-1 Vpr combats the PU.1-driven antiviral response in primary human macrophages.

HIV-1 Vpr promotes efficient spread of HIV-1 from macrophages to T cells by transcriptionally downmodulating restriction factors that target HIV-1 Envelope protein (Env). Here we find that Vpr induces broad transcriptomic changes by targeting PU.1, a transcription factor necessary for expression of host innate immune response genes, including those that target Env. Consistent with this, we find silencing PU.1 in infected macrophages lacking Vpr rescues Env. Vpr downmodulates PU.1 through a proteasomal degradation pathway that depends on physical interactions with PU.1 and DCAF1, a component of the Cul4A E3 ubiquitin ligase. The capacity for Vpr to target PU.1 is highly conserved across primate lentiviruses. In addition to impacting infected cells, we find that Vpr suppresses expression of innate immune response genes in uninfected bystander cells, and that virion-associated Vpr can degrade PU.1. Together, we demonstrate Vpr counteracts PU.1 in macrophages to blunt antiviral immune responses and promote viral spread.

Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.

R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, Precise RNA-mediated INsertion of Transgenes (PRINT), relies on co-delivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.

The hidden professionalism curriculum: Teach it, see it, do it and repeat!

Background: Professionalism is a complex and multifaceted component of medical education. Historically, students have learned about professionalism informally and as part of the hidden curriculum. Currently, professionalism is increasingly prominent in formal curricula, but uncertainty remains regarding optimal professionalism pedagogies. In this study, the authors explored medical students' exposure to professional topics and considered factors that enabled students to correctly recognize and manage these issues. Methods: Convenience sampling was used to recruit medical students from existing clinical attachments at the authors' hospital. A semi-structured interview format was used to explore participants' awareness of professional issues within fictional vignettes created using published regulatory guidance. The interview transcripts and interview guide field notes were then analyzed. Results: The data suggest that students require a combination of didactic teaching and experiential learning to reliably recognize and manage professional issues. Didactic teaching alone enabled topic recognition, but with uncertainty about management strategies. Experiential learning alone led to erratic recognition of the subject and reliance upon role modeling to guide its management. This work stimulates faculty development to enhance teaching professionalism. Conclusions: Undergraduate medical education on professionalism must be introduced into the formal curriculum. Didactic teaching is required to scaffold experiential learning. Failure to do so renders students unable to reliably recognize or manage professional issues encountered in clinical practice. Further research questions were identified to progress this work.

Gammaherpesvirus infection triggers the formation of tRNA fragments from premature tRNAs.

Transfer RNAs (tRNAs) are fundamental for both cellular and viral gene expression during viral infection. In addition, mounting evidence supports biological function for tRNA cleavage products, including in the control of gene expression during conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus, MHV68, leads to enhanced tRNA transcription. However, whether this has any influence on tRNA transcript processing, viral replication, or the host response is not known. Here, we combined two new approaches, sequencing library preparation by Ordered Two Template Relay (OTTR) and tRNA bioinformatic analysis by tRAX, to quantitatively profile full-length tRNAs and tRNA fragment (tRF) identities during MHV68 infection. We find that MHV68 infection triggers both pre-tRNA and mature tRNA cleavage, resulting in the accumulation of specific tRFs. OTTR-tRAX revealed not only host tRNAome changes, but also the expression patterns of virally-encoded tRNAs (virtRNAs) and virtRFs made from the MHV68 genome, including their base modification signatures. Because the transcript ends of several host tRFs matched tRNA splice junctions, we tested and confirmed the role of tRNA splicing factors TSEN2 and CLP1 in MHV68-induced tRF biogenesis. Further, we show that CLP1 kinase, and by extension tRNA splicing, is required for productive MHV68 infection. Our findings provide new insight into how gammaherpesvirus infection both impacts and relies on tRNA transcription and processing.

Conserved and divergent DNA recognition specificities and functions of R2 retrotransposon N-terminal domains.

