Objectives/Goals: The overall goal of this project is to determine bacterial transcriptional signatures from clinical sputum and assess their potential to monitor treatment response and predict the outcome of drug therapy in patients with tuberculosis (TB). Methods/Study Population: We are developing a novel transcript capture sequencing (TC-Seq) approach to sequence the mRNA of Mycobacterium tuberculosis (Mtb) and analyze transcriptomes from clinical samples containing minimal amounts of bacterial RNA. This protocol generates single-stranded biotinylated probes from Mtb DNA. Probes are hybridized to and allow enrichment of Mtb-specific mRNA within next-generation RNA sequencing libraries. We will apply TC-Seq to sputum samples collected throughout an 18-month Phase II clinical trial investigating response to TB treatment to compare the transcriptome of Mtb between patients whose treatment results in cure or relapse. Results/Anticipated Results: We have refined a technique to generate biotinylated probes starting from DNA of lab grown Mtb. This protocol achieves robust and unbiased sampling of the Mtb transcriptome from mixed samples containing both human and Mtb RNA. Preliminary sequencing of clinical sputum collected pretreatment has generated 1–4 million Mtb-specific reads, a sequencing depth that allows examination of the entire bacterial transcriptome. We will measure differential gene expression before and during treatment as well as between cure and relapse cases. These results will allow us to characterize bacterial response to treatment and identify bacterial markers that correlate with relapse. Discussion/Significance of Impact: Understanding Mtb activity during treatment will offer new ways to assess the efficacy of different treatment regimens. Crucially, identifying clear bacterial markers that demarcate a cure or relapse outcome will have a significant impact on determining patient eligibility for shorter drug therapy.

Microbial reduction of clay mineral structural Fe(III) decreases the swelling of nontronite gels, most importantly at intermediate oxidation states (40 to 80 cmol Fe(II) kg−1 clay). The purpose of this study was to establish whether microbial reduction of structural Fe(III) decreased the swelling of other Fe-bearing smectites and to discern the influence that organic compounds of microbial origin (bacterial cells, cell fragments and/or exudates) may have on clay swelling and texture. Structural Fe(III) was reduced by incubating smectite suspensions with either a combination of Pseudomonas bacteria or a mixture of anaerobic bacteria. The influence of organics on clay swelling was estimated on smectites suspended in either organic or inorganic media in the absence of bacteria. The gravimetric water content of the reduced clay gels equilibrated at various applied pressures was recorded as a function of Fe oxidation state. Transmission electron microscopy (TEM) was employed to determine the influence of bacteria and type of media on the texture of reduced smectite gels. Reduction of structural Fe(III) by bacteria decreased the swelling pressure of all Fe-bearing smectites. Increased clay swelling, due to the presence of organics (organic medium, exudates or cell fragments), was correlated to the total Fe content, the extent of structural Fe reduction, as well as the initial swelling characteristics of the Fe-bearing smectites. High structural Fe(II) contents (>50 cmol Fe(II) kg−1) resulted in increased attractive forces between clay platelets that decreased clay swelling, even in organic medium suspensions. Microbial reduction resulted in increased face-face association of individual clay layers, forming larger and more distinct crystallite subunits than in nonreduced clay gels. But, perhaps more importantly, microbial reduction of structural Fe(III) resulted in an increased association between crystallite subunits and, thus, an overall larger particle size and pore size distribution, due to the interaction of bacteria ceils, cell fragments and organic exudates.

Structural Fe in ferruginous smectite (sample SWa-1, Source Clays Repository of the Clay Minerals Society) was reduced by a mixture of five Pseudomonas species of bacteria in a defined Fe-free medium to determine the effect of microbial reduction on clay swelling. Iron(II), total Fe, and gravimetric water content (mw/mc) were determined in clay gels equilibrated at applied pressures of 0.1, 0.3, and 0.5 MPa. The water content of microbially reduced SWa-1 decreased at all three applied pressures as the Fe(II) content approached about 0.8 mmol Fe(II)/g-clay. As Fe(II) increased from 0.8 mmol/g-clay, however, further change in mw/mc was negligible. Concurrent with microbial reduction of structural Fe was a significant decrease in the swelling pressure (PI) of SWa-1: for example, when mw/mc = 1.2 (g/g), PI changed from 0.47 MPa at Fe(II) = 0.2, to 0.19 MPa at Fe(II) = 0.9 mmol/g-clay. Both biologically and chemically reduced smectites displayed lower values of mw/mc and a concurrent decrease in II as Fe(II) content increased, but the effect of Fe(II) on mw/mc was greater for the microbially reduced smectites at all applied pressures.

