The T-lock: automated compensation of radio-frequency induced sample heating

Modern high-field NMR spectrometers can stabilize the nominal sample temperature at a precision of less than 0.1 K. However, the actual sample temperature may differ from the nominal value by several degrees because the sample heating caused by high-power radio frequency pulses is not readily detected by the temperature sensors. Without correction, transfer of chemical shifts between different experiments causes problems in the data analysis. In principle, the temperature differences can be corrected by manual procedures but this is cumbersome and not fully reliable. Here, we introduce the concept of a “T-lock”, which automatically maintains the sample at the same reference temperature over the course of different NMR experiments. The T-lock works by continuously measuring the resonance frequency of a suitable spin and simultaneously adjusting the temperature control, thus locking the sample temperature at the reference value. For three different nuclei, ¹³C, ¹⁷O and ³¹P in the compounds alanine, water, and phosphate, respectively, the T-lock accuracy was found to be <0.1 K. The use of dummy scan periods with variable lengths allows a reliable establishment of the thermal equilibrium before the acquisition of an experiment starts.     

Inconstancy of Taylor'sb: Simulated sampling with different quadrat sizes and spatial distributions

Taylor 's power law, s²=am^b, provides a precise summary of the relationship between sample variance (s²) and sample mean (m) for many organisms. The coefficient b has been interpreted as an index of aggregation, with a characteristic value for a given species in a particular environment, and has been thought to be independent of the sample unit. Simulation studies were conducted that demonstrate that the value of b may vary with the size of the sample unit in quadrat sampling, and this relationship, in turn, depends on the underlying spatial distribution of the population. For example, simulated populations with hierarchical aggregation on a large scale produced values of b that increased with the size of the sample unit. In contrast, for a simulated population with randomly distributed clusters of individuals, the value of b eventually decreased with increasing quadrat size, as sample counts became more uniform. A single value ofTaylor 's b, determined with a particular sample unit, provides neither a fixed index of aggregation nor a complete picture of a species' spatial distribution. Rather, it describes a consistent relationship between sample variance and sample mean over a range of densities, on a spatial scale related to the size of the sample unit. This relationship may reflect, but not uniquely define, density-dependent population and behavioral processes governing the spatial distribution of the organism. Interpretation ofTaylor 'sb for a particular organism should be qualified by reference to the sample unit, and comparisons should not be made between cases in which different sample units were used. Whenever possible, a range of sample units should be used to provide information about the pattern of distribution of a population on various spatial scales.     

Otoliths speak out: why the Pacific halibut in Puget sound are different

Pacific halibut, Hippoglossus stenolepis, is one of the most important commercial groundfish and is managed as a single coast-wide population from Alaska to northern California. Nevertheless, genetic investigations did not show success in detecting the population structure of the species. Here I report stable oxygen and carbon isotope analyses (δ¹⁸O and δ¹³C) in otoliths to discriminate the stock differences from two sample locations between the Washington coast (WC) and the northern Puget Sound (PS), and two sample years in 2007 and 2008. In general the δ¹⁸O values of halibut otoliths from WC ranged from ?0.2 to 1.8‰, higher than the PS samples from ?0.5 to 1.4‰. In contrast, the δ¹³C values from WC ranged from ?3.6 to ?1.0‰, lower than the PS samples from ?3.2 to ?1.2‰. Results from the otolith nuclei (age-0 halibut) and the 8^th (the earliest maturity age for male halibut) and edge otolith rings (the latest location where the fish lived) showed significant differences between halibut samples from PS and WC. In particular, the sample location difference (between PS and WC) in both δ¹³C and δ¹⁸O data was significant and markedly larger than the sample year difference (between 2007 and 2008). These isotopic signatures provide evidence that the PS halibut may belong to a distinct stock that is significantly different from WC halibut.     

Minireview: Fluorescence Correlation Spectroscopy (FCS) - A Highly Sensitive Method to Analyze Drug/Target Interactions

Abstract

Fluorescence Correlation Spectroscopy (FCS), a new analytical technology, allows binding properties to be determined very accurately in biological assays at the level of single molecules. At concentrations of ≥ 10^-12 M, binding constants, on/off-rates, and even reaction/enzyme kinetics can be determined in real-time, and in sample volumes as low as 10^-9 μl. The FCS technology can be applied to study molecular and cellular interactions in homogeneous assays. Assay times in the range of seconds in combination with nanoliter sample volumes allow FCS to be used for high throughput screening to identify new pharmaceutical lead structures or new pharmacological targets. FCS is fully compatible with standard microtiter plate formats. However, for high throughput screening, specially designed sample carriers containing many thousand sub-microliter sample wells may be used in combination with a nanopipetting and sample retrieval system.     