R2 non-long terminal repeat (non-LTR) retrotransposons are among the most extensively distributed mobile genetic elements in multicellular eukaryotes and show promise for applications in transgene supplementation of the human genome. They insert new gene copies into a conserved site in 28S ribosomal DNA with exquisite specificity. R2 clades are defined by the number of zinc fingers (ZFs) at the N terminus of the retrotransposon-encoded protein, postulated to additively confer DNA site specificity. Here, we illuminate general principles of DNA recognition by R2 N-terminal domains across and between clades, with extensive, specific recognition requiring only one or two compact domains. DNA-binding and protection assays demonstrate broadly shared as well as clade-specific DNA interactions. Gene insertion assays in cells identify the N-terminal domains sufficient for target-site insertion and reveal roles in second-strand cleavage or synthesis for clade-specific ZFs. Our results have implications for understanding evolutionary diversification of non-LTR retrotransposon insertion mechanisms and the design of retrotransposon-based gene therapies.

ER-associated degradation adapter Sel1L is required for CD8+ T cell function and memory formation following acute viral infection.

The maintenance of antigen-specific CD8+ T cells underlies the efficacy of vaccines and immunotherapies. Pathways contributing to CD8+ T cell loss are not completely understood. Uncovering the pathways underlying the limited persistence of CD8+ T cells would be of significant benefit for developing novel strategies of promoting T cell persistence. Here, we demonstrate that murine CD8+ T cells experience endoplasmic reticulum (ER) stress following activation and that the ER-associated degradation (ERAD) adapter Sel1L is induced in activated CD8+ T cells. Sel1L loss limits CD8+ T cell function and memory formation following acute viral infection. Mechanistically, Sel1L is required for optimal bioenergetics and c-Myc expression. Finally, we demonstrate that human CD8+ T cells experience ER stress upon activation and that ER stress is negatively associated with improved T cell functionality in T cell-redirecting therapies. Together, these results demonstrate that ER stress and ERAD are important regulators of T cell function and persistence.

Structure-Activity Relationships of Natural and Semisynthetic Plecomacrolides Suggest Distinct Pathways for HIV-1 Immune Evasion and Vacuolar ATPase-Dependent Lysosomal Acidification.

The human immunodeficiency virus (HIV)-encoded accessory protein Nef enhances pathogenicity by reducing major histocompatibility complex I (MHC-I) cell surface expression, protecting HIV-infected cells from immune recognition. Nef-dependent downmodulation of MHC-I can be reversed by subnanomolar concentrations of concanamycin A (1), a well-known inhibitor of vacuolar ATPase, at concentrations below those that interfere with lysosomal acidification or degradation. We conducted a structure-activity relationship study that assessed 76 compounds for Nef inhibition, 24 and 72 h viability, and lysosomal neutralization in Nef-expressing primary T cells. This analysis demonstrated that the most potent compounds were natural concanamycins and their derivatives. Comparison against a set of new, semisynthetic concanamycins revealed that substituents at C-8 and acylation of C-9 significantly affected Nef potency, target cell viability, and lysosomal neutralization. These findings provide important progress toward understanding the mechanism of action of these compounds and the identification of an advanced lead anti-HIV Nef inhibitory compound.

Distinct and overlapping RNA determinants for binding and target-primed reverse transcription by Bombyx mori R2 retrotransposon protein.

Eukaryotic retrotransposons encode a reverse transcriptase that binds RNA to template DNA synthesis. The ancestral non-long terminal repeat (non-LTR) retrotransposons encode a protein that performs target-primed reverse transcription (TPRT), in which the nicked genomic target site initiates complementary DNA (cDNA) synthesis directly into the genome. The best understood model system for biochemical studies of TPRT is the R2 protein from the silk moth Bombyx mori. The R2 protein selectively binds the 3' untranslated region of its encoding RNA as template for DNA insertion to its target site in 28S ribosomal DNA. Here, binding and TPRT assays define RNA contributions to RNA-protein interaction, template use for TPRT and the fidelity of template positioning for TPRT cDNA synthesis. We quantify both sequence and structure contributions to protein-RNA interaction. RNA determinants of binding affinity overlap but are not equivalent to RNA features required for TPRT and its fidelity of template positioning for full-length TPRT cDNA synthesis. Additionally, we show that a previously implicated RNA-binding protein surface of R2 protein makes RNA binding affinity dependent on the presence of two stem-loops. Our findings inform evolutionary relationships across R2 retrotransposon RNAs and are a step toward understanding the mechanism and template specificity of non-LTR retrotransposon mobility.

Gammaherpesvirus infection alters transfer RNA splicing and triggers tRNA cleavage.

