Asymmetric unwrapping of nucleosomes under tension directed by DNA local flexibility.

Dynamics of the nucleosome and exposure of nucleosomal DNA play key roles in many nuclear processes, but local dynamics of the nucleosome and its modulation by DNA sequence are poorly understood. Using single-molecule assays, we observed that the nucleosome can unwrap asymmetrically and directionally under force. The relative DNA flexibility of the inner quarters of nucleosomal DNA controls the unwrapping direction such that the nucleosome unwraps from the stiffer side. If the DNA flexibility is similar on two sides, it stochastically unwraps from either side. The two ends of the nucleosome are orchestrated such that the opening of one end helps to stabilize the other end, providing a mechanism to amplify even small differences in flexibility to a large asymmetry in nucleosome stability. Our discovery of DNA flexibility as a critical factor for nucleosome dynamics and mechanical stability suggests a novel mechanism of gene regulation by DNA sequence and modifications.

Measuring DNA mechanics on the genome scale.

Mechanical deformations of DNA such as bending are ubiquitous and have been implicated in diverse cellular functions1. However, the lack of high-throughput tools to measure the mechanical properties of DNA has limited our understanding of how DNA mechanics influence chromatin transactions across the genome. Here we develop 'loop-seq'-a high-throughput assay to measure the propensity for DNA looping-and determine the intrinsic cyclizabilities of 270,806 50-base-pair DNA fragments that span Saccharomyces cerevisiae chromosome V, other genomic regions, and random sequences. We found sequence-encoded regions of unusually low bendability within nucleosome-depleted regions upstream of transcription start sites (TSSs). Low bendability of linker DNA inhibits nucleosome sliding into the linker by the chromatin remodeller INO80, which explains how INO80 can define nucleosome-depleted regions in the absence of other factors2. Chromosome-wide, nucleosomes were characterized by high DNA bendability near dyads and low bendability near linkers. This contrast increases for deeper gene-body nucleosomes but disappears after random substitution of synonymous codons, which suggests that the evolution of codon choice has been influenced by DNA mechanics around gene-body nucleosomes. Furthermore, we show that local DNA mechanics affect transcription through TSS-proximal nucleosomes. Overall, this genome-scale map of DNA mechanics indicates a 'mechanical code' with broad functional implications.

Patients taking benralizumab, dupilumab, or mepolizumab have lower postvaccination SARS-CoV-2 immunity.

BACKGROUND: Biologic therapies inhibiting the IL-4 or IL-5 pathways are very effective in the treatment of asthma and other related conditions. However, the cytokines IL-4 and IL-5 also play a role in the generation of adaptive immune responses. Although these biologics do not cause overt immunosuppression, their effect in primary severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunization has not been studied completely. OBJECTIVE: Our aim was to evaluate the antibody and cellular immunity after SARS-CoV-2 mRNA vaccination in patients on biologics (PoBs). METHODS: Patients with severe asthma or atopic dermatitis who were taking benralizumab, dupilumab, or mepolizumab and had received the initial dose of the 2-dose adult SARS-CoV-2 mRNA vaccine were enrolled in a prospective, observational study. As our control group, we used a cohort of immunologically healthy subjects (with no significant immunosuppression) who were not taking biologics (NBs). We used a multiplexed immunoassay to measure antibody levels, neutralization assays to assess antibody function, and flow cytometry to quantitate Spike-specific lymphocytes. RESULTS: We analyzed blood from 57 patients in the PoB group and 46 control subjects from the NB group. The patients in the PoB group had lower levels of SARS-CoV-2 antibodies, pseudovirus neutralization, live virus neutralization, and frequencies of Spike-specific B and CD8 T cells at 6 months after vaccination. In subgroup analyses, patients with asthma who were taking biologics had significantly lower pseudovirus neutralization than did subjects with asthma who were not taking biologics. CONCLUSION: The patients in the PoB group had reduced SARS-CoV-2-specific antibody titers, neutralizing activity, and virus-specific B- and CD8 T-cell counts. These results have implications when considering development of a more individualized immunization strategy in patients who receive biologic medications blocking IL-4 or IL-5 pathways.