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology ¹. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes ^{2, 3}. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice ⁴, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast ⁵. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes ⁶, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.     

Sampling volume in root studies: the pitfalls of under‐sampling exposed using accumulation curves

Root systems are important for global models of below‐ground carbon and nutrient cycling. Notoriously difficult sampling methods and the fractal distribution of root diameters in the soil make data being used in these models especially susceptible to error resulting from under‐sampling. We applied the concept of species accumulation curves to root data to quantify the extent of under‐sampling inherent to minirhizotron and soil coring sampling for both root uptake and carbon content studies. Based on differences in sample size alone, minirhizotron sampling missed approximately one third of the root diameters observed by soil core sampling. Sample volumes needed to encounter 90% of root diameters averaged 2481 cm³ for uptake studies and 5878 cm³ for root carbon content studies. These results show that small sample volumes encounter a non‐representative sample of the overall root pool, and provide future guidelines for determining optimal sample volumes in root studies.     

Solid-state NMR spectroscopy of 10% 13C labeled ubiquitin: spectral simplification and stereospecific assignment of isopropyl groups

We describe the simplification of ¹³C–¹³C correlation spectra obtained from a microcrystalline protein sample expressed on a growth medium of 10% fully ¹³C labeled glucose diluted in 90% natural abundance glucose as compared to a fully labeled sample. Such a labeling scheme facilitates the backbone and side-chain resonance assignment of Phe, Tyr, His, Asp, Asn, Ile, Lys and Pro and yields an unambiguous stereospecific assignment of the valine Cγ1, Cγ2 ¹³C resonances and of Leucine Cδ2.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Individual closed chamber: an alternative method for quantifying 14C in both labeled organic and inorganic carbon substrates

Incubation of ¹⁴C-labeled substrates continues to be a widely used procedure in soil organic matter (OM) research due to its sensitiveness. When the labeling is found in liquid fractions (soil extracts, hydrolysates), ¹⁴C can be easily quantified by using an aliquot for scintillation counting. For this reason, converting a solid carbon sample into liquid form is a typical step for accurate ¹⁴C analysis. We have developed an alternative method to carry out this step, which uses standard glass hardware and does not require complex laboratory facilities. Carbon (both in organic or inorganic forms) is converted into CO₂ within a reaction vessel connected to a Twisselmann’s extractor with an alkali trap inside. This forms an individual closed chamber (ICC) for each sample, thus eliminating the risk of cross-contaminations. The alkali solution adsorbs the evolved CO₂ within the closed system, and the excess of pressure is easily overcome by the use of a balloon. We tested the procedure on a set of substrates and two contrasting soils, checking also the effect of different sample loads (from 20 to 160 mg C) on the CO₂ recovery of the process. The percentage of carbon recovered into the alkali (i.e. the efficiency of the process) ranged from 92% for the inorganic C to 93–95% for the organic C method, the latter being sensitive to the amount of sample used for analysis. The ICC method can be successfully applied to analyze ¹⁴C-labeling in both carbonates and OM from solid samples, thus representing an alternative method to some established protocols, and it is suitable for substrates with low or very low ¹⁴C contents, in which high volumes of sample must be analyzed in order to guarantee representative results.     

Common polymorphism of peptidase A: Formal genetics and population data

Summary Peptidase A polymorphism was investigated in 83 mother-child pairs and in a population sample of 182 unrelated individuals from southwestern Germany. Hereby the formal hypothesis that there are two common alleles, PepA¹ and PepA⁸, at an autosomal locus PepA, was confirmed. The frequency of the PepA⁸ allele was calculated to be 0.27±0.02.     

Towards Monitoring Biodiversity in Amazonian Forests: How Regular Samples Capture Meso-Scale Altitudinal Variation in 25 km2 Plots

Background

Ecological monitoring and sampling optima are context and location specific. Novel applications (e.g. biodiversity monitoring for environmental service payments) call for renewed efforts to establish reliable and robust monitoring in biodiversity rich areas. As there is little information on the distribution of biodiversity across the Amazon basin, we used altitude as a proxy for biological variables to test whether meso-scale variation can be adequately represented by different sample sizes in a standardized, regular-coverage sampling arrangement.

