Mad Honey Ingestion Leading to Grayanotoxin Poisoning During the Burning Man Music Festival: A Case Series.

This is a case series of three patients who presented to the medical facilities at Burning Man, an annual week-long gathering in the Black Rock Desert of Nevada, for recreational grayanotoxin ingestion. Grayanotoxin, also known as "mad honey," caused the patients to present with varying degrees of dizziness, nausea, vomiting, and diarrhea based on the quantity ingested. Vital signs showed significant bradycardia and hypotension and were successfully treated with atropine and intravenous fluids. Patients were later discharged after a period of observation and resolution of symptoms.

Assessment of Cardiovascular Disease Among Predominantly Black Gout Patients.

INTRODUCTION: Although the association between gout and cardiovascular disease (CVD) has been extensively studied, scarce data are available for the Black population. We aimed to assess the association between gout and CVD in a predominantly Black urban population with gout. METHODS: A cross-sectional analysis was performed between a gout cohort and an age-/sex-matched control group. Clinical parameters and 2D echocardiograms were reviewed for the patients with gout and heart failure (HF). The primary outcome studied includes the prevalence and strength of association between gout and CVD. Secondary outcomes studied includes strength of association of gout and HF categorized by ejection fraction, mortality, and HF readmissions. RESULTS: Four hundred seventy-one patients with gout had a mean age of 63.7 ± 0.5 years; 89% were Black, 63% were men, and mean body mass index was 31.3 ± 0.4 kg/m 2 . Hypertension, diabetes mellitus, and dyslipidemia were present in 89%, 46%, and 52%, respectively. Compared with controls, patients with gout had significantly higher rates of angina, arrhythmia, coronary artery disease/stents, myocardial infarction, coronary artery bypass graft surgery, cerebrovascular accident, and peripheral vascular disease. The adjusted odds ratio for CVD was 2.9 (95% confidence interval, 1.9-4.5; p < 0.001). Gout patients had a higher prevalence of HF with 45% (n = 212) compared with controls with 9.4% (n = 44). Adjusted odds ratio for HF risk was 7.1 (95% confidence interval, 4.7-10.6; p < 0.01). CONCLUSIONS: Gout in a predominantly Black population confers 3 times the CVD risk and 7 times HF-specific risk compared with age- and sex-matched cohort. Further research is needed to confirm our findings and to develop interventions to reduce morbidity associated with gout.

A general mechanism for transcription bubble nucleation in bacteria.

Bacterial transcription initiation requires σ factors for nucleation of the transcription bubble. The canonical housekeeping σ factor, σ70, nucleates DNA melting via recognition of conserved bases of the promoter -10 motif, which are unstacked and captured in pockets of σ70. By contrast, the mechanism of transcription bubble nucleation and formation during the unrelated σN-mediated transcription initiation is poorly understood. Herein, we combine structural and biochemical approaches to establish that σN, like σ70, captures a flipped, unstacked base in a pocket formed between its N-terminal region I (RI) and extra-long helix features. Strikingly, RI inserts into the nascent bubble to stabilize the nucleated bubble prior to engagement of the obligate ATPase activator. Our data suggest a general paradigm of transcription initiation that requires σ factors to nucleate an early melted intermediate prior to productive RNA synthesis.

Structural origins of Escherichia coli RNA polymerase open promoter complex stability.

The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms "open" complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λPR (t1/2 ∼10 h), T7A1 (t1/2 ∼4 min), and a point mutant in λPR (λPR-5C) (t1/2 ∼2 h). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand "scrunches" inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.

Native Mass Spectrometry-Based Screening for Optimal Sample Preparation in Single-Particle Cryo-EM.

Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well suited to routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.

Stepwise Promoter Melting by Bacterial RNA Polymerase.

Transcription initiation requires formation of the open promoter complex (RPo). To generate RPo, RNA polymerase (RNAP) unwinds the DNA duplex to form the transcription bubble and loads the DNA into the RNAP active site. RPo formation is a multi-step process with transient intermediates of unknown structure. We use single-particle cryoelectron microscopy to visualize seven intermediates containing Escherichia coli RNAP with the transcription factor TraR en route to forming RPo. The structures span the RPo formation pathway from initial recognition of the duplex promoter in a closed complex to the final RPo. The structures and supporting biochemical data define RNAP and promoter DNA conformational changes that delineate steps on the pathway, including previously undetected transient promoter-RNAP interactions that contribute to populating the intermediates but do not occur in RPo. Our work provides a structural basis for understanding RPo formation and its regulation, a major checkpoint in gene expression throughout evolution.

E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation.

TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp, but the structural basis for regulation by these factors remains unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis revealed TraR-induced changes in RNAP conformational heterogeneity. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the ßlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.