Rationale
Evidence‐based medicine (EBM), the dominant approach to assessing the effectiveness of clinical and public health interventions, focuses on the results of association studies. EBM+ is a ...development of EBM that systematically considers mechanistic studies alongside association studies.
Aims and objectives
To explore examples of the importance of mechanistic evidence to coronavirus research.
Methods
We have reviewed the mechanistic evidence in four major areas that are relevant to the management of COVID‐19.
Results and conclusions
(a) Assessment of combination therapy for MERS highlights the need for systematic assessment of mechanistic evidence. (b) That hypertension is a risk factor for severe disease in the case of SARS‐CoV‐2 suggests that altering hypertension treatment might alleviate disease, but the mechanisms are complex, and it is essential to consider and evaluate multiple mechanistic hypotheses. (c) Confidence that public health interventions will be effective requires a detailed assessment of social and psychological components of the mechanisms of their action, in addition to mechanisms of disease. (d) In particular, if vaccination programmes are to be effective, they must be carefully tailored to the social context; again, mechanistic evidence is crucial. We conclude that coronavirus research is best situated within the EBM+ evaluation framework.
Background
Efficacy of COVID‐19 convalescent plasma (CCP) is hypothesized to be associated with the concentration of neutralizing antibodies (nAb) to SARS‐CoV‐2. High capacity serologic assays ...detecting binding antibodies (bAb) have been developed; nAb assays are not adaptable to high‐throughput testing. We sought to determine the effectiveness of using surrogate bAb signal‐to‐cutoff ratios (S/Co) in predicting nAb titers using a pseudovirus reporter viral particle neutralization (RVPN) assay.
Methods
CCP donor serum collected by three US blood collectors was tested with a bAb assay (Ortho Clinical Diagnostics VITROS Anti‐SARS‐CoV‐2 Total, CoV2T) and a nAb RVPN assay. Prediction effectiveness of various CoV2T S/Co criteria was evaluated for RVPN nAb NT50 titers using receiver operating characteristics.
Results
Seven hundred and fifty‐three CCPs were tested with median CoV2T S/Co and NT50 of 71.2 of 527.5. Proportions of donors with NT50 over target nAb titers were 86% ≥1:80, 76% ≥1:160, and 62% ≥1:320. Increasing CoV2T S/Co criterion reduced the sensitivity to predict NT50 titers, while specificity to identify those below increased. As target NT50 titers increase, the CoV2T assay becomes less accurate as a predictor with a decline in positive predictive value and rise in negative predictive value.
Conclusion
Selection of a clinically effective nAb titer will impact availability of CCP. Product release with CoV2T assay S/Co criterion must balance the risk of releasing products below target nAb titers with the cost of false negatives. A two‐step testing scheme may be optimal, with nAb testing on CoV2T samples with S/Cos below criterion.
Two manufacturers, Maxim Biomedical and Sedia Biosciences Corporation, supply CDC-approved versions of the HIV-1 Limiting Antigen Avidity EIA (LAg) for detecting 'recent' HIV infection in ...cross-sectional incidence estimation. This study assesses and compares the performance of the two assays for incidence surveillance.
We ran both assays on a panel of 2,500 well-characterized HIV-1-infected specimens. We analysed concordance of assay results, assessed reproducibility using repeat testing and estimated mean durations of recent infection (MDRIs) and false-recent rates (FRRs) for a range of normalized optical density (ODn) thresholds, alone and in combination with viral load thresholds. We defined three hypothetical surveillance scenarios, similar to the Kenyan and South African epidemics, and a concentrated epidemic. These scenarios allowed us to evaluate the precision of incidence estimates obtained by means of various recent infection testing algorithms (RITAs) based on each of the two assays.
The Maxim assay produced lower ODn values than the Sedia assay on average, largely as a result of higher calibrator readings (mean OD of 0.749 vs. 0.643), with correlation of normalized readings lower (R2 = 0.908 vs. R2 = 0.938). Reproducibility on blinded control specimens was slightly better for Maxim. The MDRI of a Maxim-based algorithm at the 'standard' threshold (ODn ≤1.5 & VL >1,000) was 201 days (95% CI: 180,223) and for Sedia 171 (152,191). The difference Differences in MDRI were estimated at 32.7 (22.9,42.8) and 30.9 days (21.7,40.7) for the two algorithms, respectively. Commensurately, the Maxim algorithm had a higher FRR in treatment-naive subjects (1.7% vs. 1.1%). The two assays produced similar precision of incidence estimates in the three surveillance scenarios.