Methodology/Principal Findings

We used Shuttle-Radar-Topography-Mission digital elevation values to evaluate if the regular sampling arrangement in standard RAPELD (rapid assessments (“RAP”) over the long-term (LTER [“PELD” in Portuguese])) grids captured patters in meso-scale spatial variation. The adequacy of different sample sizes (n = 4 to 120) were examined within 32,325 km²/3,232,500 ha (1293×25 km² sample areas) distributed across the legal Brazilian Amazon. Kolmogorov-Smirnov-tests, correlation and root-mean-square-error were used to measure sample representativeness, similarity and accuracy respectively. Trends and thresholds of these responses in relation to sample size and standard-deviation were modeled using Generalized-Additive-Models and conditional-inference-trees respectively. We found that a regular arrangement of 30 samples captured the distribution of altitude values within these areas. Sample size was more important than sample standard deviation for representativeness and similarity. In contrast, accuracy was more strongly influenced by sample standard deviation. Additionally, analysis of spatially interpolated data showed that spatial patterns in altitude were also recovered within areas using a regular arrangement of 30 samples.

Conclusions/Significance

Our findings show that the logistically feasible sample used in the RAPELD system successfully recovers meso-scale altitudinal patterns. This suggests that the sample size and regular arrangement may also be generally appropriate for quantifying spatial patterns in biodiversity at similar scales across at least 90% (≈5 million km²) of the Brazilian Amazon.     

Within-tree distribution of Ricania shantungensis (Hemiptera: Ricaniidae) eggs on chestnuts

Within-tree distribution of an insect pest is useful to determine sample unit, sampling location, and required sample number for this pest within a tree. This study was conducted to characterize the within-tree distribution pattern and determine an efficient sample unit for eggs of Ricania shantungensis (Hemiptera: Ricaniidae) on a chestnut tree. A total of 60 chestnut trees were randomly selected from chestnut fields in Gongju-Si and Buyeo-Gun, Korea in 2019. For each tree, 12 branches were cut at three vertical heights (low, middle, and high) in four cardinal directions (east, west, south, and north). The cut branches were observed for counting the number of egg masses per 10 cm from the branch tip. Coefficient of variation (CV) and coefficient of determination (r²) were calculated and used to determine an optimal sample unit for eggs of R. shantungensis on chestnut trees. Numbers of R. shantungensis egg masses were not significantly different (P > 0.05) vertically and horizontally within a tree. Based on CV and r² values, the 50 cm branch tip would be the optimal sample unit for R. shantungensis eggs. Using this small sample unit for sampling of R. shantungensis eggs could be economical and efficient.     

DNA and its counterions: a molecular dynamics study

The behaviour of mobile counterions, Na⁺ and K⁺, was analysed around a B-DNA double helix with the sequence CCATGCGCTGAC in aqueous solution during two 50 ns long molecular dynamics trajectories. The movement of both monovalent ions remains diffusive in the presence of DNA. Ions sample the complete space available during the simulation time, although individual ions sample only about one-third of the simulation box. Ions preferentially sample electronegative sites around DNA, but direct binding to DNA bases remains a rather rare event, with highest site occupancy values of <13%. The location of direct binding sites depends greatly on the nature of the counterion. While Na⁺ binding in both grooves is strongly sequence-dependent with the preferred binding site in the minor groove, K⁺ mainly visits the major groove and binds close to the centre of the oligomer. The electrostatic potential of an average DNA structure therefore cannot account for the ability of a site to bind a given cation; other factors must also play a role. An extensive analysis of the influence of counterions on DNA conformation showed no evidence of minor groove narrowing upon ion binding. A significant difference between the conformations of the double helix in the different simulations can be attributed to extensive α/γ transitions in the phosphate backbone during the simulation with Na⁺. These transitions, with lifetimes over tens of nanoseconds, however, appear to be correlated with ion binding to phosphates. The ion-specific conformational properties of DNA, hitherto largely overlooked, may play an important role in DNA recognition and binding.     