Impact of an Emergency Department Saturation Tool on a Pediatric Hospital's Capacity Management Policy.

OBJECTIVE: We sought to describe how the Emergency Department Work Index (EDWIN) saturation tool (1) correlates with PED overcrowding during a capacity management activation policy, known internally as Purple Alert and (2) compare overall hospital-wide capacity metrics on days in which the alert was instituted versus days it was not. METHODS: This study was conducted between January 1, 2017 and December 31, 2019 in a 30-bed academic quaternary care, urban PED within a university hospital. The EDWIN tool was implemented in January 2019 and objectively measured the busyness of the PED. To determine correlation with overcrowding, EDWIN scores were calculated at alert initiation. Mean alert hours per month were plotted on a control chart before and after EDWIN implementation. We also compared daily numbers of PED visits, inpatient admissions, and patients left without being seen (LWBS) for days with and without alert initiation to assess whether or not Purple Alert correlated with high PED usage. RESULTS: During the study period, the alert was activated a total of 146 times; 43 times after EDWIN implementation. Mean EDWIN score was 2.5 (SD 0.5, min 1.5, max 3.8) at alert initiation. There were no alert occurrences for EDWIN scores less than 1.5 (not overcrowded). There was no statistically significant difference for mean alert hours per month before and after EDWIN was instituted (21.4 vs 20.2, P = 0.08). Mean numbers of PED visits, inpatient admissions, and patients left without being seen were higher on days with alert activation ( P < 0.001 for all). CONCLUSIONS: The EDWIN score correlated with PED busyness and overcrowding during alert activation and correlated with high PED usage. Future studies could include implementing a real-time Web-based EDWIN score as a prediction tool to prevent overcrowding and verifying EDWIN generalizability at other PED sites.

Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy.

G-quadruplexes (GQs) can adopt diverse structures and are functionally implicated in transcription, replication, translation, and maintenance of telomere. Their conformational diversity under physiological levels of mechanical stress, however, is poorly understood. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to measure human telomeric sequences under tension. Abrupt GQ unfolding with K+ in solution occurred at as many as four discrete levels of force. Added to an ultrastable state and a gradually unfolding state, there were six mechanically distinct structures. Extreme mechanical diversity was also observed with Na+, although GQs were mechanically weaker. Our ability to detect small conformational changes at low forces enabled the determination of refolding forces of about 2 pN. Refolding was rapid and stochastically redistributed molecules to mechanically distinct states. A single guanine-to-thymine substitution mutant required much higher ion concentrations to display GQ-like unfolding and refolded via intermediates, contrary to the wild type. Contradicting an earlier proposal, truncation to three hexanucleotide repeats resulted in a single-stranded DNA-like mechanical behavior under all conditions, indicating that at least four repeats are required to form mechanically stable structures.

Virtual Interviewing for Pediatric Emergency Medicine Fellowship-A National Survey.

OBJECTIVE: This study aimed to evaluate both applicant and interviewer satisfaction with the virtual interviewing process for pediatric emergency medicine (PEM) fellowship in hopes to improve the fellowship interviewing process. It was proposed that fellowship programs and applicants would prefer virtual interviews over traditional interviews. METHODS: A survey developed in collaboration with UT Southwestern PEM fellowship leaders and national PEM leaders was sent to all PEM fellowship applicants and programs at the conclusion of the 2020 interview season and rank list submission. The applicant survey obtained information on ease of virtual interviews and whether applicants felt that they obtained adequate information from virtual interviews to make informed program selections. Program director surveys collected data on thoughts and feelings about virtual interviews and obstacles encountered during the recruitment season. Both surveys asked about costs for interviews and interview type preference. RESULTS: A response rate of 49% from applicants and 47% from programs was obtained. Virtual interview days were similar in the amount of time and staff hours used compared with traditional days. Applicants spent less on virtual interviews compared with those who underwent traditional interviews (average $725 vs $4312). Programs received more applications than the prior year and spent less money during the virtual cycle. The majority of the applicants (90%) were comfortable with the virtual interview platform, and most (66%) agreed that virtual interviews provided adequate information to determine program rank. Geography was the number 1 rank determining factor. Programs and applicants preferred a form of in-person interviews. CONCLUSIONS: Virtual interviews provide cost savings for both applicants and programs. Despite this, both parties prefer a form of in-person interviews.

