Small-Molecule-Based Fluorescent Sensors for Selective Detection of Reactive Oxygen Species in Biological Systems.

Reactive oxygen species (ROS) encompass a collection of intricately linked chemical entities characterized by individually distinct physicochemical properties and biological reactivities. Although excessive ROS generation is well known to underpin disease development, it has become increasingly evident that ROS also play central roles in redox regulation and normal physiology. A major challenge in uncovering the relevant biological mechanisms and deconvoluting the apparently paradoxical roles of distinct ROS in human health and disease lies in the selective and sensitive detection of these transient species in the complex biological milieu. Small-molecule-based fluorescent sensors enable molecular imaging of ROS with great spatial and temporal resolution and have thus been appreciated as excellent tools for aiding discoveries in modern redox biology. We review a selection of state-of-the-art sensors with demonstrated utility in biological systems. By providing a systematic overview based on underlying chemical sensing mechanisms, we wish to highlight the strengths and weaknesses in prior sensor works and propose some guiding principles for the development of future probes.

Reversible, tunable epigenetic silencing of TCF1 generates flexibility in the T cell memory decision.

The immune system encodes information about the severity of a pathogenic threat in the quantity and type of memory cells it forms. This encoding emerges from lymphocyte decisions to maintain or lose self-renewal and memory potential during a challenge. By tracking CD8+ T cells at the single-cell and clonal lineage level using time-resolved transcriptomics, quantitative live imaging, and an acute infection model, we find that T cells will maintain or lose memory potential early after antigen recognition. However, following pathogen clearance, T cells may regain memory potential if initially lost. Mechanistically, this flexibility is implemented by a stochastic cis-epigenetic switch that tunably and reversibly silences the memory regulator, TCF1, in response to stimulation. Mathematical modeling shows how this flexibility allows memory T cell numbers to scale robustly with pathogen virulence and immune response magnitudes. We propose that flexibility and stochasticity in cellular decisions ensure optimal immune responses against diverse threats.

Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment.

During T cell development, multipotent progenitors relinquish competence for other fates and commit to the T cell lineage by turning on Bcl11b, which encodes a transcription factor. To clarify lineage commitment mechanisms, we followed developing T cells at the single-cell level using Bcl11b knock-in fluorescent reporter mice. Notch signaling and Notch-activated transcription factors collaborate to activate Bcl11b expression irrespectively of Notch-dependent proliferation. These inputs work via three distinct, asynchronous mechanisms: an early locus 'poising' function dependent on TCF-1 and GATA-3, a stochastic-permissivity function dependent on Notch signaling, and a separate amplitude-control function dependent on Runx1, a factor already present in multipotent progenitors. Despite their necessity for Bcl11b expression, these inputs act in a stage-specific manner, providing a multitiered mechanism for developmental gene regulation.

Pancreatic Enzyme Use Reduces Pancreatitis Frequency in Children With Acute Recurrent or Chronic Pancreatitis: A Report From INSPPIRE.

INTRODUCTION: Among children who suffer from acute recurrent pancreatitis (ARP) or chronic pancreatitis (CP), acute pancreatitis (AP) episodes are painful, often require hospitalization, and contribute to disease complications and progression. Despite this recognition, there are currently no interventions to prevent AP episodes. In this retrospective cohort study, we assessed the impact of pancreatic enzyme therapy (PERT) use on clinical outcomes among children with pancreatic-sufficient ARP or CP. METHODS: Children with pancreatic-sufficient ARP or CP in the INSPPIRE-2 cohort were included. Clinical outcomes were compared for those receiving vs not receiving PERT, as well as frequency of AP before and after PERT. Logistic regression was used to study the association between development of AP episodes after starting PERT and response predictors. RESULTS: Among 356 pancreatic-sufficient participants, 270 (76%) had ARP, and 60 (17%) received PERT. Among those on PERT, 42% did not have a subsequent AP episode, during a mean 2.1 years of follow-up. Children with a SPINK1 mutation ( P = 0.005) and those with ARP (compared with CP, P = 0.008) were less likely to have an AP episode after starting PERT. After initiation of PERT, the mean AP annual incidence rate decreased from 3.14 down to 0.71 ( P < 0.001). DISCUSSION: In a retrospective analysis, use of PERT was associated with a reduction in the incidence rate of AP among children with pancreatic-sufficient ARP or CP. These results support the need for a clinical trial to evaluate the efficacy of PERT to improve clinical outcomes among children with ARP or CP.