Sample size in the monitoring of benthic macrofauna in the profundal of lakes: evaluation of the precision of estimates

We discuss here the influence of sample size (number of replicates) on the accuracy and precision of the results when sampling profundal benthos with an Ekman grab according to the Finnish standard, SFS 5076, which is equivalent to the Swedish and Norwegian standards. The aim was to find criteria for choosing a sample size which would avoid any powerful influence of chance on the results without entailing an unreasonable amount of work for monitoring purposes.Lake Haukivesi (area 620 km², total phosphorus 13 µg l^–1 and colour 35 Pt mg l^–1), Lake Paasivesi (116 km², 5 µg l^–1 and 35 Pt mg l^–1) and Lake Puruvesi (322 km², 4 µg l^–1 and 5 Pt mg l^–1) were sampled randomly in June and October 1991. 25 Replicate samples were taken on each occasion from the deep profundal area of each lake, defined here as 60-100% of the maximum depth. The sedimentation areas studied were fairly homogeneous, since the animal communities were not markedly affected by the variations in depth. Distribution estimates for the statistics studied, such as number of individuals, expected number of species, diversity and benthic quality indices, were calculated for a large set of random samples taken from the empirical data by computer (bootstrap sampling). The sample variance, s ², correlated with the mean animal density, m (ind. m^–2), according to the equation s ² = 31.77 m ^1.247. The sample size required to achieve the desired precision in mean animal density (D, expressed as the ratio standard error/mean) can thus be estimated as n = 31.77 m ^–0.753 D ^–2. The number of replicate samples needed to achieve a standard error of 20% of the mean density was 10 in Lake Haukivesi, seven in Lake Paasivesi and 11 in Lake Puruvesi. The accuracy and precision of the estimated number of species, Shannon's diversity and Benthic Quality Index improved markedly as the sample size was increased to 10 replicates. As a compromise between work load and statistical reliability, a figure of 10 replicate Ekman samples is proposed here for the monitoring of profundal benthos. The proposed sample size usually produces individual numbers which are high enough for practical purposes, probably at least 100 individuals, which is recommended as a minimum in the standard. The lower number of replicate samples recommended in recent Finnish handbook, 3–5, usually produces inadequate data, and this may detract from the comparability of the results and leave the changes in profundal communities undetected.     

Emerging Lyngbya wollei toxins: A new high resolution mass spectrometry method to elucidate a potential environmental threat

Mass spectrometric methods for the quantitative and qualitative analyses of algal biotoxins are often complicated by co-eluting compounds that present analytically as interferences. This issue is particularly critical for organic polyamines, where co-eluting materials can suppress the formation of cations during electrospray ionization. Here we present an extraction procedure designed specifically to overcome matrix-derived ion suppression of algal toxins in samples of Lyngbya wollei, a filamentous benthic algae known to produce several saxitoxin analogues. Lyngbya wollei samples were collected from a large, persistent harmful algal bloom in Lake Wateree, SC. Six known Lyngbya wollei-specific toxins (LWT1–6) were successfully resolved and quantified against saxitoxin using hydrophilic interaction liquid chromatography coupled with triple quadrupole and quadrupole time-of-flight mass spectrometry. The parent ions [M²⁺ – H⁺]⁺ were observed for LWTs 1–6 and the [M]²⁺ ion was observed for LWT5. High resolution mass spectra and unique fragmentation ions were obtained for LWTs 1–6. A dilution factor of 50 resulted in a linear calibration of saxitoxin in the algae matrix. Ion suppression was resolved by sample dilution, which led to linear, positive correlations between peak area and mass of the extracted sample (R² > 0.96). Optimized sample extraction method and instrument parameters are presented.     

Educational attainment: a genome wide association study in 9538 Australians

Background

Correlations between Educational Attainment (EA) and measures of cognitive performance are as high as 0.8. This makes EA an attractive alternative phenotype for studies wishing to map genes affecting cognition due to the ease of collecting EA data compared to other cognitive phenotypes such as IQ.

Methodology

In an Australian family sample of 9538 individuals we performed a genome-wide association scan (GWAS) using the imputed genotypes of ∼2.4 million single nucleotide polymorphisms (SNP) for a 6-point scale measure of EA. Top hits were checked for replication in an independent sample of 968 individuals. A gene-based test of association was then applied to the GWAS results. Additionally we performed prediction analyses using the GWAS results from our discovery sample to assess the percentage of EA and full scale IQ variance explained by the predicted scores.

