Taurine is one of the most abundant amino acids in mammalian tissues. It is obtained from the diet and by de novo synthesis from cysteic acid or hypotaurine. Despite the discovery in 1954 that the oxygenation of hypotaurine produces taurine, the identification of an enzyme catalyzing this reaction has remained elusive. In large part, this is due to the incorrect assignment, in 1962, of the enzyme as an NAD-dependent hypotaurine dehydrogenase. For more than 55 years, the literature has continued to refer to this enzyme as such. Here we show, both in vivo and in vitro, that the enzyme that oxygenates hypotaurine to produce taurine is flavin-containing monooxygenase (FMO) 1. Metabolite analysis of the urine of Fmo1-null mice by ¹H NMR spectroscopy revealed a buildup of hypotaurine and a deficit of taurine in comparison with the concentrations of these compounds in the urine of wild-type mice. In vitro assays confirmed that human FMO1 catalyzes the conversion of hypotaurine to taurine, utilizing either NADPH or NADH as cofactor. FMO1 has a wide substrate range and is best known as a xenobiotic- or drug-metabolizing enzyme. The identification that the endogenous molecule hypotaurine is a substrate for the FMO1-catalyzed production of taurine resolves a long-standing mystery. This finding should help establish the role FMO1 plays in a range of biologic processes in which taurine or its deficiency is implicated, including conjugation of bile acids, neurotransmitter, antioxidant and anti-inflammatory functions, and the pathogenesis of obesity and skeletal muscle disorders.

Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80–2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD+-dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 in FdsB and Fe2S2 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH•, not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH−, initially formed via NADH-mediated reduction, to the Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in Mycobacterium tuberculosis, which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites. Furthermore, this allosteric mechanism is co-opted by pathway branchpoint enzyme chorismate mutase upon complex formation. In this study, using statistical coupling analysis, site-directed mutagenesis, isothermal calorimetry, small-angle X-ray scattering, and X-ray crystallography analyses, we have pinpointed a critical node within the complex dynamic communication network responsible for this sophisticated allosteric machinery. Through a facile Gly to Pro substitution, we have altered backbone dynamics, completely severing the allosteric signal yet remarkably, generating a nonallosteric enzyme that retains full catalytic activity. We also identified a second residue of prime importance to the inter-enzyme communication with chorismate mutase. Our results reveal that highly complex dynamic allostery is surprisingly vulnerable and provide further insights into the intimate link between catalysis and allostery.

Discovery of the Breagh gas field in the Southern North Sea (SNS) has demonstrated the potential that the Lower Carboniferous (Visean, 346.7–330.9 Ma) Farne Group reservoirs have to contribute to the UK's future energy mix. New biostratigraphic correlations provide a basis to compare Asbian and Brigantian sedimentary cores from the Breagh Field and age-equivalent sediments exposed on the Northumberland Coast, which has proved critical in gaining an understanding of exploration and development opportunities. Thirteen facies associations characterize the mixed carbonate–siliciclastic system, grouped into: marine, delta front, delta shoreface, lower delta plain and upper delta plain gross depositional environments. The facies associations are interpreted as depositing in a mixed carbonate and siliciclastic fluvio-deltaic environment, and are arranged into coarsening- and cleaning-upward cycles (parasequences) bounded by flooding surfaces. Most cycles are characterized by mouth bars, distributary channels, interdistributary bays and common braided rivers, interpreted as river-dominated deltaic deposits. Some cycles include rare shoreface and tidally-influenced deposits, interpreted as river-dominated and wave- or tide-influenced deltaic deposits. The depositional processes that formed each cycle have important implications for the reservoir net/gross ratio (where this ratio indicates the proportion of sandstone beds in a cycle), thickness and lateral extent. The deltaic deposits were controlled by a combination of tectonic and eustatic (allocyclic) events and delta avulsion (autocyclic) processes, and are likely to reflect a changing tectonic regime, from extension within elongate fault-bounded basins (synrift) to passive regional thermal subsidence (post-rift). Deep incision by the Base Permian Unconformity across the Breagh Field has removed the Westphalian, Namurian and upper Visean, to leave the more prospective thicker clastic reservoirs within closure.