Liver transplant for primary biliary tract neuroendocrine tumor in a nine-year-old girl.

BACKGROUND: Neuroendocrine tumors (NETs) are rare epithelial neoplasms that arise most commonly from the gastrointestinal tract. In pediatrics, the most common site of origin is in the appendix, with the liver being the most common site of metastasis. Neuroendocrine tumors arising from the biliary tract are extremely rare. METHODS: We describe a case of a nine-year-old girl who presented with obstructive cholestasis and was found to have multiple liver masses identified on biopsy as well-differentiated neuroendocrine tumor with an unknown primary tumor site. RESULT: The patient underwent extensive investigation to identify a primary tumor site, including endoscopy, endoscopic ultrasound, and capsule endoscopy. The patient ultimately underwent definitive management with liver transplant, and on explant was discovered to have multiple well-differentiated neuroendocrine tumors, WHO Grade 1, with extensive infiltration into the submucosa of bile duct, consistent with primary biliary tract neuroendocrine tumor. CONCLUSION: Identifying the site of the primary tumor in NETs found within the liver can be challenging. To determine if an extrahepatic primary tumor exists, workup should include endoscopy, EUS, and capsule endoscopy. Children with well-differentiated hepatic NETs, with no identifiable primary tumor, and an unresectable tumor, are considered favorable candidates for liver transplantation.

Chromatin accessibility and gene expression during adipocyte differentiation identify context-dependent effects at cardiometabolic GWAS loci.

Chromatin accessibility and gene expression in relevant cell contexts can guide identification of regulatory elements and mechanisms at genome-wide association study (GWAS) loci. To identify regulatory elements that display differential activity across adipocyte differentiation, we performed ATAC-seq and RNA-seq in a human cell model of preadipocytes and adipocytes at days 4 and 14 of differentiation. For comparison, we created a consensus map of ATAC-seq peaks in 11 human subcutaneous adipose tissue samples. We identified 58,387 context-dependent chromatin accessibility peaks and 3,090 context-dependent genes between all timepoint comparisons (log2 fold change>1, FDR<5%) with 15,919 adipocyte- and 18,244 preadipocyte-dependent peaks. Adipocyte-dependent peaks showed increased overlap (60.1%) with Roadmap Epigenomics adipocyte nuclei enhancers compared to preadipocyte-dependent peaks (11.5%). We linked context-dependent peaks to genes based on adipocyte promoter capture Hi-C data, overlap with adipose eQTL variants, and context-dependent gene expression. Of 16,167 context-dependent peaks linked to a gene, 5,145 were linked by two or more strategies to 1,670 genes. Among GWAS loci for cardiometabolic traits, adipocyte-dependent peaks, but not preadipocyte-dependent peaks, showed significant enrichment (LD score regression P<0.005) for waist-to-hip ratio and modest enrichment (P < 0.05) for HDL-cholesterol. We identified 659 peaks linked to 503 genes by two or more approaches and overlapping a GWAS signal, suggesting a regulatory mechanism at these loci. To identify variants that may alter chromatin accessibility between timepoints, we identified 582 variants in 454 context-dependent peaks that demonstrated allelic imbalance in accessibility (FDR<5%), of which 55 peaks also overlapped GWAS variants. At one GWAS locus for palmitoleic acid, rs603424 was located in an adipocyte-dependent peak linked to SCD and exhibited allelic differences in transcriptional activity in adipocytes (P = 0.003) but not preadipocytes (P = 0.09). These results demonstrate that context-dependent peaks and genes can guide discovery of regulatory variants at GWAS loci and aid identification of regulatory mechanisms.