Transfer RNAs (tRNAs) are fundamental for both cellular and viral gene expression during viral infection. Moreover, mounting evidence supports a noncanonical role for tRNA cleavage products in the control of gene expression during diverse conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus, MHV68, leads to altered tRNA transcription, suggesting that tRNA regulation may play an important role in mediating viral replication or the host response. To better understand how viral infection alters tRNA expression, we combined Ordered Two Template Relay (OTTR) with tRNA-specific bioinformatic software called tRAX to profile full-length tRNAs and fragmented tRNA-derived RNAs (tDRs) during infection with MHV68. We find that OTTR-tRAX is a powerful sequencing strategy for combined tRNA/tDR profiling and reveals that MHV68 infection triggers pre-tRNA and mature tRNA cleavage, resulting in the accumulation of specific tDRs. Fragments of virally-encoded tRNAs (virtRNAs), as well as virtRNA base modification signatures are also detectable during infection. We present evidence that tRNA splicing factors are involved in the biogenesis of MHV68-induced cleavage products from pre-tRNAs and, in the case of CLP1 kinase, impact infectious virus production. Our data offers new insights into the importance of tRNA processing during gammaherpesvirus infection.

Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci.

Current approaches for inserting autonomous transgenes into the genome, such as CRISPR-Cas9 or virus-based strategies, have limitations including low efficiency and high risk of untargeted genome mutagenesis. Here, we describe precise RNA-mediated insertion of transgenes (PRINT), an approach for site-specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus. PRINT uses delivery of two in vitro transcribed RNAs: messenger RNA encoding avian R2 retroelement-protein and template RNA encoding a transgene of length validated up to 4 kb. The R2 protein coordinately recognizes the target site, nicks one strand at a precise location and primes complementary DNA synthesis for stable transgene insertion. With a cultured human primary cell line, over 50% of cells can gain several 2 kb transgenes, of which more than 50% are full-length. PRINT advantages include no extragenomic DNA, limiting risk of deleterious mutagenesis and innate immune responses, and the relatively low cost, rapid production and scalability of RNA-only delivery.

HIV-1 Vpr combats the PU.1-driven antiviral response in primary human macrophages.

HIV-1 Vpr promotes efficient spread of HIV-1 from macrophages to T cells by transcriptionally downmodulating restriction factors that target HIV-1 Envelope protein (Env). Here we find that Vpr induces broad transcriptomic changes by targeting PU.1, a transcription factor necessary for expression of host innate immune response genes, including those that target Env. Consistent with this, we find silencing PU.1 in infected macrophages lacking Vpr rescues Env. Vpr downmodulates PU.1 through a proteasomal degradation pathway that depends on physical interactions with PU.1 and DCAF1, a component of the Cul4A E3 ubiquitin ligase. The capacity for Vpr to target PU.1 is highly conserved across primate lentiviruses. In addition to impacting infected cells, we find that Vpr suppresses expression of innate immune response genes in uninfected bystander cells, and that virion-associated Vpr can degrade PU.1. Together, we demonstrate Vpr counteracts PU.1 in macrophages to blunt antiviral immune responses and promote viral spread.

Disease Course, Treatments, and Outcomes of Children With Systemic Juvenile Idiopathic Arthritis-Associated Lung Disease.

OBJECTIVE: Systemic juvenile idiopathic arthritis-associated lung disease (SJIA-LD) is a life-threatening disease complication. Key questions remain regarding clinical course and optimal treatment approaches. The objectives of the study were to detail management strategies after SJIA-LD detection, characterize overall disease courses, and measure long-term outcomes. METHODS: This was a prospective cohort study. Clinical data were abstracted from the electronic medical record, including current clinical status and changes since diagnosis. Serum biomarkers were determined and correlated with presence of LD. RESULTS: We enrolled 41 patients with SJIA-LD, 85% with at least one episode of macrophage activation syndrome and 41% with adverse reactions to a biologic. Although 93% of patients were alive at last follow-up (median 2.9 years), 37% progressed to requiring chronic oxygen or other ventilator support, and 65% of patients had abnormal overnight oximetry studies, which changed over time. Eighty-four percent of patients carried the HLA-DRB1*15 haplotype, significantly more than patients without LD. Patients with SJIA-LD also showed markedly elevated serum interleukin-18 (IL-18), variable C-X-C motif chemokine ligand 9 (CXCL9), and significantly elevated matrix metalloproteinase 7. Treatment strategies showed variable use of anti-IL-1/6 biologics and addition of other immunomodulatory treatments and lung-directed therapies. We found a broad range of current clinical status independent of time from diagnosis or continued biologic treatment. Multidomain measures of change showed imaging features were the least likely to improve with time. CONCLUSION: Patients with SJIA-LD had highly varied courses, with lower mortality than previously reported but frequent hypoxia and requirement for respiratory support. Treatment strategies were highly varied, highlighting an urgent need for focused clinical trials.

