Evolution of the substrate specificity of an RNA ligase ribozyme from phosphorimidazole to triphosphate activation.

The acquisition of new RNA functions through evolutionary processes was essential for the diversification of RNA-based primordial biology and its subsequent transition to modern biology. However, the mechanisms by which RNAs access new functions remain unclear. Do RNA enzymes need completely new folds to support new but related functions, or is reoptimization of the active site sufficient? What are the roles of neutral and adaptive mutations in evolutionary innovation? Here, we address these questions experimentally by focusing on the evolution of substrate specificity in RNA-catalyzed RNA assembly. We use directed in vitro evolution to show that a ligase ribozyme that uses prebiotically relevant 5'-phosphorimidazole-activated substrates can be evolved to catalyze ligation with substrates that are 5'-activated with the biologically relevant triphosphate group. Interestingly, despite catalyzing a related reaction, the new ribozyme folds into a completely new structure and exhibits promiscuity by catalyzing RNA ligation with both triphosphate and phosphorimidazole-activated substrates. Although distinct in sequence and structure, the parent phosphorimidazolide ligase and the evolved triphosphate ligase ribozymes can be connected by a series of point mutations where the intermediate sequences retain at least some ligase activity. The existence of a quasi-neutral pathway between these distinct ligase ribozymes suggests that neutral drift is sufficient to enable the acquisition of new substrate specificity, thereby providing opportunities for subsequent adaptive optimization. The transition from RNA-catalyzed RNA assembly using phosphorimidazole-activated substrates to triphosphate-activated substrates may have foreshadowed the later evolution of the protein enzymes that use monomeric triphosphates (nucleoside triphosphates, NTPs) for RNA synthesis.

A potential role for RNA aminoacylation prior to its role in peptide synthesis.

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid-specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here, we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture that is found in many noncoding RNAs, including the modern tRNA. We demonstrate that the T-loop-assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of amino acid-bridged chimeric ribozymes provides a direct and facile route for the covalent incorporation of amino acids into RNA. A greater functionality of covalently incorporated amino acids could contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid-specific aminoacyl-RNA synthetase ribozymes in the RNA World. The synthesis of specifically aminoacylated RNAs, an unlikely prospect for nonenzymatic reactions but a likely one for ribozymes, could have set the stage for the subsequent evolution of coded protein synthesis.

Single-cell transcriptomic profiling of human pancreatic islets reveals genes responsive to glucose exposure over 24 h.

AIMS/HYPOTHESIS: Disruption of pancreatic islet function and glucose homeostasis can lead to the development of sustained hyperglycaemia, beta cell glucotoxicity and subsequently type 2 diabetes. In this study, we explored the effects of in vitro hyperglycaemic conditions on human pancreatic islet gene expression across 24 h in six pancreatic cell types: alpha; beta; gamma; delta; ductal; and acinar. We hypothesised that genes associated with hyperglycaemic conditions may be relevant to the onset and progression of diabetes. METHODS: We exposed human pancreatic islets from two donors to low (2.8 mmol/l) and high (15.0 mmol/l) glucose concentrations over 24 h in vitro. To assess the transcriptome, we performed single-cell RNA-seq (scRNA-seq) at seven time points. We modelled time as both a discrete and continuous variable to determine momentary and longitudinal changes in transcription associated with islet time in culture or glucose exposure. Additionally, we integrated genomic features and genetic summary statistics to nominate candidate effector genes. For three of these genes, we functionally characterised the effect on insulin production and secretion using CRISPR interference to knock down gene expression in EndoC-ßH1 cells, followed by a glucose-stimulated insulin secretion assay. RESULTS: In the discrete time models, we identified 1344 genes associated with time and 668 genes associated with glucose exposure across all cell types and time points. In the continuous time models, we identified 1311 genes associated with time, 345 genes associated with glucose exposure and 418 genes associated with interaction effects between time and glucose across all cell types. By integrating these expression profiles with summary statistics from genetic association studies, we identified 2449 candidate effector genes for type 2 diabetes, HbA1c, random blood glucose and fasting blood glucose. Of these candidate effector genes, we showed that three (ERO1B, HNRNPA2B1 and RHOBTB3) exhibited an effect on glucose-stimulated insulin production and secretion in EndoC-ßH1 cells. CONCLUSIONS/INTERPRETATION: The findings of our study provide an in-depth characterisation of the 24 h transcriptomic response of human pancreatic islets to glucose exposure at a single-cell resolution. By integrating differentially expressed genes with genetic signals for type 2 diabetes and glucose-related traits, we provide insights into the molecular mechanisms underlying glucose homeostasis. Finally, we provide functional evidence to support the role of three candidate effector genes in insulin secretion and production. DATA AVAILABILITY: The scRNA-seq data from the 24 h glucose exposure experiment performed in this study are available in the database of Genotypes and Phenotypes (dbGap; https://www.ncbi.nlm.nih.gov/gap/ ) with accession no. phs001188.v3.p1. Study metadata and summary statistics for the differential expression, gene set enrichment and candidate effector gene prediction analyses are available in the Zenodo data repository ( https://zenodo.org/ ) under accession number 11123248. The code used in this study is publicly available at https://github.com/CollinsLabBioComp/publication-islet_glucose_timecourse .