Involvement of the Streptococcus mutans PgfE and GalE 4-epimerases in protein glycosylation, carbon metabolism, and cell division.

Streptococcus mutans is a key pathogen associated with dental caries and is often implicated in infective endocarditis. This organism forms robust biofilms on tooth surfaces and can use collagen-binding proteins (CBPs) to efficiently colonize collagenous substrates, including dentin and heart valves. One of the best characterized CBPs of S. mutans is Cnm, which contributes to adhesion and invasion of oral epithelial and heart endothelial cells. These virulence properties were subsequently linked to post-translational modification (PTM) of the Cnm threonine-rich repeat region by the Pgf glycosylation machinery, which consists of 4 enzymes: PgfS, PgfM1, PgfE, and PgfM2. Inactivation of the S. mutans pgf genes leads to decreased collagen binding, reduced invasion of human coronary artery endothelial cells, and attenuated virulence in the Galleria mellonella invertebrate model. The present study aimed to better understand Cnm glycosylation and characterize the predicted 4-epimerase, PgfE. Using a truncated Cnm variant containing only 2 threonine-rich repeats, mass spectrometric analysis revealed extensive glycosylation with HexNAc2. Compositional analysis, complemented with lectin blotting, identified the HexNAc2 moieties as GlcNAc and GalNAc. Comparison of PgfE with the other S. mutans 4-epimerase GalE through structural modeling, nuclear magnetic resonance, and capillary electrophoresis demonstrated that GalE is a UDP-Glc-4-epimerase, while PgfE is a GlcNAc-4-epimerase. While PgfE exclusively participates in protein O-glycosylation, we found that GalE affects galactose metabolism and cell division. This study further emphasizes the importance of O-linked protein glycosylation and carbohydrate metabolism in S. mutans and identifies the PTM modifications of the key CBP, Cnm.

Esophageal Dilation in Children: A State of the Art Review.

Esophageal dilations in children are performed by several pediatric and adult professionals. We aim to summarize improvements in safety and new technology used for the treatment of complex and refractory strictures, including triamcinolone injection, endoscopic electro-incisional therapy, topical mitomycin-C application, stent placement, functional lumen imaging probe assisted dilation, and endoscopic vacuum-assisted closure in the pediatric population.

Esophageal dilation with EsoFLIP is faster than CRE balloon dilation combined with EndoFLIP in children.

BACKGROUND: Controlled radial expansion (CRE) balloon dilators are traditionally used to dilate esophageal strictures during an esophagogastroduodenoscopy (EGD). EndoFLIP is a diagnostic tool used during an EGD to measure important parameters of the gastrointestinal lumen, capable of assessing treatment before and after dilation. EsoFLIP is a related device that combines a balloon dilator with high-resolution impedance planimetry to provide some of the luminal parameters in real time during dilation. We sought to compare procedure time, fluoroscopy time, and safety profile of esophageal dilation using either CRE balloon dilation combined with EndoFLIP (E + CRE) versus EsoFLIP alone. METHODS: A single-center retrospective review was performed to identify patients ≤ 21 years of age who underwent an EGD with biopsy and esophageal stricture dilation using E + CRE or EsoFLIP between October 2017 and May 2022. RESULTS: Twenty-nine EGDs with esophageal stricture dilation were performed in 23 patients (19 E + CRE and 10 EsoFLIP). The two groups did not differ in age, gender, race, chief complaint, type of esophageal stricture, or history of prior gastrointestinal procedures (all p > 0.05). The most common medical history in the E + CRE and EsoFLIP groups were eosinophilic esophagitis and epidermolysis bullosa, respectively. Median procedures times were shorter in the EsoFLIP cohort compared to E + CRE balloon dilation (40.5 min [IQR 23-57 min] for the EsoFLIP group; 64 min [IQR 51-77 min] for the E + CRE group; p < 0.01). Median fluoroscopy times were also shorter for patients who underwent EsoFLIP (0.16 min [IQR 0-0.30 min] for EsoFLIP dilation; 0.30 min [IQR 0.23-0.55] for the E + CRE group; p = 0.003). There were no complications or unplanned hospitalizations in either group. CONCLUSION: EsoFLIP dilation of esophageal strictures was faster and required less fluoroscopy than CRE balloon dilation combined with EndoFLIP in children, while being equally as safe. Prospective studies are needed to further compare the two modalities.

