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 .

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.

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.

Delayed and Attenuated Antibody Responses to Coronavirus Disease 2019 Vaccination With Poor Cross-Variant Neutralization in Solid-Organ Transplant Recipients-A Prospective Longitudinal Study.

Background: Therapeutically immunosuppressed transplant recipients exhibit attenuated responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines. To elucidate the kinetics and variant cross-protection of vaccine-induced antibodies in this population, we conducted a prospective longitudinal study in heart and lung transplant recipients receiving the SARS-CoV-2 messenger RNA (mRNA) 3-dose vaccination series. Methods: We measured longitudinal serum antibody and neutralization responses against the ancestral and major variants of SARS-CoV-2 in SARS-CoV-2-uninfected lung (n = 18) and heart (n = 17) transplant recipients, non-lung-transplanted patients with cystic fibrosis (n = 7), and healthy controls (n = 12) before, during, and after the primary mRNA vaccination series. Results: Among healthy controls, strong anti-spike responses arose immediately following vaccination and displayed cross-neutralization against all variants. In contrast, among transplant recipients, after the first 2 vaccine doses, increases in antibody concentrations occurred gradually, and cross-neutralization was completely absent against the Omicron B.1.1.529 variant. However, most (73%) of the transplant recipients had a significant response to the third vaccine dose, reaching levels comparable to those of healthy controls, with improved but attenuated neutralization of immune evasive variants, particularly Beta, Gamma, and Omicron. Responses in non-lung-transplanted patients with cystic fibrosis paralleled those in healthy controls. Conclusions: In this prospective, longitudinal analysis of variant-specific antibody responses, lung and heart transplant recipients display delayed and defective responses to the first 2 SARS-CoV-2 vaccine doses but significantly augmented responses to a third dose. Gaps in antibody-mediated immunity among transplant recipients are compounded by decreased neutralization against Omicron variants, leaving many patients with substantially weakened immunity against currently circulating variants.

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.

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.

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.

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.

Invasive Fungal Infections and Targeted Therapies in Hematological Malignancies.

The use of targeted biologic therapies for hematological malignancies has greatly expanded in recent years. These agents act upon specific molecular pathways in order to target malignant cells but frequently have broader effects involving both innate and adaptive immunity. Patients with hematological malignancies have unique risk factors for infection, including immune dysregulation related to their underlying disease and sequelae of prior treatment regimens. Determining the individual risk of infection related to any novel agent is challenging in this setting. Invasive fungal infections (IFIs) represent one of the most morbid infectious complications observed in hematological malignancy. In recent years, growing evidence suggests that certain small molecule inhibitors, such as BTK inhibitors and PI3K inhibitors, may cause an increased risk of IFI in certain patients. It is imperative to better understand the impact that novel targeted therapies might have on the development of IFIs in this high-risk patient population.

Vaccine serologic responses among transplant patients associate with COVID-19 infection and T peripheral helper cells.

BACKGROUND: Therapeutically immunosuppressed transplant recipients exhibit attenuated responses to COVID-19 vaccines. To better understand the immune alterations that determined poor vaccine response, we correlated quantities of circulating T and B cell subsets at baseline with longitudinal serologic responses to SARS-CoV-2 mRNA vaccination in heart and lung transplant recipients. METHODS: Samples at baseline and at approximately 8 and 30 days after each vaccine dose for 22 heart and lung transplant recipients with no history of COVID-19, four heart and lung transplant recipients with prior COVID-19 infection, and 12 healthy controls undergoing vaccination were analyzed. Anti-spike protein receptor binding domain (RBD) IgG and pseudovirus neutralization activity were measured. Proportions of B and T cell subsets at baseline were comprehensively quantitated. RESULTS: At 8-30 days post vaccination, healthy controls displayed robust anti-RBD IgG responses, whereas heart and lung transplant recipients showed minimally increased responses. A parallel absence of activity was observed in pseudovirus neutralization. In contrast, three of four (75%) transplant recipients with prior COVID-19 infection displayed robust responses at levels comparable to controls. Baseline levels of activated PD-1 + HLA-DR + CXCR5 - CD4 + T cells (also known as T peripheral helper [T PH ] cells) and CD4+ T cells strongly predicted the ability to mount a response. CONCLUSIONS: Immunosuppressed patients have defective vaccine responses but can be induced to generate neutralizing antibodies after SARS-CoV-2 infection. Strong correlations of vaccine responsiveness with baseline T PH and CD4 + T cell numbers highlights a role for T helper activity in B cell differentiation into antibody secreting cells during vaccine response.

Breath-Based Diagnosis of Infectious Diseases: A Review of the Current Landscape.

Various analytical methods can be applied to concentrate, separate, and examine trace volatile organic metabolites in the breath, with the potential for noninvasive, rapid, real-time identification of various disease processes, including an array of microbial infections. Although biomarker discovery and validation in microbial infections can be technically challenging, it is an approach that has shown great promise, especially for infections that are particularly difficult to identify with standard culture and molecular amplification-based approaches. This article discusses the current state of breath analysis for the diagnosis of infectious diseases.

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.

The Evolving Landscape of Fungal Diagnostics, Current and Emerging Microbiological Approaches.

Invasive fungal infections are increasingly recognized in immunocompromised hosts. Current diagnostic techniques are limited by low sensitivity and prolonged turnaround times. We review emerging diagnostic technologies and platforms for diagnosing the clinically invasive disease caused by Candida, Aspergillus, and Mucorales.

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.

Omadacycline for the Treatment of Mycobacterium abscessus Disease: A Case Series.

BACKGROUND: Omadacycline is an aminomethylcycline antimicrobial approved by the US Food and Drug Administration in 2018 for community-acquired bacterial pneumonia and acute bacterial skin and skin structure infections. It has in vitro activity against nontuberculous mycobacteria, including Mycobacterium abscessus complex, but clinical data for this indication are lacking. METHODS: Omadacycline use was reviewed at an 804-bed academic medical center. Patients were included if they received omadacycline for culture-proven M abscessus disease in 2019. RESULTS: Four patients received omadacycline for the treatment of culture-positive M abscessus disease in 2019. Two patients had cutaneous disease, 1 had pulmonary disease, and 1 had osteomyelitis and bacteremia. The patients received omadacycline for a median duration of 166 days (range, 104-227) along with a combination of other antimicrobial agents. Omadacycline-containing regimens were associated with a clinical cure in 3 of 4 patients, with 1 patient improving on ongoing treatment. Omadacycline's tolerability was acceptable for patients with M abscessus disease, with 1 patient discontinuing therapy in month 6 due to nausea. CONCLUSIONS: Omadacycline is a novel oral option for the treatment of M abscessus disease, for which safe and effective options are needed. Although this case series is promising, further data are required to determine omadacycline's definitive role in the treatment of M abscessus disease.