Thematic collection: This article is part of the Under-explored plays and frontier basins of the UK continental shelf collection available at: https://www.lyellcollection.org/cc/under-explored-plays-and-frontier-basins-of-the-uk-continental-shelf

Exploration success at Breagh demonstrates that western parts of the Mid North Sea High area are prospective despite the absence of an Upper Permian (Rotliegend Group) Leman Sandstone Formation reservoir and source rocks belonging to the Upper Carboniferous Westphalian Coal Measures Group. Detailed seismic and well interpretation shows that the Breagh trap was a long-lived footwall high, the prospectivity of which was enhanced by Variscan folding and uplift, leading to the truncation (subcrop) of Lower Carboniferous reservoirs beneath the Base Permian Unconformity. Its drape (supra-crop) by Upper Permian (Zechstein Super Group) evaporites creates the seal. The complexity of its overburden means that an accurate picture of the Breagh structure only emerges after accurate depth-conversion that takes the effects of the Mesozoic graben into account. Pronounced easterly tilting during the Cenozoic affected the area and controlled gas migration into the structure from palaeostructures lying to the east. However, evidence that Breagh is not filled to spill point (underfill) suggests that charge limitation remained an issue. The study demonstrates that a poorly-documented and under-explored Lower Carboniferous play exists in Southern North Sea, which relies upon careful structural mapping and basin modelling to be undertaken for the play to be understood and its further potential to be realized.

Thematic collection: This article is part of the Under-explored plays and frontier basins of the UK continental shelf collection available at: https://www.lyellcollection.org/cc/under-explored-plays-and-frontier-basins-of-the-uk-continental-shelf

The Dukla Nappe in the Skrzydlna area exposes two types of mélange reflecting two different phases of basinal and tectonic evolution of the Outer West Carpathian orogen in its Polish sector. The Oligocene-age sedimentary mélange (olistostrome) is related to growth of the accretionary wedge, whereas the Miocene-age diapiric mélange postdates the orogenic thrusting. Textural and structural features of the very coarse-grained sedimentary mélange suggest non-cohesive debris flows and high-density turbidity currents as predominant emplacement mechanisms. Growth strata, associated with progressive unconformities, and facies contrast between the underlying fine-grained unit and the overlying olistostrome reflect a considerable uplift of the source area and rotation of the adjacent part of the basin floor. The olistostrome and the overlying turbidite succession form a retrogressive sequence interpreted as a submarine canyon infill grading to a small submarine fan. The diapiric mélange, injected into the Oligocene-age succession of the Dukla Nappe, contains the Early and Late Cretaceous-age blocks and matrix derived from the underlying Silesian Nappe. The features reflecting diapiric emplacement include matrix proportion increase and block content decrease towards the mélange margins, scaly fabric and shear zones. Both mélanges, interpreted in the past as chaotic bodies, upon detailed examination reveal genesis-related subtle internal organization.

The Lower and Middle Ordovician of the Yangtze Platform, China, is characterized by a sedimentary succession dominated by carbonate rocks. Three sections spanning the Nantsinkuan/Lunshan, Fenhsiang, Hunghuayuan, and Dawan/Zitai formations, corresponding to the Tremadocian–Dapingian in age, have been sampled for high-resolution ¹³C chemostratigraphy (542 samples in total). Our new ¹³C data reveal five tie-points with the potential for global correlation: (1) a positive ¹³C excursion in the lower Nantsinkuan Formation within the Tremadocian Rossodus manitouensis Zone; (2) an excursion with two peaks roughly within the late Tremadocian Paltodus ‘deltifer’ Zone; (3) a positive ¹³C shift in the lower Hunghuayuan Formation, within the early Floian Serratognathus diversus Zone; (4) a gradual positive ¹³C shift in the late Floian, ranging from the uppermost S. diversus Zone to the basal Oepikodus evae Zone; (5) a minor negative shift in the lower Dawan/Zitai Formation, within the early Dapingian Baltoniodus triangularis Zone. These excursions are herein used for correlation of the Yangtze Platform strata with successions from South China, North China, the Argentine Precordillera, North America and Baltica. From a palaeogeographical perspective the Gudongkou, Xiangshuidong and Daling sections represent depositional environments along an inner to outer ramp profile. ¹³C data from these sections show successively heavier (higher) ¹³C values with increasing depositional depth. This is interpreted as due to remineralization of organic carbon within the carbonate rocks.