Differences between the assays can be primarily attributed to the calibrators supplied by the manufacturers. Performance for surveillance was extremely similar, although different thresholds were optimal (i.e. produced the lowest variance of incidence estimates) and at any given ODn threshold, different estimates of MDRI and FRR were obtained. The two assays cannot be treated as interchangeable: assay and algorithm-specific performance characteristic estimates must be used for survey planning and incidence estimation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Polyphenols (tannins) in the diet not only precipitate oral proteins, producing an astringent sensation, but also interact with dietary proteins and digestive enzymes in the gut, resulting in a ...variety of antinutritive and toxic effects. Salivary proline-rich proteins (PRPs), which are secreted into the oral cavity, form complexes with and precipitate dietary polyphenols, and thus, they constitute the primary mammalian defense directed against ingested tannins. In order to characterize the interaction, NMR studies were performed which involved titrating a series of polyphenols into a synthetic 19-residue PRP fragment. The results show that the predominant mode of association is a hydrophobic stacking of the polyphenol ring against the pro-S face of proline and that the first proline residue of a Pro-Pro sequence is a particularly favored binding site. Measurement of dissociation constants indicates that the larger and more complex polyphenols interact more strongly with the PRP fragment; the order of binding affinity was determined as procyanidin dimer B-2 > pentagalloylglucose > trigalloylglucose >> proanthocyanidin monomer (−)-epicatechin ≈ propyl gallate. Smaller polyphenols can bind with one phenolic ring stacked against each proline residue, whereas larger polyphenols occupy two or three consecutive prolines. The more complex polyphenols interact with the PRP fragment in a multidentate fashion; moreover, they self-associate or stack when bound. Thus, a model is proposed in which multiple polyphenol/polyphenol and polyphenol/PRP interactions act cooperatively to achieve precipitation.
Background
Zika virus (ZIKV) epidemics with infections in pregnant women are associated with severe neurological disease in newborns. Although an arbovirus, ZIKV is also blood transfusion‐transmitted ...(TT). Greater knowledge of the efficiency of ZIKV TT would aid decisions on testing and pathogen reduction technologies (PRT).
Study Design and Methods
Plasma units from ZIKV RNA‐reactive blood donors were used to study infectivity in vitro, in mice, and in macaques. Furthermore, plasma units were subjected to PRT using amotosalen/ultraviolet light A (A/UVA) before transfusion.
Results
In vitro infectivity of ZIKV RNA‐reactive plasma varied between 100 and 1000 international units (IU) of ZIKV RNA. Immunodeficient mice were more sensitive with as low as 32 IU sufficient to infect 50% of mice. 50–5500 IU of RNA led to TT in macaques using dose escalation of three different RNA‐positive, seronegative plasma units. In contrast, RNA‐reactive units collected postseroconversion were not infectious in macaques, even at a dose of 9 million IU RNA. After A/UVA PRT, transfusion of plasma containing up to 18 million IU was no longer infectious in vitro and did not result in ZIKV TT in macaques.
Conclusion
Significant risks of ZIKV TT are likely confined to a relatively short viremic window before seroconversion, and that sensitive nucleic acid amplification testing likely identifies the majority of infectious plasma. PRT was demonstrated to be effective at preventing ZIKV TT. Considering that there is no approved ZIKV vaccine, these data are relevant to mitigate the risk of TT during the future ZIKV outbreaks.