Screening Wetland and Forage Plants for Phytoremediation of Salt-Affected Soils in the Vietnamese Mekong Delta.

This study evaluated the salt tolerance and sodium (Na) bioaccumulation of Typha orientalis, Lepironia articulata, Eleocharis dulcis, Scirpus littoralis, Brachiaria mutica, Paspalum atratum and Setaria sphacelata at five salinity levels of 0, 5, 10, 15 and 20‰ (corresponding to 0, 2.4, 6.9, 12.6 and 18 g NaCl L-1). S. littoralis showed zero-reduction in total dry biomass and was classified as a salt tolerant plant based on the membership function value. The highest Na+ accumulation was observed in S. sphacelata (307.9 mg plant-1) in spite of its salt sensitivity, followed by S. littoralis and T. orientalis at concentration of 155 mg plant-1. Consequently, the Na+ phytoextraction potential of these species can be estimated as 46.2, 23.3 and 23.3 kg ha-1 over 49 days, respectively. Taken together, they show high potential as Na+ hyperaccumulators, and can be selected in the national reclamation program for salt-affected soils in the context adaptation to climate change.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Multiomics modeling of the immunome, transcriptome, microbiome, proteome and metabolome adaptations during human pregnancy.

Motivation: Multiple biological clocks govern a healthy pregnancy. These biological mechanisms produce immunologic, metabolomic, proteomic, genomic and microbiomic adaptations during the course of pregnancy. Modeling the chronology of these adaptations during full-term pregnancy provides the frameworks for future studies examining deviations implicated in pregnancy-related pathologies including preterm birth and preeclampsia. Results: We performed a multiomics analysis of 51 samples from 17 pregnant women, delivering at term. The datasets included measurements from the immunome, transcriptome, microbiome, proteome and metabolome of samples obtained simultaneously from the same patients. Multivariate predictive modeling using the Elastic Net (EN) algorithm was used to measure the ability of each dataset to predict gestational age. Using stacked generalization, these datasets were combined into a single model. This model not only significantly increased predictive power by combining all datasets, but also revealed novel interactions between different biological modalities. Future work includes expansion of the cohort to preterm-enriched populations and in vivo analysis of immune-modulating interventions based on the mechanisms identified. Availability and implementation: Datasets and scripts for reproduction of results are available through: https://nalab.stanford.edu/multiomics-pregnancy/. Supplementary information: Supplementary data are available at Bioinformatics online.

Getah virus epizootic among wild boars in Japan around 2012.

In 2014, an outbreak of Getah virus (GETV) infection occurred in Japan in a horse population that was inoculated with a vaccine against GETV. In this study, we investigated the seroprevalence of GETV infection among wild boars in Japan. Interestingly, the highest rate of anti-GETV-positive wild boars was observed in 2013, which gradually decreased during 2014-2016. The results suggested that GETV spread among wild boars around 2012, resulting in the 2014 outbreak.

Quantitative analysis of the dose-response of white spot syndrome virus in shrimp.

White spot syndrome virus (WSSV) is an important cause of mortality and economic losses in shrimp farming. Although WSSV-induced mortality is virus dose dependent and WSSV infection does not necessarily lead to mortality, the relationships between virus-particle dose, infection and mortality have not been analysed quantitatively. Here, we explored WSSV dose-response by a combination of experiments, modelling and meta-analysis. We performed dose-response experiments in Penaeus vannamei postlarvae, recorded host mortality and detected WSSV infection. When we fitted infection models to these data, two models-differing in whether they incorporated heterogeneous host susceptibility to the virus or not-were supported for two independent experiments. To determine the generality of these results, we reanalysed published data sets and then performed a meta-analysis. We found that WSSV dose-response kinetics is indeed variable over experiments. We could not clearly identify which specific infection model has the most support by meta-analysis, but we argue that these results also are most concordant with a model incorporating varying levels of heterogeneous host susceptibility to WSSV. We have identified suitable models for analysing WSSV dose-response, which can elucidate the most basic virus-host interactions and help to avoid underestimating WSSV infection at low virus doses.