Supplementary material: Carbon and oxygen isotope data are available at: https://doi.org/10.6084/m9.figshare.c.4767080

The Northern Paraguai Belt, at the SE border of the Amazonian Craton, central Brazil, has been interpreted as a Brasiliano–Pan-African (c. 650–600 Ma) belt with a foreland basin, recording collisional polyphase tectonism and greenschist-facies metamorphism extending from the late Precambrian to the Cambrian–Ordovician. New structural investigations indicate that the older metasedimentary rocks of the Cuiabá Group represent a Tonian–Cryogenian basement assemblage deformed in two contemporaneous fault-bounded structural sub-domains of wrench-dominated (rake <10°) and contraction-dominated (rake ~30–40°) sinistral transpression, with tectonic vergence towards the SE. The younger late Cryogenian to early Cambrian sedimentary rocks lying to the NW of the Cuiabá Group are non-metamorphic and display only pervasive brittle transtension characterized by normal-oblique faults, fractures and forced drag folds with no consistent vergence pattern. Our analyses suggest that an unconformity separates the metasedimentary Cuiabá Group basement of the Northern Paraguai Belt from the unmetamorphosed sedimentary cover. It is proposed that the latter units were deposited during a post-glacial marine transgression (after c. 635 Ma) in a post-collisional basin. The Paraguai Belt basement and its post-collisional sedimentary cover share a number of significant geological similarities with sequences in the Bassarides Belt and Taoudéni Basin in the SW portion of the West African Craton.

The Tabaquito batholith (Frontal Cordillera, western Argentina), is mainly composed of shallowly emplaced granodiorite to minor monzogranite with abundant mafic microgranular enclaves. New sensitive high-resolution ion microprobe U–Pb zircon ages of c. 337 Ma (biotite granodiorite) and c. 284 Ma (mafic dyke) along with previously published geochronological data suggest that a long-lived magmatic system formed through at least two magmatic pulses at c. 337 and c. 322 Ma with later superimposition of Permian magmatism. The Tabaquito granitoids are metaluminous, calc-alkalic and magnesian with I-type affinity. Elevated Th/Nb, Y/Nb and La/Nb ratios along with negative Nb–Ta and positive Pb anomalies are consistent with a continental arc setting. Hf, Nd and Sr isotopic composition of the Tabaquito granitoids suggests that their source could result from mixing of an old felsic crustal component and a juvenile mafic to intermediate component. New geochronological and geochemical data together with published data reveal a continuous arc setting from the Carboniferous to the Permian in Argentina, and important magmatic compositional variations through time and space controlled by episodic fluctuations in the subduction angle of the oceanic plate. Reported and compiled data allow us to infer the continuity of the Carboniferous magmatic arc along the west margin of Gondwana.

Supplementary material: Detailed petrography, analytical methods and data, zircon cathodoluminescence images and supplementary figures are available at https://doi.org/10.6084/m9.figshare.c.4763993

Bovine tuberculosis (TB), caused by Mycobacterium bovis, remains an important zoonotic disease posing a serious threat to livestock and wildlife. The current TB tests relying on cell-mediated and humoral immune responses in cattle have performance limitations. To identify new serodiagnostic markers of bovine TB, we screened a panel of 101 recombinant proteins, including 10 polyepitope fusions, by a multiantigen print immunoassay (MAPIA) with well-characterized serum samples serially collected from cattle with experimental or naturally acquired M. bovis infection. A novel set of 12 seroreactive antigens was established. Evaluation of selected proteins in the dual-path platform (DPP) assay showed that the highest diagnostic accuracy (~95%) was achieved with a cocktail of five best-performing antigens, thus demonstrating the potential for development of an improved and more practical serodiagnostic test for bovine TB.