Multiplexed single-cell profiling of chromatin states at genomic loci by expansion microscopy.

Proper regulation of genome architecture and activity is essential for the development and function of multicellular organisms. Histone modifications, acting in combination, specify these activity states at individual genomic loci. However, the methods used to study these modifications often require either a large number of cells or are limited to targeting one histone mark at a time. Here, we developed a new method called Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE) that uses Expansion Microscopy (ExM) to visualize and quantify multiple histone modifications at non-repetitive genomic regions in single cells at a spatial resolution of ∼75 nm. Using SCEPTRE, we distinguished multiple histone modifications at a single housekeeping gene, quantified histone modification levels at multiple developmentally-regulated genes in individual cells, and evaluated the relationship between histone modifications and RNA polymerase II loading at individual loci. We find extensive variability in epigenetic states between individual gene loci hidden from current population-averaged measurements. These findings establish SCEPTRE as a new technique for multiplexed detection of combinatorial chromatin states at single genomic loci in single cells.

Factors That Impact the Timing and Removal of Gastrostomy Placement/Nissen Fundoplication in Children with Bronchopulmonary Dysplasia.

OBJECTIVE: The study aimed to identify factors that impact timing of gastrostomy placement/removal and Nissen fundoplication (NF) in infants with bronchopulmonary dysplasia (BPD). STUDY DESIGN: Clinical data were reviewed retrospectively from patients recruited from the Johns Hopkins Bronchopulmonary Dysplasia Clinic (January 1, 2014-December 31, 2018). RESULTS: Patients with gastrostomy tubes (GTs) placed in the neonatal intensive care unit (NICU) were older at discharge (p < 0.001) and less likely to have abnormal upper gastrointestinal series findings (p = 0.005) than those with GTs placed after NICU discharge. Patients with NF had lower mean gestational ages (p = 0.011), longer NICU stays (p = 0.019), more frequent home ventilation requirements (p = 0.005), and greater likelihood of pulmonary hypertension (p = 0.032) compared with those without. Median age of GT removal was 61.6 months. Patients with GTs were weaned from supplemental oxygen and/or home ventilation before GT removal (p < 0.001). CONCLUSION: Patients with GT/NF were more medically complex than those with GT alone. Patients were more likely to be weaned from home respiratory support before GT removal. KEY POINTS: · Patients with GT/NF were more medically complex than those with GT alone.. · Patients were more likely to be weaned from home respiratory support before GT removal.. · Patients with GTs placed in NICU were older at discharge and less likely to have abnormal upper gastrointestinal series result..

Structural studies of codeinone reductase reveal novel insights into aldo-keto reductase function in benzylisoquinoline alkaloid biosynthesis.

Benzylisoquinoline alkaloids (BIAs) are a class of specialized metabolites with a diverse range of chemical structures and physiological effects. Codeine and morphine are two closely related BIAs with particularly useful analgesic properties. The aldo-keto reductase (AKR) codeinone reductase (COR) catalyzes the final and penultimate steps in the biosynthesis of codeine and morphine, respectively, in opium poppy (Papaver somniferum). However, the structural determinants that mediate substrate recognition and catalysis are not well defined. Here, we describe the crystal structure of apo-COR determined to a resolution of 2.4 Å by molecular replacement using chalcone reductase as a search model. Structural comparisons of COR to closely related plant AKRs and more distantly related homologues reveal a novel conformation in the ß1α1 loop adjacent to the BIA-binding pocket. The proximity of this loop to several highly conserved active-site residues and the expected location of the nicotinamide ring of the NADP(H) cofactor suggest a model for BIA recognition that implies roles for several key residues. Using site-directed mutagenesis, we show that substitutions at Met-28 and His-120 of COR lead to changes in AKR activity for the major and minor substrates codeinone and neopinone, respectively. Our findings provide a framework for understanding the molecular basis of substrate recognition in COR and the closely related 1,2-dehydroreticuline reductase responsible for the second half of a stereochemical inversion that initiates the morphine biosynthesis pathway.