REVERSE: a user-friendly web server for analyzing next-generation sequencing data from in vitro selection/evolution experiments.

Next-generation sequencing (NGS) enables the identification of functional nucleic acid sequences from in vitro selection/evolution experiments and illuminates the evolutionary process at single-nucleotide resolution. However, analyzing the vast output from NGS can be daunting, especially with limited programming skills. We developed REVERSE (Rapid EValuation of Experimental RNA Selection/Evolution) (https://www.reverseserver.org/), a web server that implements an integrated computational pipeline through a graphical user interface, which performs both pre-processing and detailed sequence level analyses within minutes. Raw FASTQ files are quality-filtered, dereplicated, and trimmed before being analyzed by either of two pipelines. The first pipeline counts, sorts, and tracks enrichment of unique sequences and user-defined sequence motifs. It also identifies mutational intermediates present in the sequence data that connect two input sequences. The second pipeline sorts similar sequences into clusters and tracks enrichment of peak sequences. It also performs nucleotide conservation analysis on the cluster of choice and generates a consensus sequence. Both pipelines generate downloadable spreadsheets and high-resolution figures. Collectively, REVERSE is a one-stop solution for the rapid analysis of NGS data obtained from in vitro selection/evolution experiments that obviates the need for computational expertise.

Hidden in plain sight: urinary Cryptococcus neoformans missed by routine diagnostics in a patient with acute leukemia.

Cryptococcuria is a rare manifestation of localized cryptococcal disease. We present a case of Cryptococcus neoformans urinary tract infection in an immunocompromised host missed by routine laboratory workup. The patient had negative blood cultures, a negative serum cryptococcal antigen (CrAg), and "non-Candida yeast" growing in urine culture that was initially dismissed as non-pathogenic. The diagnosis was ultimately made by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) from a repeat urine culture after transfer to a tertiary care center. Cryptococcus should be considered in the differential of refractory urinary tract infections growing non-Candida yeast.

A eulogy for Dr Francisco Miguel Marty Forero.

As some of those who were lucky enough to have been mentored by Dr Francisco Marty in transplant infectious diseases, we stand with the larger medical community in mourning his untimely death and in commemorating him as a uniquely exceptional and talented physician, investigator, teacher, mentor, friend, artist, and human being.

Validation of pneumonia prognostic scores in a statewide cohort of hospitalised patients with COVID-19.