The global burden of disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) is increasing as the prevalence of multidrug-resistant strains rises. A multivalent ExPEC O-antigen bioconjugate vaccine could have a substantial impact in preventing bacteremia and urinary tract infections. Development of an ExPEC vaccine requires a readout to assess the functionality of antibodies. We developed an opsonophagocytic killing assay (OPA) for four ExPEC serotypes (serotypes O1A, O2, O6A, and O25B) based on methods established for pneumococcal conjugate vaccines. The performance of the assay was assessed with human serum by computing the precision, linearity, trueness, total error, working range, and specificity. Serotypes O1A and O6A met the acceptance criteria for precision (coefficient of variation for repeatability and intermediate precision, ≤50%), linearity (90% confidence interval of the slope of each strain, 0.80, 1.25), trueness (relative bias range, –30% to 30%), and total error (total error range, –65% to 183%) at five serum concentrations and serotypes O2 and O25B met the acceptance criteria at four concentrations (the lowest concentration for serotypes O2 and O25B did not meet the system suitability test of maximum killing of ≥85% of E. coli cells). All serotypes met the acceptance criteria for specificity (opsonization index value reductions of ≤20% for heterologous serum preadsorption and ≥70% for homologous serum preadsorption). The assay working range was defined on the basis of the lowest and highest concentrations at which the assay jointly fulfilled the target acceptance criteria for linearity, precision, and accuracy. An OPA suitable for multiple E. coli serotypes has been developed, qualified, and used to assess the immunogenicity of a 4-valent E. coli bioconjugate vaccine (ExPEC4V) administered to humans.

South Africa is a mineral-rich country with a diverse geology and a long history of mining. The rich history of mining activities includes the extraction of coal from the Ecca Group Sediments of the Karoo Supergroup (250 Ma), gold and uranium from the Witwatersrand Supergroup (2900 Ma), as well as platinum, uranium, tin and lead from the layered Bushveld Igneous Complex (BIC) (2150 Ma). The extraction of gold, copper, tin, lead and rare earth minerals also took place in the Archean rocks of Swazium age (3500–3000 Ma). The historical mining records have either not been accurately recorded or have been lost over time. This has resulted in significant geohazard risk during infrastructure development, especially in and around historical mining towns, such as Johannesburg and Ermelo. These geohazard risks require careful appraisal and quantification prior to any infrastructure design or construction.

This case study aims to set out the development aspects of the Multi-Faceted Geophysical Modelling Systems approach, which was used by the South African National Roads Agency SOC Ltd (SANRAL) during an investigation of undermined ground for the historical coal-mining town of Ermelo in South Africa. The N11/N2 ring road was planned to go around Ermelo to ensure mobility between major routes, whilst still maintaining town access.

The systems approach used a combination of airborne geophysics (Versatile Time Domain Electromagnetic System (VTEM^TM) and magnetics), generally used in mining exploration, land-based and borehole geophysics, borehole water testing, and ground-truthing. The approach was continuous and iterative, building on the data at hand and reducing unnecessary investigations while eliminating the possibility of anomalies being missed, as in the case of conventional discrete drilling. The investigation ensured that 100% of the route was comprehensively investigated with a high confidence in the geological and geophysical data, and concomitant mitigation of infrastructure risk.