Probable Animal-to-Human Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Delta Variant AY.127 Causing a Pet Shop-Related Coronavirus Disease 2019 (COVID-19) Outbreak in Hong Kong.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect human and other mammals, including hamsters. Syrian (Mesocricetus auratus) and dwarf (Phodopus sp.) hamsters are susceptible to SARS-CoV-2 infection in the laboratory setting. However, pet shop-related Coronavirus Disease 2019 (COVID-19) outbreaks have not been reported. METHODS: We conducted an investigation of a pet shop-related COVID-19 outbreak due to Delta variant AY.127 involving at least 3 patients in Hong Kong. We tested samples collected from the patients, environment, and hamsters linked to this outbreak and performed whole genome sequencing analysis of the reverse transcription polymerase chain reaction (RT-PCR)-positive samples. RESULTS: The patients included a pet shop keeper (Patient 1), a female customer of the pet shop (Patient 2), and the husband of Patient 2 (Patient 3). Investigation showed that 17.2% (5/29) and 25.5% (13/51) environmental specimens collected from the pet shop and its related warehouse, respectively, tested positive for SARS-CoV-2 RNA by RT-PCR. Among euthanized hamsters randomly collected from the storehouse, 3% (3/100) tested positive for SARS-CoV-2 RNA by RT-PCR and seropositive for anti-SARS-CoV-2 antibody by enzyme immunoassay. Whole genome analysis showed that although all genomes from the outbreak belonged to the Delta variant AY.127, there were at least 3 nucleotide differences among the genomes from different patients and the hamster cages. Genomic analysis suggests that multiple strains have emerged within the hamster population, and these different strains have likely transmitted to human either via direct contact or via the environment. CONCLUSIONS: Our study demonstrated probable hamster-to-human transmission of SARS-CoV-2. As pet trading is common around the world, this can represent a route of international spread of this pandemic virus.

Polygenic early-onset colorectal cancer in pediatric patients.

Colorectal cancer in the pediatric population is a rare but transpirable phenomenon. The occurrence should prompt suspicion for underlying genetic mutations in the setting of a hereditary cancer predisposition syndrome. In this series, we outline three pediatric patients with colonic adenocarcinoma who were found to have one or more germline mutations. The presence of compound mutations may lead to a hypermutator phenotype resulting in earlier presentation of colorectal cancer in childhood and adolescence. The diagnosis of colorectal cancer in pediatric patients warrants timely recognition, multigene panel testing, genetic counseling for the patient and family, and increased surveillance for intestinal and extra-intestinal tumors.

Endoluminal Functional Lumen Imaging Probe Is Safe in Children Under Five Years Old.

BACKGROUND: Endoluminal functional lumen imaging probe (EndoFLIP) is a minimally invasive, novel device that uses high-resolution impedance planimetry to measure important parameters of the gastrointestinal lumen that aid in the diagnosis of esophageal disorders. EndoFLiP is approved by the US Food and Drug Administration (FDA) for children 5 years and older. We sought to compare its safety and luminal characteristics between children under 5 years of age with children 5 years and older. METHODS: A single-center retrospective review was performed to identify all patients < 21 years of age who underwent esophagogastroduodenoscopy (EGD) with EndoFLIP between October 2017 and November 2020. Results: Sixty-seven EGDs with EndoFLIP were performed in 56 patients, including 14 that were done in children < 5 years and 53 in children ≥5years. The median age in the < 5-year group was 1.7years (interquartile range [IQR], 0.9-4.4) and the youngest patient was 1 month old. The median age in ≥5-year group was 14.3 years (IQR, 8.2-16.2). Median procedure times were similar (32 minutes [IQR, 25-48] for the < 5-year group; 28 minutes [IQR, 20-33] for ≥5-year group; P  = 0.08). There were no adverse events or unplanned hospitalizations in either group. At 30 mL inflation, the pressure of the lower esophageal sphincter (LES) was statistically different between the 2 groups (P = 0.02). CONCLUSION: EndoFLIP appears to be safe for use in our small cohort of children < 5 years of age and may be an important tool in the management of esophageal disorders in this age group. There was no difference in procedure time between our 2 age groups and there were no procedure-related complications.