OBJECTIVE: We aimed to externally validate the predictive performance of two recently developed COVID-19-specific prognostic tools, the COVID-GRAM and CALL scores, and prior prognostic scores for community-acquired pneumonia (CURB-65), viral pneumonia (MuBLSTA) and H1N1 influenza pneumonia (Influenza risk score) in a contemporary US cohort. METHODS: We included 257 hospitalised patients with laboratory-confirmed COVID-19 pneumonia from three teaching hospitals in Rhode Island. We extracted data from within the first 24 hours of admission. Variables were excluded if values were missing in >20% of cases, otherwise, missing values were imputed. One hundred and fifteen patients with complete data after imputation were used for the primary analysis. Sensitivity analysis was performed after the exclusion of one variable (LDH) in the complete dataset (n = 257). Primary and secondary outcomes were in-hospital mortality and critical illness (mechanical ventilation or death), respectively. RESULTS: Only the areas under the receiver-operating characteristic curves (RO-AUC) of COVID-GRAM (RO-AUC = 0.775, 95% CI 0.525-0.915) for in-hospital death, and CURB65 for in-hospital death (RO-AUC = 0.842, 95% CI 0.674-0.932) or critical illness (RO-AUC = 0.766, 95% CI 0.584-0.884) were significantly better than random. Sensitivity analysis yielded similar trends. Calibration plots showed better agreement between the estimated and observed probability of in-hospital death for CURB65, compared with COVID-GRAM. The negative predictive value (NPV) of CURB65 ≥2 was 97.2% for in-hospital death and 88.1% for critical illness. CONCLUSIONS: The COVID-GRAM score demonstrated acceptable predictive performance for in-hospital death. The CURB65 score had better prognostic utility for in-hospital death and critical illness. The high NPV of CURB65 values ≥2 may be useful in triaging and allocation of resources.

Correction to: Clinical experience with a novel assay measuring cytomegalovirus (CMV)-specific CD4+ and CD8+ T-cell immunity by flow cytometry and intracellular cytokine staining to predict clinically significant CMV events.

After publication of the original article [1], we were notified that an author's family name has been erroneously spelled. Aditya Chandrokar should be replaced with Aditya Chandorkar.

Clinical experience with a novel assay measuring cytomegalovirus (CMV)-specific CD4+ and CD8+ T-cell immunity by flow cytometry and intracellular cytokine staining to predict clinically significant CMV events.

BACKGROUND: Cytomegalovirus (CMV) infection is one of the most common opportunistic infections following organ transplantation, despite administration of CMV prophylaxis. CMV-specific T-cell immunity (TCI) has been associated with reduced rates of CMV infection. We describe for the first time clinical experience using the CMV T-Cell Immunity Panel (CMV-TCIP), a commercially available assay which measures CMV-specific CD4+ and CD8+ T-cell responses, to predict clinically significant CMV events. METHODS: Adult (> 18-year-old) patients with CMV-TCIP results and ≥ 1 subsequent assessment for CMV DNAemia were included at Brown University and the University of Maryland Medical Center-affiliated hospitals between 4/2017 and 5/2019. A clinically significant CMV event was defined as CMV DNAemia prompting initiation of treatment. We excluded indeterminate results, mostly due to background positivity, allogeneic hematopoetic cell transplant (HCT) recipients, or patients who were continued on antiviral therapy against CMV irrespective of the CMV-TCIP result, because ongoing antiviral therapy could prevent a CMV event. RESULTS: We analyzed 44 samples from 37 patients: 31 were solid organ transplant recipients, 4 had hematologic malignancies, 2 had autoimmune disorders. The CMV-protection receiver operating characteristic (ROC) area under the curve (AUC) was significant for %CMV-specific CD4+ (AUC: 0.78, P < 0.001) and borderline for CD8+ (AUC: 0.66, P = 0.064) T-cells. At a cut-off value of 0.22% CMV-specific CD4+ T-cells, positive predictive value (PPV) for protection against CMV was 85% (95%CI 65-96%), and negative predictive value (NPV) was 67% (95%CI 41-87%). CONCLUSIONS: The CMV-TCIP, in particular %CMV-specific CD4+ T-cells, showed good diagnostic performance to predict CMV events. The CMV-TCIP may be a useful test in clinical practice, and merits further validation in larger prospective studies.

Opportunities Revealed for Antimicrobial Stewardship and Clinical Practice with Implementation of a Rapid Respiratory Multiplex Assay.