Simultaneously Monitoring Immune Response and Microbial Infections during Pregnancy through Plasma cfRNA Sequencing.

BACKGROUND: Plasma cell-free RNA (cfRNA) encompasses a broad spectrum of RNA species that can be derived from both human cells and microbes. Because cfRNA is fragmented and of low concentration, it has been challenging to profile its transcriptome using standard RNA-seq methods. METHODS: We assessed several recently developed RNA-seq methods on cfRNA samples. We then analyzed the dynamic changes of both the human transcriptome and the microbiome of plasma during pregnancy from 60 women. RESULTS: cfRNA reflects a well-orchestrated immune modulation during pregnancy: an up-regulation of antiinflammatory genes and an increased abundance of antimicrobial genes. We observed that the plasma microbiome remained relatively stable during pregnancy. The bacteria Ureaplasma shows an increased prevalence and increased abundance at postpartum, which is likely to be associated with postpartum infection. We demonstrated that cfRNA-seq can be used to monitor viral infections. We detected a number of human pathogens in our patients, including an undiagnosed patient with a high load of human parvovirus B19 virus (B19V), which is known to be a potential cause of complications in pregnancy. CONCLUSIONS: Plasma cfRNA-seq demonstrates the potential to simultaneously monitor immune response and microbial infections during pregnancy.

Influence of the Gastrointestinal Environment on the Bioavailability of Ethinyl Estradiol Sorbed to Single-Walled Carbon Nanotubes.

Recent evidence suggests that, because of their sorptive nature, if single-walled carbon nanotubes (SWCNTs) make their way into aquatic environments, they may reduce the toxicity of other waterborne contaminants. However, few studies have examined whether contaminants remain adsorbed following ingestion by aquatic organisms. The objective of this study was to examine the bioavailability and bioactivity of ethinyl estradiol (EE2) sorbed onto SWCNTs in a fish gastrointestinal (GI) tract. Sorption experiments indicated that SWCNTs effectively adsorbed EE2, but the chemical was still able to bind and activate soluble estrogen receptors (ERs) in vitro. However, centrifugation to remove SWCNTs and adsorbed EE2 significantly reduced ER activity compared to that of EE2 alone. Additionally, the presence of SWCNTs did not reduce the extent of EE2-driven induction of vitellogenin 1 in vivo compared to the levels in organisms exposed to EE2 alone. These results suggest that while SWCNTs adsorb EE2 from aqueous solutions, under biological conditions EE2 can desorb and retain bioactivity. Additional results indicate that interactions with gastrointestinal proteins may decrease the level of adsorption of estrogen to SWCNTs by 5%. This study presents valuable data for elucidating how SWCNTs interact with chemicals that are already present in our aquatic environments, which is essential for determining their potential health risk.

Nucleosomes undergo slow spontaneous gaping.

In eukaryotes, DNA is packaged into a basic unit, the nucleosome which consists of 147 bp of DNA wrapped around a histone octamer composed of two copies each of the histones H2A, H2B, H3 and H4. Nucleosome structures are diverse not only by histone variants, histone modifications, histone composition but also through accommodating different conformational states such as DNA breathing and dimer splitting. Variation in nucleosome structures allows it to perform a variety of cellular functions. Here, we identified a novel spontaneous conformational switching of nucleosomes under physiological conditions using single-molecule FRET. Using FRET probes placed at various positions on the nucleosomal DNA to monitor conformation of the nucleosome over a long period of time (30-60 min) at various ionic conditions, we identified conformational changes we refer to as nucleosome gaping. Gaping transitions are distinct from nucleosome breathing, sliding or tightening. Gaping modes switch along the direction normal to the DNA plane through about 5-10 angstroms and at minutes (1-10 min) time scale. This conformational transition, which has not been observed previously, may be potentially important for enzymatic reactions/transactions on nucleosomal substrate and the formation of multiple compression forms of chromatin fibers.