Direct numerical simulations of stratified open channel flows subject to a varying surface heat flux are performed. The influence of the diurnal heating time on the spatial and temporal variation of ...mixing in the flow and the characteristics of the mean flow state are examined. The control parameters are the bulk stability parameter
λ
B
, defined through the ratio of the channel height
δ
and a bulk Obukhov length scale
L
B
, and the diurnal time scale
t
^
, defined as the ratio of the heating time to an eddy turnover time. The Prandtl number
Pr
and Reynolds number
R
e
τ
have values of 1 and 400. Simulations are performed over
t
^
=
1
to 24 and
λ
B
=
0.6
to 26. Two key flow features are used to classify the flow regimes observed, namely the laminar layer depth (LLD) and stratified layer depth (SLD) where the LLD is defined as the depth from the free surface when the buoyancy Reynolds number
R
e
B
≈
7
and the SLD is the depth from the free surface when the turbulent Froude number
F
r
≈
1
. This study attempts to characterise how these length scales vary across the diel cycle. The LLD is a viscous length scale and a regime map of a viscous parameter, the bulk Obhukov Reynolds number
R
e
L
, and
t
^
is presented to classify the LLD behaviour. A regime map of
λ
B
and
t
^
is presented to classify the behaviour of the SLD. Three classifications for each layer depth behaviour within a diel cycle form the basis of the regime maps for this paper: a neutral flow where the LLD or SLD does not exist (denoted by NL and NS), a stratified flow where the LLD or SLD are diurnally varying (denoted as DL and DS) and a persistent layer of the LLD or SLD (denoted as PL and PS). The transition between the NL to DL is
t
^
∝
R
e
L
4.5
, DL to PL is
t
^
∝
R
e
L
-
0.5
, NS to DS is
t
^
∝
λ
B
0
and DS to PS is
t
^
∝
λ
B
1
. The regime maps may be used as a predictive tool to determine when suppressed mixing regimes occur in rivers. At each flow depth, the flow sweeps though a range of mixing states across the diel cycle. The local mixing efficiency are briefly assessed and found to scale well with the instantaneous
Fr
number according to the regimes proposed by Garanaik and Venayagamoorthy (
J. Fluid Mech.
, vol. 867, 2019, pp. 323-333).
Article Highlights
This paper reports on direct numerical simulations of stratified open channel flows subject to a varying surface heat flux. The results have found that:
Increasing the diurnal time scale allows the flow to sweep through a wider range of flow states from turbulent to strongly stratified,
Simulation data of the mixing efficiency and turbulent Froude number from this temporally varying and spatially inhomogenous flow that undergoes strong temporal forcing collapses well onto the parameterisation scheme of Garanaik & Venayagamoorthy (
J. Fluid Mech.
, vol. 867, 2019, pp. 323–333) found for homogenous stratified flows, and
Three distinct classifications (a persistent layer, a diurnal layer and one where the layer does not exist) of the laminar layer depth (LLD) and stratified layer depth (SLD) behaviour persists throughout a diel cycle and form a regime map given a
λ
B
,
R
e
τ
and
t
^
value. The relationship between each transitions are: from no LLD (NL) to a diurnal LLD (DL)
t
^
∝
R
e
L
4.5
, DL to a persistent LLD (PL)
t
^
∝
R
e
L
-
0.5
, no SLD (NS) to a diurnal SLD (DS)
t
^
∝
λ
B
0
and DS to a persistent SLD (PS) is
t
^
∝
λ
B
1
. The regime maps may used as a predictive model to calculate when suppressed mixing transpires in rivers.
Zipper-interacting protein kinase (ZIPK) has been implicated in Ca2+-independent smooth muscle contraction, although its specific role is unknown. The addition of ZIPK to demembranated rat caudal ...arterial strips induced an increase in force, which correlated with increases in LC20 and MYPT1 phosphorylation. However, because of the number of kinases capable of phosphorylating LC20 and MYPT1, it has proven difficult to identify the mechanism underlying ZIPK action. Therefore, we set out to identify bona fide ZIPK substrates using a chemical genetics method that takes advantage of ATP analogs with bulky substituents at the N6 position and an engineered ZIPK capable of utilizing such substrates. 32P-Labeled 6-phenyl-ATP and ZIPK-L93G mutant protein were added to permeabilized rat caudal arterial strips, and substrate proteins were detected by autoradiography following SDS-PAGE. Mass spectrometry identified LC20 as a direct target of ZIPK in situ for the first time. Tissues were also exposed to 6-phenyl-ATP and ZIPK-L93G in the absence of endogenous ATP, and putative ZIPK substrates were identified by Western blotting. LC20 was thereby confirmed as a direct target of ZIPK; however, no phosphorylation of MYPT1 was detected. We conclude that ZIPK is involved in the regulation of smooth muscle contraction through direct phosphorylation of LC20.
It is becoming increasingly clear that proteins transiently populate high-energy excited states as a necessary requirement for function. Here, we demonstrate that rational mutation based on the ...characteristics of the structure and dynamics of proteins obtained from pressure experiments is a new strategy for amplifying particular fluctuations in proteins. We have previously shown that ubiquitin populates a high-energy conformer, N2, at high pressures. Here, we show that the Q41N mutation favors N2: high-pressure nuclear magnetic resonance (NMR) shows that N2 is ∼70% populated in Q41N but only ∼20% populated in the wild type at ambient pressure. This allows us to characterize the structure of N2, in which α1-helix, the following loop, β3-strand, and β5-strand change their orientations relative to the remaining regions. Conformational fluctuation on the microsecond time scale, characterized by 15N spin relaxation NMR analysis, is markedly increased for these regions of the mutant. The N2 conformers produced by high pressure and by the Q41N mutation are quite similar in both structure and dynamics. The conformational change to produce N2 is proposed to be a novel dynamic feature beyond the known recognition dynamics of the protein. Indeed, it is orthogonal to that seen when proteins containing a ubiquitin-interacting motif bind at the hydrophobic patch of ubiquitin but matches changes seen on binding to the E2 conjugating enzyme. More generally, structural and dynamic effects of hydrodynamic pressure are shown to be useful for characterizing functionally important intermediates.