Few studies assess the utility of rapid multiplex molecular respiratory panels in adult patients. Previous multiplex PCR assays took hours to days from order time to result. We analyze the clinical impact of switching to a molecular assay with a 3-h test-turnaround-time (TAT). We performed a retrospective review of adult patients who presented to our emergency departments with respiratory symptoms and had a respiratory viral panel (xTAG RVP; RVP) or respiratory pathogen panel (ePlex RP; RPP) within 48 h of presentation. The average TATs for the RVP and RPP were 27.9 and 3.0 h, respectively (P < 0.0001). In RVP-positive and RPP-positive patients, 68.9 and 44.5% of those with normal chest imaging received antibiotics (P = 0.013), while 95.4 and 89.6% of those with abnormal imaging received antibiotics, respectively (P = 0.187). There was no difference in antibiotic duration in RVP-positive and RPP-positive patients with abnormal chest imaging (6.2 and 6.0 days, respectively; P = 0.923) and normal chest imaging (4.5 and 4.3 days, respectively; P = 0.922). Fewer patients were admitted in the RPP-positive compared to the RVP-positive group (76.9 and 88.6%, respectively; P = 0.013), while the proportion of admissions were similar among RPP-negative and RVP-negative patients (85.3 and 87.1%, P = 0.726). Switching to a multiplex respiratory panel with a clinically actionable TAT is associated with reduced hospital admissions and, in admitted adults without focal radiographic findings, reduced antibiotic initiation. Opportunities to further mitigate inappropriate antibiotic use may be realized by combining rapid multiplex PCR with provider education, clinical decision-care algorithms, and active antibiotic stewardship.

False positive bronchoalveolar lavage galactomannan: Effect of host and cut-off value.

OBJECTIVES: Bronchoalveolar lavage galactomannan (BAL-GM) is a mycological criterion for diagnosis of probable invasive aspergillosis (IA) per European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORT-MSG) consensus criteria, but its real-world positive predictive value (PPV) has not been well-studied. Our aim was to estimate the PPV of BAL-GM in a contemporary cohort of patients with positive BAL-GM. METHODS: We identified consecutive patients with ≥1 positive BAL-GM value (index ≥ 0.5) at Brigham and Women's Hospital from 11/2009 to 3/2016. We classified patients as having no, possible, probable, or proven IA, excluding BAL-GM as mycological criterion. RESULTS: We studied 134 patients: 54% had hematologic malignancy (HM), and 10% were solid organ transplant (SOT) recipients. A total of 42% of positive (≥0.5) BAL-GM results were falsely positive (PPV 58%). The number of probable IA cases was increased by 23% using positive BAL-GM as mycologic criterion alone. PPV was higher in patients with HM or SOT (P < 0.001) and with use of higher thresholds for positivity (BAL-GM ≥ 1 vs 1-0.8 vs 0.8-0.5: P = 0.002). CONCLUSIONS: 42% of positive BAL-GM values were falsely positive. We propose a critical reassessment of BAL-GM cutoff values in different patient populations. Accurate noninvasive tests for diagnosis of IA are urgently needed.

Heterotopic Ossification of the Abdomen: A Rare Sequela Following Trauma and Damage Control Laparotomy.

Heterotopic ossification (HO) of the abdomen is a rare yet highly morbid complication following blunt and penetrating trauma requiring damage control laparotomy. We present the case of a 22-year-old man, 20 months after life-threatening motor vehicle crash with major vascular injury requiring multiple abdominal surgeries. The patient was initially treated at a community hospital and subsequently developed a chronic left lower quadrant enterocutaneous fistula, accompanied by a gradually worsening diffuse abdominal pain. He was referred to our tertiary care center with extensive skin breakdown and an inability to control the fistula despite numerous wound care consultations. He also had severe abdominal deformities due to HO in the abdominal wall, peritoneum, paraspinal muscles, and parapelvic regions. As HO is largely underreported, it is crucial to refer those patients, once medically stabilized, to tertiary care centers for surveillance and possible treatment when symptomatic.

Structure-guided aminoacylation and assembly of chimeric RNAs.

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture found in modern tRNA. We demonstrate that the T-loop assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of such chimeric ribozymes would have allowed the greater functionality of amino acids to contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid specific aminoacyl-RNA synthetase enzymes prior to their role in protein synthesis.

A decade of clinical microbiology: top 10 advances in 10 years: what every infection preventionist and antimicrobial steward should know.