This study aims to understand the time-to-treatment initiation pre and post DAA access to inform strategies to improve HCV care. The data for our study were derived from the SuperMIX cohort study of people who inject drugs in Melbourne, Australia. Time-to-event analysis using Weibull accelerated failure time was performed for data collected between 2009 and 2021, among a cohort of HCV-positive participants. Among 223 participants who tested positive for active hepatitis C infection, 102 people (45.7%) reported treatment initiation, with a median time-to-treatment of 7 years. However, the median time-to-treatment reduced to 2.3 years for those tested positive after 2016. The study found that treatment with Opioid Agonist Therapy (TR 0.7, 95% CI 0.6–0.9), engagement with health or social services (TR 0.7, 95% CI 0.6–0.9), and having a first positive HCV RNA test after March 2016 (TR 0.3, 95% CI 0.2–0.3) were associated with a reduced time-to-treatment initiation. The study highlights the need for strategies to improve engagement with health services, including drug treatment services into routine HCV care to achieve timely treatment.

This paper estimates the effect of environmental remediation on housing prices in the Milwaukee Estuary Area of Concern (AOC) using a hedonic analysis of individual arms-length sales before and after a major remediation action between 2011 and 2015. Our design leverages this before-after comparison as well as the proximity of homes to remediation and the AOC boundary. Measuring the effect of AOCs in the housing market has always been a difficult task, given that water in AOCs can provide a mix of amenities and disamenities. Indeed, we find little evidence of a negative proximity effect when applying hedonic analysis to cross-section data. However, when we apply the analysis to a repeated cross section in a quasi-experimental framework, we find statistically significant evidence that living near the affected part of the AOC became more desirable after cleanup.

Effective management of uncertainty can lead to better, more informed decisions. However, many decision makers and their advisers do not always face up to uncertainty, in part because there is little constructive guidance or tools available to help. This paper outlines six Uncertainty Principles to manage uncertainty.

• Face up to uncertainty

• Deconstruct the problem

• Don’t be fooled (un/intentional biases)

• Models can be helpful, but also dangerous

• Think about adaptability and resilience

• Bring people with you

‘Munchausen's syndrome by proxy’ characteristically describes women alleged to have fabricated or induced illnesses in children under their care, purportedly to attract attention. Where conclusive evidence exists the condition's aetiology remains speculative, where such evidence is lacking diagnosis hinges upon denial of wrong-doing (conduct also compatible with innocence). How might investigators obtain objective evidence of guilt or innocence? Here, we examine the case of a woman convicted of poisoning a child. She served a prison sentence but continues to profess her innocence. Using a modified fMRI protocol (previously published in 2001) we scanned the subject while she affirmed her account of events and that of her accusers. We hypothesized that she would exhibit longer response times in association with greater activation of ventrolateral prefrontal and anterior cingulate cortices when endorsing those statements she believed to be false (i.e., when she ‘lied’). The subject was scanned 4 times at 3 Tesla. Results revealed significantly longer response times and relatively greater activation of ventrolateral prefrontal and anterior cingulate cortices when she endorsed her accusers' version of events. Hence, while we have not ‘proven’ that this subject is innocent, we demonstrate that her behavioural and functional anatomical parameters behave as if she were.

Mill's On Liberty is centrally concerned with avoiding social tyranny. But Mill's Principle of Liberty defines interfering, in the context of social pressure, as intentionally punishing and it seems to allow speech and actions that critics have thought would conflict with liberty in self-regarding matters. To critics, Mill draws distinctions among social influences where no genuine difference is to be found and he permits more social pressure than can be accepted by someone who values liberty highly. In this article, I explain where and why Mill draws the line he does between permitted and forbidden influences and show the line is coherent and tracks a genuine difference. I also show that although the Principle leaves residual social pressure, Mill has resources besides the Principle that can prevent social influences that threaten individuality while retaining beneficial social influences.

Sandstones of the Palaeocene Montrose Group were deposited in a deepwater fan environment, and form a major oil reservoir in the North Sea. Calcite concretions occur commonly within thick-bedded and structureless sandstones. These concretions have been identified by sonic logs and well reports, and were cross-checked with available core data. Regionally, 101 wells have been examined and carbonate concretions form 0.6–7.2% of the core. Concretions are most abundant along the flank of the Fladen Ground Spur, the north Witch Ground Graben (WGG), the east south Viking Graben and East Central Graben (ECG). Concretions of the ECG formed at deep burial, with C from decarboxylation. Geochemical inheritance of Mn and Sr from Cretaceous chalk clasts may occur. Concretion growth may also have been influenced by vertical expulsion of fluids (leak-off) localized above salt tectonics. Isotopic and petrographic evidence indicates that much carbonate C in the WGG was derived from biodegradation of migrating oil in meteoric water at shallow depth. The locations of abundant carbonate with characteristic negative C isotope signatures can be used as shallow exploration guides to leak-off points located above deep overpressured structures.