Template and target-site recognition by human LINE-1 in retrotransposition.

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease1. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs2. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.

Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells.

Ribosome profiling has unveiled diverse regulation and perturbations of translation through a transcriptome-wide survey of ribosome occupancy, read out by sequencing of ribosome-protected messenger RNA fragments. Generation of ribosome footprints and their conversion into sequencing libraries is technically demanding and sensitive to biases that distort the representation of physiological ribosome occupancy. We address these challenges by producing ribosome footprints with P1 nuclease rather than RNase I and replacing RNA ligation with ordered two-template relay, a single-tube protocol for sequencing library preparation that incorporates adaptors by reverse transcription. Our streamlined approach reduced sequence bias and enhanced enrichment of ribosome footprints relative to ribosomal RNA. Furthermore, P1 nuclease preserved distinct juxtaposed ribosome complexes informative about yeast and human ribosome fates during translation initiation, stalling and termination. Our optimized methods for mRNA footprint generation and capture provide a richer translatome profile with low input and fewer technical challenges.

Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism.

Eukaryotic reverse transcriptases (RTs) can have essential or deleterious roles in normal human physiology and disease. Compared to well-studied helicases, it remains unclear how RTs overcome the ubiquitous RNA structural barriers during reverse transcription. Herein, we describe the development of a Mycobacterium smegmatis porin A (MspA) nanopore technique to sequence RNA to quantify the single-molecule kinetics of an RT from Bombyx mori with single-nucleotide resolution. By establishing a quadromer map that correlates RNA sequence and MspA ion current, we were able to quantify the RT's dwell time at every single nucleotide step along its RNA template. By challenging the enzyme with various RNA structures, we found that during cDNA synthesis the RT can sense and actively destabilize RNA structures 11-12 nt downstream of its front boundary. The ability to sequence single molecules of RNA with nanopores paves the way to investigate the single-nucleotide activity of other processive RNA translocases.

The impact of nursing skill-mix on adverse events in intensive care: a single centre cohort study.

BACKGROUND: The highly complex and technological environment of critical care manages the most critically unwell patients in the hospital system, as such there is a need for a highly trained nursing workforce. Intensive care is considered a high-risk area for errors and adverse events (AE) due to the severity of illness and number of procedures performed. OBJECTIVE: To investigate if the percentage of Critical Care Registered Nurses (CCRN) within an Intensive Care Unit (ICU) is associated with an increased risk of patients experiencing an AE. DESIGN & SETTING: We conducted a retrospective cohort study of patients admitted between January 2016 and December 2020 to a tertiary ICU in Australia. Descriptive statistics and multivariable logistic regression were used to investigate the relationship between the proportion of CCRNs each month and the occurrence of an AE defined as any one of a medication error, fall, pressure injury or unplanned removal of a central venous catheter or endotracheal tube per patient. RESULTS: A total of 13,560 patients were included in the study, with 854 (6.3%) experiencing one AE. Patients with an AE were associated with higher illness severity and frailty scores. They were more commonly admitted after medical emergency team response calls and were less commonly elective ICU admissions. Those with an AE had longer ICU and in-hospital length of stay, and higher ICU and in-hospital mortality, on average. After adjusting for ICU LOS and acute severity of illness, being admitted during a month of higher critical care nursing skill-mix was associated with a statistically significant lower odds of having a subsequent AE (OR 0.966 [95% CI: 0.944-0.988], p 0.003). CONCLUSION: An increasing percentage of CCRNs is independently associated with a lower risk-adjusted likelihood of an AE. Increasing the skill-mix of the ICU nursing staff may reduce the occurrence of AEs and lead to improved patient outcomes.