The Multi-Faceted Geophysical Modelling Systems approach was successfully used to identify a previously unknown 1 x 1 m mining stope cavity at 90 m depth and a 3 x 5 m access tunnel at 24 m depth in a timely and cost-effective manner. Seven reverse-circulation percussion boreholes confirmed the structural integrity of these underground cavities, as well as the structural geology along the centreline. Based on the great success achieved in identifying shallow anomalies, this Multi-Faceted Geophysical Modelling Systems approach is now being considered for field trails on the dolomitic formations and the Wild Coast greenfields road project where there are large historical slumps and many fault lines.

Thematic collection: This article is part of the Ground-related risk to transportation infrastructure collection available at https://www.lyellcollection.org/cc/Ground-related-risk-to-transportation-infrastructure

This case study aims to identify the cubic capacity and geometry of the geological body of silt–organic sediments in the environment of a former gravel pit situated in a drainless depression of the alluvium of the Čierna voda River. It is located in the well-known geotourism locality of Slnečné Jazera in Senec, in the SW of Slovakia. To identify the body, electrical resistivity tomography was combined with the use of sonar. The research shows that this approach is appropriate for a number of activities that are subjects of engineering-geological investigations. The cubic capacity and geometry of specific aqueous engineering-geological environments must be determined in connection with the need for the management, control, quantification and extraction of selected sedimentary bodies. In addition, the management of sustainable development of reservoirs, sedimentation basins, industrial ponds, settling pits and natural pools for recreation (the subject of the case study) is important to control the limit amounts of sediments in such environments. The results of this study may be applied in analogous engineering-geological conditions. The drainless depression Slnečné Jazera contained a body of silt–organic sediments amounting to 23 000 m³ (41 Olympic-size pools of 25 m x 12.5 m x 1.8 m). The maximum thickness of the bottom sediments was about 6.3 m on the alluvium with an articulated morphology owing to the submerged digging of gravel. The proposed approach improved the control of extraction of the sedimentary body and optimized the engineering-geological conditions in this geotourism locality.

Routine identification of fungal pathogens from positive blood cultures by culture-based methods can be time-consuming, delaying treatment with appropriate antifungal agents. The GenMark Dx ePlex investigational use only blood culture identification fungal pathogen panel (BCID-FP) rapidly detects 15 fungal targets simultaneously in blood culture samples positive for fungi by Gram staining. We aimed to determine the performance of the BCID-FP in a multicenter clinical study. Blood culture samples collected at 10 United States sites and tested with BCID-FP at 4 sites were compared to the standard-of-care microbiological and biochemical techniques, fluorescence in situ hybridization using peptide nucleic acid probes (PNA-FISH) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Discrepant results were analyzed by bi-directional PCR/sequencing of residual blood culture samples. A total of 866 clinical samples, 120 retrospectively and 21 prospectively collected, along with 725 contrived samples were evaluated. Sensitivity and specificity of detection of Candida species (C. albicans, C. auris, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. kefyr, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis) ranged from 97.1 to 100% and 99.8 to 100%, respectively. For the other organism targets, sensitivity and specificity were as follows: 100% each for Cryptococcus neoformans and C. gattii, 98.6% and 100% for Fusarium spp., and 96.2% and 99.9% for Rhodotorula spp., respectively. In 4 of the 141 clinical samples, the BCID-FP panel correctly identified an additional Candida species, undetected by standard-of-care methods. The BCID-FP panel offers a faster turnaround time for identification of fungal pathogens in positive blood cultures that may allow for earlier antifungal interventions and includes C. auris, a highly multidrug-resistant fungus.

Infections due to methicillin-resistant Staphylococcus aureus (MRSA) are present worldwide and represent a major public health concern. The capability of PCR followed by high-resolution melt (HRM) curve analysis for the detection of community-associated and livestock-associated MRSA strains and the identification of staphylococcal protein A (spa) locus was evaluated in 74 MRSA samples which were isolated from the environment, humans, and pigs on a single piggery. PCR-HRM curve analysis identified four spa types among MRSA samples and differentiated MRSA strains accordingly. A nonsubjective differentiation model was developed according to genetic confidence percentage values produced by tested samples, which did not require visual interpretation of HRM curve results. The test was carried out at different settings, and result data were reanalyzed and confirmed with DNA sequencing. PCR-HRM curve analysis proved to be a robust and reliable test for spa typing and can be used as a tool in epidemiological studies.