Photosensitizer to the rescue: in planta and field application of photodynamic inactivation against plant pathogenic bacteria.

Control of plant pathogens using chemical and synthetic pesticides raises a major safety concern for humans and the environment. Despite the ongoing exploration of sustainable alternative methods, management practices for pathogens, especially bacteria, have remained almost unchanged over decades, whereby long-term uses of copper and antibiotics has led to widespread bacterial resistance in the field. Antimicrobial photodynamic inactivation (aPDI) of bacteria is emerging as an alternative strategy to combat resistant plant pathogens. aPDI utilizes light-sensitive molecules (photosensitizers) that upon illumination produce reactive oxygen species able to kill pathogens. Here we explore the potential of an anionic semisynthetic water-soluble derivative of chlorophyl (Sodium Magnesium Chlorophyllin: Mg-chl), as an antibacterial agent in planta, by simulating processes naturally occurring in the field. Mg-chl in combination with Na2EDTA (cell wall permeabilizing agent) was able to effectively inhibit Pseudomonas syringae pv. tomato DC3000 in vitro and in planta in both tomato and N. benthamiana. Notably, Mg-chl in combination with Na2EDTA and the common surfactant Morwet D-400 significantly reduced Xanthomonas hortorum pv. gardneri and Xanthomonas fragarie, respectively, in a commercial greenhouse trial against bacterial spot disease in tomato and in field experiments against angular leaf spot disease in strawberries.

Rapid Broad Spectrum Detection of Carbapenemases with a Dual Fluorogenic-Colorimetric Probe.

Carbapenems stand as one of the last-resort antibiotics; however, their efficacy is threatened by the rising number and rapid spread of carbapenemases. Effective antimicrobial stewardship thus calls for rapid tests for these enzymes to aid appropriate prescription and infection control. Herein, we report the first effective pan-carbapenemase reporter CARBA-H with a broad scope covering all three Ambler classes. Using a chemical biology approach, we demonstrated that the absence of the 1ß-substituent in the carbapenem core is key to pan-carbapenemase recognition, which led to our rational design and probe development. CARBA-H provides a dual colorimetric-fluorogenic response upon carbapenemase-mediated hydrolysis. A clear visual readout can be obtained within 15 min when tested against a panel of carbapenemase-producing Enterobacteriaceae (CPE) clinical isolates that notably includes OXA-48 and OXA-181-producing strains. Furthermore, CARBA-H can be applied to the detection of carbapemenase activity in CPE-spiked urine samples.

Any day, split halfway: Flexibility in scheduling high-dose cisplatin-A large retrospective review from a high-volume cancer center.

High-dose (HD) cisplatin remains the standard of care with chemoradiation for locally advanced oropharyngeal cancer (OPC). Cooperative group trials mandate bolus-HD (100 mg/m2 × 1 day, every 3 weeks) cisplatin administration at the beginning of the week to optimize radiosensitization-a requirement which may be unnecessary. This analysis evaluates the impact of chemotherapy administration day of week (DOW) on outcomes. We also report our institutional experience with an alternate dosing schedule, split-HD (50 mg/m2 × 2 days, every 3 weeks). We retrospectively reviewed 435 definitive chemoradiation OPC patients from 10 December 2001 to 23 December 2014. Those receiving non-HD cisplatin regimens or induction chemotherapy were excluded. Data collected included DOW, dosing schedule (bolus-HD vs split-HD), smoking, total cumulative dose (TCD), stage, Karnofsky Performance Status, human papillomavirus status and creatinine (baseline, peak and posttreatment baseline). Local failure (LF), regional failure (RF), locoregional failure (LRF), distant metastasis (DM), any failure (AF, either LRF or DM) and overall survival (OS) were calculated from radiation therapy start. Median follow-up was 8.0 years (1.8 months-17.0 years). DOW, dosing schedule and TCD were not associated with any outcomes in univariable or multivariable regression models. There was no statistically significant difference in creatinine or association with TCD in split-HD vs bolus-HD. There was no statistically significant association between DOW and outcomes, suggesting that cisplatin could be administered any day. Split-HD had no observed differences in outcomes, renal toxicity or TCD compared to bolus-HD cisplatin. Our data suggest that there is some flexibility of when and how to give HD cisplatin compared to clinical trial mandates.

