Identifying individual functional B cell receptors (BCRs) is common, but two-dimensional analysis of B cell frequency versus BCR potency would delineate both quantity and quality of antigen-specific ...memory B cells. We efficiently determine quantitative BCR neutralizing activities using a single-cell-derived antibody supernatant analysis (SCAN) workflow and develop a frequency-potency algorithm to estimate B cell frequencies at various neutralizing activity or binding affinity cutoffs. In an HIV-1 fusion peptide (FP) immunization study, frequency-potency curves elucidate the quantity and quality of FP-specific immunoglobulin G (IgG)+ memory B cells for different animals, time points, and antibody lineages at single-cell resolution. The BCR neutralizing activities are mainly determined by their affinities to soluble envelope trimer. Frequency analysis definitively demonstrates dominant neutralizing antibody lineages. These findings establish SCAN and frequency-potency analyses as promising approaches for general B cell analysis and monoclonal antibody (mAb) discovery. They also provide specific rationales for HIV-1 FP-directed vaccine optimization.
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•Frequency-potency of antigen-specific memory B cells at single-cell resolution•Affinities to soluble trimer dominate vaccine-induced, FP-directed neutralization•96% Ig sequence recovery definitively demonstrates dominant neutralizing lineages•SCAN efficiently identifies cross-neutralizing monoclonal antibodies
Tenggara et al. use a single-cell-derived antibody supernatant analysis (SCAN) workflow to efficiently generate quantitative BCR neutralizing IC50 data against HIV-1, on top of which a two-dimensional frequency-potency analysis delineates quantity and quality of IgG+ memory B cells simultaneously at single-cell resolution.
The human telomere contains tandem repeat of (TTAGG) capable of forming a higher order DNA structure known as G-quadruplex. Porphyrin molecules such as TMPyP4 bind and stabilize G-quadruplex ...structure.
Isothermal titration calorimetry (ITC), circular dichroism (CD), and mass spectroscopy (ESI/MS), were used to investigate the interactions between TMPyP4 and the Co(III), Ni(II), Cu(II), and Zn(II) complexes of TMPyP4 (e.g. Co(III)-TMPyP4) and a model human telomere G-quadruplex (hTel22) at or near physiologic ionic strength (Na+ or K+≈0.15M).
The apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 all formed complexes having a saturation stoichiometry of 4:1, moles of ligand per mole of DNA. Binding of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 is described by a “four-independent-sites model”. The two highest-affinity sites exhibit a K in the range of 108 to 1010M−1 with the two lower-affinity sites exhibiting a K in the range of 104 to 105M−1. Binding of Co(III)-TMPyP4, and Zn(II)-TMPyP4, is best described by a “two-independent-sites model” in which only the end-stacking binding mode is observed with a K in the range of 104 to 105M−1.
In the case of apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4, the thermodynamic signatures for the two binding modes are consistent with an “end stacking” mechanism for the higher affinity binding mode and an “intercalation” mechanism for the lower affinity binding mode. In the case of Co(III)-TMPyP4 and Zn(II)-TMPyP4, both the lower affinity for the “end-stacking” mode and the loss of the intercalative mode for forming the 2:1 complexes with hTel22 are attributed to the preferred metal coordination geometry and the presence of axial ligands.
The preferred coordination geometry around the metal center strongly influences the energetics of the interactions between the metallated-TMPyP4 and the model human telomeric G-quadruplex. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.
•apo-TMPyP4, Ni(II)-TMPyP4, and Cu(II)-TMPyP4 bind tightly to hTel22 G-quadruplex.•Co(III)-TMPyP4 and Zn(II)-TMPyP4 bind poorly to hTel22 G-quadruplex.•The metal center coordination geometry strongly influences the binding energetics.
A COVID-19 outbreak occurred among Cameron Peak Fire responders in Colorado, USA, during August 2020-January 2021. The Cameron Peak Fire was the largest recorded wildfire in Colorado history, lasting ...August-December 2020. At least 6,123 responders were involved, including 1,260 firefighters in 63 crews who mobilized to the fire camps. A total of 79 COVID-19 cases were identified among responders, and 273 close contacts were quarantined. State and local public health investigated the outbreak and coordinated with wildfire management teams to prevent disease spread. We performed whole-genome sequencing and applied social network analysis to visualize clusters and transmission dynamics. Phylogenetic analysis identified 8 lineages among sequenced specimens, implying multiple introductions. Social network analysis identified spread between and within crews. Strategies such as implementing symptom screening and testing of arriving responders, educating responders about overlapping symptoms of smoke inhalation and COVID-19, improving physical distancing of crews, and encouraging vaccinations are recommended.
What is already known about this topic? Toxigenic Corynebacterium ulcerans can cause diphtheria-like illness in humans. Transmission of this uncommon zoonotic pathogen between humans and animals is ...poorly understood. What is added by this report? Investigations in Utah and Colorado provide evidence of the risk for C. ulcerans transmission between humans and household pets. Treatment based on antibiotic susceptibility testing results led to successful infection control. What are the implications for public health practice? A One Health (human, animal, and environmental health) approach can be used to control the transmission of and infection with C. ulcerans.