Microscopy and rapid diagnostic tests (RDTs) are the main diagnostic tools for malaria but fail to detect low-density parasitemias that are important for maintaining malaria transmission. To complement existing diagnostic methods, an isothermal reverse transcription-recombinase polymerase amplification and lateral flow assay (RT-RPA) was developed. We compared the performance with that of ultrasensitive reverse transcription-quantitative PCR (uRT-qPCR) using nucleic acid extracts from blood samples (n = 114) obtained after standardized controlled human malaria infection (CHMI) with Plasmodium falciparum sporozoites. As a preliminary investigation, we also sampled asymptomatic individuals (n = 28) in an area of malaria endemicity (Lambaréné, Gabon) to validate RT-RPA and assess its performance with unprocessed blood samples (dbRT-RPA). In 114 samples analyzed from CHMI trials, the positive percent agreement to uRT-qPCR was 90% (95% confidence interval [CI], 80 to 96). The negative percent agreement was 100% (95% CI, 92 to 100). The lower limit of detection was 64 parasites/ml. In Gabon, RT-RPA was 100% accurate with asymptomatic volunteers (n = 28), while simplified dbRT-RPA showed 89% accuracy. In a subgroup analysis, RT-RPA detected 9/10 RT-qPCR-positive samples, while loop-mediated isothermal amplification (LAMP) detected 2/10. RT-RPA is a reliable diagnostic test for asymptomatic low-density infections. It is particularly useful in settings where uRT-qPCR is difficult to implement.

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum. The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum. Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

Limited treatment options contribute to high morbidity/mortality rates with carbapenem-resistant, Gram-negative bacterial infections. New approaches for carbapenemase-producing organism (CPO) detection may help inform clinician decision-making on patient treatment and infection control. BD Phoenix CPO detect (CPO detect) detects and classifies carbapenemases in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa during susceptibility testing. The clinical performance of CPO detect is reported here. Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa isolates were evaluated across three sites using CPO detect and a composite reference method (RM); the latter was comprised of the modified carbapenem inactivation method and a MIC screen for ertapenem, imipenem, and meropenem. Multiplex PCR testing was also utilized for Ambler class determination. Positive and negative percentages of agreement (PPA and NPA, respectively) between CPO detect and the RM were determined. The PPA and NPA for Enterobacterales were 98.5% (confidence intervals, 96.6%, 99.4%) and 97.2% (95.8%, 98.2%), respectively. The A. baumannii PPA and NPA, respectively, were 97.1% (90.2%, 99.2%) and 97.1% (89.9%, 99.2%). The P. aeruginosa PPA and NPA, respectively, were 95.9% (88.6%, 98.6%) and 92.3% (86.7%, 95.6%). The PPA values for carbapenemase class designations for all organisms combined and Enterobacterales alone, respectively, were 95.3% (90.2%, 97.8%) and 94.6% (88.8%, 97.5%) for class A, 94.0% (88.7%, 96.6%) and 96.4% (90.0%, 98.8%) for class B, and 95.0% (90.1%, 97.6%) and 99.0% (94.4%, 99.8%) for class D carbapenemases. NPA values for all organisms and Enterobacterales alone ranged from 98.5% to 100%. CPO detect provided accurate detection and classification of CPOs for the majority of isolates of Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa tested.