Repeated sampling of spatially distributed river
chemistry can be used to assess the location, scale, and persistence of
carbon and nutrient contributions to watershed exports. Here, we provide a
...comprehensive set of water chemistry measurements and ecohydrological
metrics describing the biogeochemical conditions of permafrost-affected
Arctic watersheds. These data were collected in watershed-wide synoptic
campaigns in six stream networks across northern Alaska. Three watersheds
are associated with the Arctic Long-Term Ecological Research site at Toolik
Field Station (TFS), which were sampled seasonally each June and August from
2016 to 2018. Three watersheds were associated with the National Park
Service (NPS) of Alaska and the U.S. Geological Survey (USGS) and were
sampled annually from 2015 to 2019. Extensive water chemistry
characterization included carbon species, dissolved nutrients, and major
ions. The objective of the sampling designs and data acquisition was to
characterize terrestrial–aquatic linkages and processing of material in
stream networks. The data allow estimation of novel ecohydrological metrics
that describe the dominant location, scale, and overall persistence of
ecosystem processes in continuous permafrost. These metrics are (1)
subcatchment leverage, (2) variance collapse, and (3) spatial persistence.
Raw data are available at the National Park Service Integrated Resource Management Applications portal (O'Donnell et al., 2021, https://doi.org/10.5066/P9SBK2DZ) and within the Environmental Data Initiative (Abbott, 2021, https://doi.org/10.6073/pasta/258a44fb9055163dd4dd4371b9dce945).
Serological surveillance studies of infectious diseases provide population-level estimates of infection and antibody prevalence, generating crucial insight into population-level immunity, risk ...factors leading to infection, and effectiveness of public health measures. These studies traditionally rely on detection of pathogen-specific antibodies in samples derived from venipuncture, an expensive and logistically challenging aspect of serological surveillance. During the COVID-19 pandemic, guidelines implemented to prevent the spread of SARS-CoV-2 infection made collection of venous blood logistically difficult at a time when SARS-CoV-2 serosurveillance was urgently needed. Dried blood spots (DBS) have generated interest as an alternative to venous blood for SARS-CoV-2 serological applications due to their stability, low cost, and ease of collection; DBS samples can be self-generated via fingerprick by community members and mailed at ambient temperatures. Here, we detail the development of four DBS-based SARS-CoV-2 serological methods and demonstrate their implementation in a large serological survey of community members from 12 cities in the East Bay region of the San Francisco metropolitan area using at-home DBS collection. We find that DBS perform similarly to plasma/serum in enzyme-linked immunosorbent assays and commercial SARS-CoV-2 serological assays. In addition, we show that DBS samples can reliably detect antibody responses months postinfection and track antibody kinetics after vaccination. Implementation of DBS enabled collection of valuable serological data from our study population to investigate changes in seroprevalence over an 8-month period. Our work makes a strong argument for the implementation of DBS in serological studies, not just for SARS-CoV-2, but any situation where phlebotomy is inaccessible.
Estimation of community-level antibody responses to SARS-CoV-2 from infection or vaccination is critical to inform public health responses. Traditional studies of antibodies rely on collection of blood via venipuncture, an invasive procedure not amenable to pandemic-related social-distancing measures. Dried blood spots (DBS) are an alternative to venipuncture, since they can be self-collected by study participants at home and do not require refrigeration for shipment or storage. However, DBS-based assays to measure antibody levels to SARS-CoV-2 have not been widely utilized. Here, we show that DBS are comparable to blood as a sampling method for antibody responses to SARS-CoV-2 infection and vaccination over time measured using four distinct serological assays. The DBS format enabled antibody surveillance in a longitudinal cohort where study participants self-collected samples, ensuring the participants' safety during an ongoing pandemic. Our work demonstrates that DBS are an excellent sampling method for measuring antibody responses whenever venipuncture is impractical.
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