Results

The best SNP fell short of having a genome-wide significant p-value (p = 9.77×10⁻⁷). In our independent replication sample six SNPs among the top 50 hits pruned for linkage disequilibrium (r²<0.8) had a p-value<0.05 but only one of these SNPs survived correction for multiple testing - rs7106258 (p = 9.7*10⁻⁴) located in an intergenic region of chromosome 11q14.1. The gene based test results were non-significant and our prediction analyses show that the predicted scores explained little variance in EA in our replication sample.

Conclusion

While we have identified a polymorphism chromosome 11q14.1 associated with EA, further replication is warranted. Overall, the absence of genome-wide significant p-values in our large discovery sample confirmed the high polygenic architecture of EA. Only the assembly of large samples or meta-analytic efforts will be able to assess the implication of common DNA polymorphisms in the etiology of EA.     

Monte Carlo-based calculation of imaging plate response to <Superscript>90</Superscript>Sr in teeth: experimental validation of the required correction on sample thickness

Recently, a numerical method was proposed to correct the imaging plate (IP) response to ⁹⁰Sr concentration in tooth samples, depending on the sample thickness. This is important to quantify any ⁹⁰Sr concentration in teeth, which in turn is necessary to determine any ⁹⁰Sr incorporation of a person retrospectively. Although the final goal will be to evaluate the (inhomogeneous) spatial distribution of ⁹⁰Sr inside tooth samples precisely, the present study was restricted—as a first step—to the evaluation of ⁹⁰Sr in teeth assuming a uniform ⁹⁰Sr distribution. A numerical method proposed earlier was validated experimentally in the present study by measuring the IP response to standard sources of various thicknesses and ⁹⁰Sr concentrations. For comparison, the energy deposition of the β-rays emitted by ⁹⁰Sr in the IP—which is considered to be proportional to the IP luminescence signal—was calculated for the various sample thicknesses involved, by means of the MCNP-4C code. As a result, the measured IP response could be reproduced by the calculations within the uncertainties, depending on the thickness of the standard sources. Thus, the validity of the proposed numerical method to correct the IP response for sample thickness has successfully been demonstrated.     

Sample Size Effects on Estimates of Population Genetic Structure: Implications for Ecological Restoration

The field of ecological restoration is growing rapidly, and the sourcing of suitable seed is a major issue. Information on the population genetic structure of a species can provide valuable information to aid in defining seed collection zones. For a practical contribution from genetics, a rapid approach to delineating seed collection zones using genetic markers (amplified fragment length polymorphisms [AFLPs]) has been developed. Here, we test the effects of sampling regime on the efficacy of this method. Genetic data were collected for an outcrossing seeder, Daviesia divaricata ssp. divaricata, an important species in urban bushland restoration in Perth, Western Australia. The effect of sample size and number of AFLP markers on estimates of genetic variation and population structure was examined in relation to implications for sourcing material for restoration. Three different sample sizes were used (n= 8, 15, and 30) from six urban bushland remnants. High levels of genetic diversity were observed in D. divaricata (87.4% polymorphic markers), with significant population differentiation detected among sampled populations (Θ^B= 0.1386, p < 0.001). Although sample size does not appear to affect the spatial pattern in principle co‐ordinates analysis (PCA) plots, the number of polymorphic loci increased with sample size and estimates of population subdivision (F_ST and Θ^B) and associated confidence intervals decreased with increasing sample size. We recommend using a minimum of 30 plants for sourcing seed for restoration projects.     

Insights into the discrepant luminescence for BaSiO3:Eu2+ phosphors prepared by solid‐state reaction and precipitation reaction methods

Two synthesis routes, solid‐state reaction and precipitation reaction, were employed to prepare BaSiO₃:Eu²⁺ phosphors in this study. Discrepancies in the luminescence green emission at 505 nm for the solid‐state reaction method sample and in the yellow emission at 570 nm for the sample prepared by the precipitation reaction method, were observed respectively. A detail investigation about the discrepant luminescence of BaSiO₃:Eu²⁺ phosphors was performed by evaluation of X‐ray diffraction (XRD), photoluminescence (PL)/photoluminescence excitation (PLE), decay time and thermal quenching properties. The results showed that the yellow emission was generated from the BaSiO₃:Eu²⁺ phosphor, while the green emission was ascribed to a small amount of Ba₂SiO₄:Eu²⁺ compound that was present in the solid‐state reaction sample. This work clarifies the luminescence properties of Eu²⁺ ions in BaSiO₃ and Ba₂SiO₄ hosts.     