The past 10 years have brought paradigm-shifting changes to clinical microbiology. This paper explores the top 10 transformative innovations across the diagnostic spectrum, including not only state of the art technologies but also preanalytic and post-analytic advances. Clinical decision support tools have reshaped testing practices, curbing unnecessary tests. Innovations like broad-range polymerase chain reaction and metagenomic sequencing, whole genome sequencing, multiplex molecular panels, rapid phenotypic susceptibility testing, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry have all expanded our diagnostic armamentarium. Rapid home-based testing has made diagnostic testing more accessible than ever. Enhancements to clinician-laboratory interfaces allow for automated stewardship interventions and education. Laboratory restructuring and consolidation efforts are reshaping the field of microbiology, presenting both opportunities and challenges for the future of clinical microbiology laboratories. Here, we review key innovations of the last decade.

Evolution of antifungals for invasive mold infections in immunocompromised hosts, then and now.

INTRODUCTION: The current armamentarium of antifungal agents for invasive mold infections (IMI) has dramatically improved over the last 50 years. Existing therapies are, however, associated with toxicities, drug interactions, and, in some cases, therapeutic failures. Novel antifungals are needed to address the increasing prevalence of IMI and the growing threat of antifungal resistance. AREAS COVERED: We review the history and development of the most commonly used antifungals. We discuss the current consensus guidelines and supporting data for treatment of invasive mold infection (IMI), the role of susceptibility testing, and the niche that novel antifungals could fill. We review the current data for aspergillosis, mucormycosis, and hyalohyphomycosis. EXPERT OPINION: Robust clinical trial data demonstrating the relative effectiveness of our current antifungal agents for treating IMI outside of A. fumigatus remains limited. Clinical trials are urgently needed to delineate the relationship between MICs and clinical outcomes for existing agents and to better evaluate the in vitro and in vivo aspects of antifungal synergy. Continued international multicenter collaboration and standardized clinical endpoints for trials evaluating both existing and new agents are necessary to advance the field.

Duped by dumping syndrome: non-endemic Vibrio cholerae bacteremia in an immunocompetent host with gastric bypass surgery, a case report.

Extra-intestinal infection with non-O1/non-O139 strains of Vibrio cholerae (NOVC) is rare, though bacteremia and hepatobiliary manifestations have been reported. Reduced stomach acid, or hypochlorhydria, can increase risk of V. cholerae infection. We describe a 42-year-old woman with hypochlorhydria due to untreated Helicobacter pylori infection, gastric-bypass surgery, and chronic proton pump inhibitors (PPI) exposure, who developed acute diarrhoea following raw oyster consumption. Her symptoms were attributed to rapid gastric emptying (dumping syndrome) after a negative limited stool work-up. She had persistent diarrhoea, weight loss, and after 5 months was admitted with acute cholecystitis and NOVC bacteremia, requiring cholecystectomy. This is the first reported case of NOVC bacteremia and cholecystitis in a patient with gastric bypass. This case highlights the potential for NOVC biliary carriage, the role of hypochlorhydria as a risk factor for Vibrio infection, and the importance of excluding infectious diarrhoea in patients with new onset of symptoms compatible with dumping syndrome and a relevant travel history.

The diagnostic and clinical utility of microbial cell-free DNA sequencing in a real-world setting.

Microbial cell free DNA sequencing is increasingly used for diagnosis of infection but few studies describe its utility in real-world settings. We performed a single-center retrospective case series of microbial cell free DNA testing using the Karius assay from 29 patient samples to define the clinical reasoning and the impact of testing. Indications fell into 3 categories, identifying a causative pathogen in patients with an infectious syndrome and negative microbiologic workup (15/29, 52%), seeking another pathogen when organisms identified by traditional diagnostics failed to explain the clinical presentation (9/29, 31%) and to "rule out" infection in patients with nonspecific symptoms and negative microbiologic workup (5/29, 17%). Clinical impact was positive in 13/29 (45%) and all were for patients with high pretest probability for infection. Impact was negative in 3/29 (10%) cases. There was no impact in 15/29 (52%) cases. Further work is needed to define the optimal timing accounting for test performance, and patient characteristics.