MALDI-TOF mass spectrometry (MS) identification of pathogenic filamentous fungi is often impaired by difficulties in harvesting hyphae embedded in the medium and long extraction protocols. The ID Fungi Plate (IDFP) is a novel culture method developed to address such difficulties and improve the identification of filamentous fungi by MALDI-TOF MS. We cultured 64 strains and 11 clinical samples on IDFP, Sabouraud agar-chloramphenicol (SAB), and ChromID Candida agar (CAN2). We then compared the three media for growth, ease of harvest, amount of material picked, and MALDI-TOF identification scores after either rapid direct transfer (DT) or a long ethanol-acetonitrile (EA) extraction protocol. Antifungal susceptibility testing and microscopic morphology after subculture on SAB and IDFP were also compared for ten molds. Growth rates and morphological aspects were similar for the three media. With IDFP, harvesting of fungal material for the extraction procedure was rapid and easy in 92.4% of cases, whereas it was tedious on SAB or CAN2 in 65.2% and 80.3% of cases, respectively. The proportion of scores above 1.7 (defined as acceptable identification) were comparable for both extraction protocols using IDFP (P = 0.256). Moreover, rates of acceptable identification after DT performed on IDFP (93.9%) were significantly higher than those obtained after EA extraction with SAB (69.7%) or CAN2 (71.2%) (P = <0.001 and P = 0.001, respectively). Morphological aspects and antifungal susceptibility testing were similar between IDFP and SAB. IDFP is a culture plate that facilitates and improves the identification of filamentous fungi, allowing accurate routine identification of molds with MALDI-TOF-MS using a rapid-extraction protocol.

The number of onsite clinical microbiology laboratories in hospitals is decreasing, likely related to the business model for laboratory consolidation and labor shortages, and this impacts a variety of clinical practices, including that of banking isolates for clinical or epidemiologic purposes. To determine the impact of these trends, infectious disease (ID) physicians were surveyed regarding their perceptions of offsite services. Clinical microbiology practices for retention of clinical isolates for future use were also determined. Surveys were sent to members of the Infectious Diseases Society of America’s (IDSA) Emerging Infections Network (EIN). The EIN is a sentinel network of ID physicians who care for adult and/or pediatric patients in North America and who are members of IDSA. The response rate was 763 (45%) of 1,680 potential respondents. Five hundred forty (81%) respondents reported interacting with the clinical microbiology laboratory. Eighty-six percent of respondents thought an onsite laboratory very important for timely diagnostic reporting and ongoing communication with the clinical microbiologist. Thirty-five percent practiced in institutions where the core microbiology laboratory has been moved offsite, and an additional 7% (n = 38) reported that movement of core laboratory functions offsite was being considered. The respondents reported that only 24% of laboratories banked all isolates, with the majority saving isolates for less than 30 days. Based on these results, the trend toward centralized core laboratories negatively impacts the practice of ID physicians, potentially delays effective implementation of prompt and targeted care for patients with serious infections, and similarly adversely impacts infection control epidemiologic investigations.

Serological testing for nasopharyngeal carcinoma (NPC) has recently been reinvigorated by the implementation of novel Epstein-Barr virus (EBV)-specific IgA and IgG antibodies from a proteome array. Although proteome arrays are well suited for comprehensive antigen selection, they are not applicable for large-scale studies. We adapted a 13-marker EBV antigen signature for NPC risk identified by proteome arrays to multiplex serology to establish an assay for large-scale studies. Taiwanese NPC cases (n = 175) and matched controls (n = 175) were used for assay validation. Spearman’s correlation was calculated, and the diagnostic value of all multiplex markers was assessed independently using the area under the receiver operating characteristic curve (AUC). Two refined signatures were identified using stepwise logistic regression and internally validated with 10-fold cross validation. Array and multiplex serology showed strong correlation for each individual EBV marker, as well as for a 13-marker combined model on continuous data. Two refined signatures with either four (LF2 and BGLF2 IgG, LF2 and BMRF1 IgA) or two (LF2 and BGLF2 IgG) antibodies on dichotomous data were identified as the most parsimonious set of serological markers able to distinguish NPC cases from controls with AUCs of 0.992 (95% confidence interval [CI], 0.983 to 1.000) and 0.984 (95% CI, 0.971 to 0.997), respectively. Neither differed significantly from the 13-marker model (AUC, 0.992; 95% CI, 0.982 to 1.000). All models were internally validated. Multiplex serology successfully validated the original EBV proteome microarray data. Two refined signatures of four and two antibodies were capable of detecting NPC with 99.2% and 98.4% accuracy.