Detection of significant antiviral drug effects on COVID-19 with reasonable sample sizes in randomized controlled trials: A modeling study

BackgroundDevelopment of an effective antiviral drug for Coronavirus Disease 2019 (COVID-19) is a global health priority. Although several candidate drugs have been identified through in vitro and in vivo models, consistent and compelling evidence from clinical studies is limited. The lack of evidence from clinical trials may stem in part from the imperfect design of the trials. We investigated how clinical trials for antivirals need to be designed, especially focusing on the sample size in randomized controlled trials.Methods and findingsA modeling study was conducted to help understand the reasons behind inconsistent clinical trial findings and to design better clinical trials. We first analyzed longitudinal viral load data for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) without antiviral treatment by use of a within-host virus dynamics model. The fitted viral load was categorized into 3 different groups by a clustering approach. Comparison of the estimated parameters showed that the 3 distinct groups were characterized by different virus decay rates (p-value < 0.001). The mean decay rates were 1.17 d⁻¹ (95% CI: 1.06 to 1.27 d⁻¹), 0.777 d⁻¹ (0.716 to 0.838 d⁻¹), and 0.450 d⁻¹ (0.378 to 0.522 d⁻¹) for the 3 groups, respectively. Such heterogeneity in virus dynamics could be a confounding variable if it is associated with treatment allocation in compassionate use programs (i.e., observational studies).Subsequently, we mimicked randomized controlled trials of antivirals by simulation. An antiviral effect causing a 95% to 99% reduction in viral replication was added to the model. To be realistic, we assumed that randomization and treatment are initiated with some time lag after symptom onset. Using the duration of virus shedding as an outcome, the sample size to detect a statistically significant mean difference between the treatment and placebo groups (1:1 allocation) was 13,603 and 11,670 (when the antiviral effect was 95% and 99%, respectively) per group if all patients are enrolled regardless of timing of randomization. The sample size was reduced to 584 and 458 (when the antiviral effect was 95% and 99%, respectively) if only patients who are treated within 1 day of symptom onset are enrolled. We confirmed the sample size was similarly reduced when using cumulative viral load in log scale as an outcome.We used a conventional virus dynamics model, which may not fully reflect the detailed mechanisms of viral dynamics of SARS-CoV-2. The model needs to be calibrated in terms of both parameter settings and model structure, which would yield more reliable sample size calculation.ConclusionsIn this study, we found that estimated association in observational studies can be biased due to large heterogeneity in viral dynamics among infected individuals, and statistically significant effect in randomized controlled trials may be difficult to be detected due to small sample size. The sample size can be dramatically reduced by recruiting patients immediately after developing symptoms. We believe this is the first study investigated the study design of clinical trials for antiviral treatment using the viral dynamics model.

Using a viral dynamics model, Shingo Iwami and colleagues investigate the sample sizes required to detect significant antiviral drug effects on COVID-19 in randomized controlled trials.     

14C in Methane and DIC in the Deep Terrestrial Subsurface: Implications for Microbial Methanogenesis

A comparison between the ¹⁴C content of the methane and dissolved inorganic carbon (DIC) in deep, terrestrial subsurface systems was used to assess the timing of microbial methanogenesis contributing to gases in fracture water samples from three mines in the Witwatersrand Basin, South Africa. The results demonstrated that the majority of methane was produced over geologic timescales. In four of the samples, the methane contained no significant radiocarbon, indicating that the estimated 90% microbial methane in these samples was produced in the geologic past by indigenous microbial communities. In two samples from different mines, methane Δ¹⁴C levels indicated a primarily ancient origin for the microbial methane with the potential for more recent contributions from ongoing indigenous microbial activities constrained to between 0 and 40%, and 0 and 24%, respectively. Microbiological evidence for methanogenic archaea was observed in both of these samples. One sample had a Δ¹⁴C CH₄ that was higher than the corresponding DIC, indicating an extreme decoupling between these species and raising concerns over the representative quality of this sample. The variations in the Δ¹⁴C of DIC and CH₄ between and within mines demonstrate the need for a thorough assessment of each sample to obtain an accurate understanding of the role and timing of microbiological gas production in these complex, heterogeneous, terrestrial subsurface systems. The approach detailed here introduces timing as a new and widely applicable signature for the recognition of a major geochemical marker of indigenous life in the deep subsurface.