Acute-Onset Chest Pain in a 17-Year-Old Female Adolescent With Systemic Lupus Erythematosus.

We report the case of a 17-year-old adolescent girl with systemic lupus erythematosus with disseminated pneumococcal infection leading to purulent pericarditis with cardiac tamponade. Although pericarditis is not an uncommon entity in autoimmune diseases such as systemic lupus erythematosus, purulent pericarditis is a rare cause (<1%) of this presentation.

Mistaken Identity: Asthma and Croup in a Previously Healthy 9-Year-Old Male.

Cough is one of the most common presenting complaints encountered in primary care settings and the emergency department. In 2010, the Centers for Disease Control and Prevention reported approximately 31 million visits to ambulatory care centers for cough, making cough the most frequent presenting complaint in ambulatory visits (2010 National Ambulatory Medical Care Survey). Significant causes of cough can often be overlooked because it is a common symptom of a myriad of pathologies. We report the case of an otherwise healthy 9-year-old male who presented with worsening cough over a month and a half, subsequently noted to have a mediastinal mass, and diagnosed with lymphoma. We discuss the challenges of diagnosing life-threatening pathologies, which present with common symptoms.

Defining Single Molecular Forces Required for Notch Activation Using Nano Yoyo.

Notch signaling, involved in development and tissue homeostasis, is activated at the cell-cell interface through ligand-receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. Here we aimed to determine the single molecular force required for Notch activation by developing a novel low tension gauge tether (LTGT). LTGT utilizes the low unbinding force between single-stranded DNA (ssDNA) and Escherichia coli ssDNA binding protein (SSB) (∼4 pN dissociation force at 500 nm/s pulling rate). The ssDNA wraps around SSB and, upon application of force, unspools from SSB, much like the unspooling of a yoyo. One end of this nano yoyo is attached to the surface though SSB, while the other end presents a ligand. A Notch receptor, upon binding to its ligand, is believed to undergo force-induced conformational changes required for activating downstream signaling. If the required force for such activation is larger than 4 pN, ssDNA will unspool from SSB, and downstream signaling will not be activated. Using these LTGTs, in combination with the previously reported TGTs that rupture double-stranded DNA at defined forces, we demonstrate that Notch activation requires forces between 4 and 12 pN, assuming an in vivo loading rate of 60 pN/s. Taken together, our study provides a direct link between single-molecular forces and Notch activation.

Primary myelofibrosis associated glomerulopathy: significant improvement after therapy with ruxolitinib.

BACKGROUND: Primary myelofibrosis (PMF) is a type of myeloproliferative neoplasm (MPN) characterized by the predominant proliferation of megakaryocytes and granulocytes in the bone marrow, leading to the deposition of fibrous tissue, and by a propensity toward extramedullary hematopoiesis. Renal involvement in PMF is rare, but kidney tissue samples from these patients reveal MPN-related glomerulopathy, a recently discovered condition, in the late stages of the disease. CASE PRESENTATION: We present the first case described in the medical literature of a patient with early renal glomerular involvement in PMF/MPN. A 60-year-old man with stage 4 chronic kidney disease and a recent diagnosis of PMF (within 4 weeks of presentation at our renal division) presented with generalized body swelling, acute kidney injury, and massive nephrotic-range proteinuria. Kidney biopsy was performed to determine the etiology of the patient's renal dysfunction and revealed early renal glomerular involvement that was histologically characteristic of MPN-related glomerulopathy. Early diagnosis and prompt medical management returned the patient's kidney functionality to the levels seen on initial presentation at our hospital. CONCLUSION: Large studies with long follow-up durations are necessary to identify and categorize the risk factors for the development of MPN-related glomerulopathy, to standardize therapeutic regimens, and to determine whether aggressive management of the myelofibrosis slows the progression of kidney disease.