Metribuzin [4-amino-6-tert-butyl-3-(methylthio)-as-triazine-5(4H)one] (0, 0.56, 1.12, 1.68 kg/ha) applied the soybean (Glycine max (L.) Merr.) cultivars ‘Bragg,’ ‘Hampton,’ ‘Bienville,’ ‘Coker 318,’ ‘Coker 102,’ and ‘Hardee’ induced significant changes in total fatty acid concentration of soybean oil. Minor changes in soybean oil quality were induced by metribuzin but percent protein was not influenced.

Growth and percentage survival was measured for slash pine (Pinus elliottii Engelm.), shortleaf pine (P. echinata Mill.), Virginia pine (P. virginiana Mill.), and loblolly pine (P. taeda L.) seedlings treated with: (a) 0.07 to 4.48 kg/ha 2,6-dichlorobenzonitrile (dichlobenil), (b) 0.28 to 8.96 kg/ha 1,1-dimethyl-3-(a,a,a-trifluoro-m-tolyl)urea (fluometuron), or (c) 1.12 to 8.96 kg/ha S-propyl butylethylthiocarbamate (pebulate). Herbicide toxicity to pine seedlings was in order of dichlobenil > fluometuron > pebulate. Acceptance of mild growth reduction would permit pebulate utilization in seed bed nurseries.

CVD accounted for 27 % of all deaths in the UK in 2014, and was responsible for 1·7 million hospital admissions in 2013/2014. This condition becomes increasingly prevalent with age, affecting 34·1 and 29·8 % of males and females over 75 years of age respectively in 2011. The dysregulation of cholesterol metabolism with age, often observed as a rise in LDL-cholesterol, has been associated with the pathogenesis of CVD. To compound this problem, it is estimated by 2050, 22 % of the world's population will be over 60 years of age, in culmination with a growing resistance and intolerance to pre-existing cholesterol regulating drugs such as statins. Therefore, it is apparent research into additional therapies for hypercholesterolaemia and CVD prevention is a growing necessity. However, it is also imperative to recognise this complex biological system cannot be studied using a reductionist approach; rather its biological uniqueness necessitates a more integrated methodology, such as that offered by systems biology. In this review, we firstly discuss cholesterol metabolism and how it is affected by diet and the ageing process. Next, we describe therapeutic strategies for hypercholesterolaemia, and finally how the systems biology paradigm can be utilised to investigate how ageing interacts with complex systems such as cholesterol metabolism. We conclude by emphasising the need for nutritionists to work in parallel with the systems biology community, to develop novel approaches to studying cholesterol metabolism and its interaction with ageing.

To identify predictive factors and mortality of patients with influenza admitted to intensive care units (ICU) we carried out a prospective cohort study of patients hospitalized with laboratory-confirmed influenza in adult ICUs in a network of Canadian hospitals between 2006 and 2012. There were 626 influenza-positive patients admitted to ICUs over the six influenza seasons, representing 17·9% of hospitalized influenza patients, 3·1/10 000 hospital admissions. Variability occurred in admission rate and proportion of hospital influenza patients who were admitted to ICUs (proportion range by year: 11·7–29·4%; 21·3% in the 2009–2010 pandemic). In logistic regression models ICU patients were younger during the pandemic and post-pandemic period, and more likely to be obese than hospital non-ICU patients. Influenza B accounted for 14·2% of all ICU cases and had a similar ICU admission rate as influenza A. Influenza-related mortality was 17·8% in ICU patients compared to 2·0% in non-ICU patients.

The mean air temperature of the Icelandic interior is below 10 °C. However, we have previously observed 16S rDNA sequences associated with thermophilic lineages in Icelandic basalts. Measurements of the temperatures of igneous rocks in Iceland showed that solar insolation of these low albedo substrates achieved a peak surface temperature of 44.5 °C. We isolated seven thermophilic Geobacillus species from basalt with optimal growth temperatures of ~65 °C. The minimum growth temperature of these organisms was ~36 °C, suggesting that they could be active in the rock environment. Basalt dissolution rates at 40 °C were increased in the presence of one of the isolates compared to abiotic controls, showing its potential to be involved in active biogeochemistry at environmental temperatures. These data raise the possibility of transient active thermophilic growth in macroclimatically cold rocky environments, implying that the biogeographical distribution of active thermophiles might be greater than previously understood. These data show that temperatures measured or predicted over large scales on a planet are not in themselves adequate to assess niches available to extremophiles at micron scales.