The structure and stability of DNA is highly dependent upon the sequence context of the bases (A, G, C, and T) and the environment under which the DNA is prepared (e.g., buffer, temperature, pH, ...ionic strength). Understanding the factors that influence structure and stability of the i-motif conformation can lead to the design of DNA sequences with highly tunable properties. We have been investigating the influence of pH and temperature on the conformations and stabilities for all permutations of the DNA sequence (CCCXXX)4, where X = A and/or T, using spectroscopic approaches. All oligomers undergo transitions from single-stranded structures at pH 7.0 to i-motif conformations at pH 5.0 as evidenced by circular dichroism (CD) studies. These folded structures possess stacked C:CH+ base pairs joined by loops of 5′-XXX-3′. Although the pH at the midpoint of the transition (pHmp) varies slightly with loop sequence, the linkage between pH and log K for the proton induced transition is highly loop sequence dependent. All oligomers also undergo the thermally induced i-motif to single-strand transition at pH 5.0 as the temperature is increased from 25 to 95 °C. The temperature at the midpoint of this transition (T m) is also highly dependent on loop sequence context effects. For seven of eight possible permutations, the pH induced, and thermally induced transitions appear to be highly cooperative and two state. Analysis of the CD optical melting profiles via a van’t Hoff approach reveals sequence-dependent thermodynamic parameters for the unfolding as well. Together, these data reveal that the i-motif conformation exhibits exquisite sensitivity to loop sequence context with respect to formation and stability.
Zika virus (ZIKV) is a mosquito-borne flavivirus that caused an epidemic in the Americas in 2016 and is linked to severe neonatal birth defects, including microcephaly and spontaneous abortion. To ...better understand the host response to ZIKV infection, we adapted the 10× Genomics Chromium single-cell RNA sequencing (scRNA-seq) assay to simultaneously capture viral RNA and host mRNA. Using this assay, we profiled the antiviral landscape in a population of human monocyte-derived dendritic cells infected with ZIKV at the single-cell level. The bystander cells, which lacked detectable viral RNA, expressed an antiviral state that was enriched for genes coinciding predominantly with a type I interferon (IFN) response. Within the infected cells, viral RNA negatively correlated with type I IFN-dependent and -independent genes (the antiviral module). We modeled the ZIKV-specific antiviral state at the protein level, leveraging experimentally derived protein interaction data. We identified a highly interconnected network between the antiviral module and other host proteins. In this work, we propose a new paradigm for evaluating the antiviral response to a specific virus, combining an unbiased list of genes that highly correlate with viral RNA on a per-cell basis with experimental protein interaction data.
Zika virus (ZIKV) remains a public health threat given its potential for re-emergence and the detrimental fetal outcomes associated with infection during pregnancy. Understanding the dynamics between ZIKV and its host is critical to understanding ZIKV pathogenesis. Through ZIKV-inclusive single-cell RNA sequencing (scRNA-seq), we demonstrate on the single-cell level the dynamic interplay between ZIKV and the host: the transcriptional program that restricts viral infection and ZIKV-mediated inhibition of that response. Our ZIKV-inclusive scRNA-seq assay will serve as a useful tool for gaining greater insight into the host response to ZIKV and can be applied more broadly to the flavivirus field.
Problem: One of the most common medical complications of pregnancy is Gestational Diabetes (GDM). After delivery, guidelines recommend follow-up glucose tolerance testing (OGTT) 4-to 12-weeks ...postpartum. Despite these recommendations, only 16% to 22% of women complete an OGTT postpartum.Methods: The purpose of this proposed quality improvement (QI) project is to pilot a postpartum outreach follow-up via telephone to reach women diagnosed with GDM; the communication will be sent using the institution’s electronic messaging system. Quantitative data were collected prospectively. Data collected included if the patient was contacted, successful patient contact, and if the patient completed the testing. This project took place in a women's health clinic. This project used a purposive sample of women, aged 18–45, diagnosed with GDM.Results: The sample was composed of a total of 24 women in the pre-implementation period and 17 women in the post-implementation period. The pre-implementation period showed seven women (29.2%) completed the OGTT, and 17 (70.8%) did not. In the post-implementation period, six women (35.3%) completed the OGTT, and 11 (64.7%) did not. The postpartum outreach achieved 100% communication from the office to the patient by telephone or electronic message.Implications for Practice: In this QI effort, the implementation of communication helped engage more patients prior to their 6-week postpartum office visit. Future recommendations for this QI project include another PDSA cycle of a longer duration to evaluate the quality of care and new knowledge in promoting responsiveness between patient and provider to achieve the goal of increasing completion of the postpartum testing in women with GDM.
Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically ...dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.
In January 2017, the Colorado Department of Public Health and Environment (CDPHE) identified four epidemiologically linked cases of mumps among persons from a Marshallese community who were members ...of the same church in the Denver metropolitan area. During 2016-2017, sizable outbreaks of mumps reported in Arkansas, Hawaii, and Washington also affected the Marshallese population (1). CDPHE, the Tri-County Health Department (TCHD), and Denver Public Health collaborated to conduct an outbreak investigation during January-March 2017 using active and passive surveillance that identified 17 confirmed and 30 probable cases. Public health actions included conducting measles-mumps-rubella (MMR) vaccination clinics at local Marshallese churches; these resulted in the vaccination of 126 persons with ≥1 doses of MMR vaccine. Implementation of active surveillance and support from local Marshallese church leaders in promoting vaccination programs likely contributed to interruption of the outbreak.
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