Hospital medication errors: a cross-sectional study.

BACKGROUND: Medication errors (MEs) are among the most common types of incidents reported in Australian and international hospitals. There is no uniform method of reporting and reducing these errors. This study aims to identify the incidence, time trends, types and factors associated with MEs in a large regional hospital in Australia. METHODS: A 5-year cross-sectional study. RESULTS: The incidence of MEs was 1.05 per 100 admitted patients. The highest frequency of errors was observed during the colder months of May-August. When distributed by day of the week, Mondays and Tuesdays had the highest frequency of errors. When distributed by hour of the day, time intervals from 7 am to 8 am and from 7 pm to 8 pm showed a sharp increase in the frequency of errors. One thousand and eighty-eight (57.8%) MEs belonged to incidence severity rating (ISR) level 4 and 787 (41.8%) belonged to ISR level 3. There were six incidents of ISR level 2 and only one incident of ISR level 1 reported during the five-year period 2014-2018. Administration-only errors were the most common accounting for 1070 (56.8%) followed by prescribing-only errors (433, 23%). High-risk medications were associated with half the number of errors, the most common of which were narcotics (17.9%) and antimicrobials (13.2%). CONCLUSIONS: MEs continue to be a problem faced by international hospitals. Inexperience of health professionals and nurse-patient ratios might be the fundamental challenges to overcome. Specific training of junior staff in prescribing and administering medication and nurse workload management could be possible solutions to reducing MEs in hospitals.

Structure-function studies of tetrahydroprotoberberine N-methyltransferase reveal the molecular basis of stereoselective substrate recognition.

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse class of plant-specialized metabolites that have been particularly well-studied in the order Ranunculales. The N-methyltransferases (NMTs) in BIA biosynthesis can be divided into three groups according to substrate specificity and amino acid sequence. Here, we report the first crystal structures of enzyme complexes from the tetrahydroprotoberberine NMT (TNMT) subclass, specifically for GfTNMT from the yellow horned poppy (Glaucium flavum). GfTNMT was co-crystallized with the cofactor S-adenosyl-l-methionine (dmin = 1.6 Å), the product S-adenosyl-l-homocysteine (dmin = 1.8 Å), or in complex with S-adenosyl-l-homocysteine and (S)-cis-N-methylstylopine (dmin = 1.8 Å). These structures reveal for the first time how a mostly hydrophobic L-shaped substrate recognition pocket selects for the (S)-cis configuration of the two central six-membered rings in protoberberine BIA compounds. Mutagenesis studies confirm and functionally define the roles of several highly-conserved residues within and near the GfTNMT-active site. The substrate specificity of TNMT enzymes appears to arise from the arrangement of subgroup-specific stereospecific recognition elements relative to catalytic elements that are more widely-conserved among all BIA NMTs. The binding mode of protoberberine compounds to GfTNMT appears to be similar to coclaurine NMT, with the isoquinoline rings buried deepest in the binding pocket. This binding mode differs from that of pavine NMT, in which the benzyl ring is bound more deeply than the isoquinoline rings. The insights into substrate recognition and catalysis provided here form a sound basis for the rational engineering of NMT enzymes for chemoenzymatic synthesis and metabolic engineering.