Recent progress has been made in identifying genomic regions implicated in trait evolution on a microevolutionary scale in many species, but whether these are relevant over macroevolutionary time remains unclear. Here, we directly address this fundamental question using bird beak shape, a key evolutionary innovation linked to patterns of resource use, divergence, and speciation, as a model trait. We integrate class-wide geometric-morphometric analyses with evolutionary sequence analyses of 10,322 protein-coding genes as well as 229,001 genomic regions spanning 72 species. We identify 1434 protein-coding genes and 39,806 noncoding regions for which molecular rates were significantly related to rates of bill shape evolution. We show that homologs of the identified protein-coding genes as well as genes in close proximity to the identified noncoding regions are involved in craniofacial embryo development in mammals. They are associated with embryonic stem cell pathways, including BMP and Wnt signaling, both of which have repeatedly been implicated in the morphological development of avian beaks. This suggests that identifying genotype-phenotype association on a genome-wide scale over macroevolutionary time is feasible. Although the coding and noncoding gene sets are associated with similar pathways, the actual genes are highly distinct, with significantly reduced overlap between them and bill-related phenotype associations specific to noncoding loci. Evidence for signatures of recent diversifying selection on our identified noncoding loci in Darwin finch populations further suggests that regulatory rather than coding changes are major drivers of morphological diversification over macroevolutionary times.

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

Decreased release of palmitic acid methyl ester (PAME), a vasodilator, from perivascular adipose tissue (PVAT) might contribute to hypertension pathogenesis. However, the PAME biosynthetic pathway remains unclear. In this study, we hypothesized that PAME is biosynthesized from palmitic acid (PA) via human catechol-O-methyltransferase (COMT) catalysis and that decreased PAME biosynthesis plays a role in hypertension pathogenesis. We compared PAME biosynthesis between age-matched normotensive Wistar Kyoto (WKY) rats and hypertensive spontaneously hypertensive rats (SHRs) and investigated the effects of losartan treatment on PAME biosynthesis. Computational molecular modeling indicated that PA binds well at the active site of COMT. Furthermore, in in vitro enzymatic assays in the presence of COMT and S-5'-adenosyl-L-methionine (AdoMet), the stable isotope [¹³C₁₆]-PA was methylated to form [¹³C₁₆]-PAME in incubation medium or the Krebs–Henseleit solution containing 3T3-L1 adipocytes or rat PVAT. The adipocytes and PVATs expressed membrane-bound (MB)-COMT and soluble (S)-COMT proteins. [¹³C₁₆]-PA methylation to form [¹³C₁₆]-PAME in 3T3-L1 adipocytes and rat PVAT was blocked by various COMT inhibitors, such as S-(5'-adenosyl)-L-homocysteine, adenosine-2',3'-dialdehyde, and tolcapone. MB- and S-COMT levels in PVATs of established SHRs were significantly lower than those in PVATs of age-matched normotensive WKY rats, with decreased [¹³C₁₆]-PA methylation to form [¹³C₁₆]-PAME. This decrease was reversed by losartan, an angiotensin II (Ang II) type 1 receptor antagonist. Therefore, PAME biosynthesis in rat PVAT is dependent on AdoMet, catalyzed by COMT, and decreased in SHRs, further supporting the role of PVAT/PAME in hypertension pathogenesis. Moreover, the antihypertensive effect of losartan might be due partly to its increased PAME biosynthesis.

We investigated whether cycloheximide (CHX) would induce amnesia for the stress-induced impairment of extinction retrieval. First, a single restraint stress session was demonstrated to impair extinction retrieval, but not fear conditioning. A second experiment showed that when CHX was administered immediately after restraint, rats exhibited significant extinction retrieval at test (i.e., retrograde amnesia for the stress). In a third experiment, the stress session impaired various amounts of extinction durations, suggesting that the stress inhibited extinction retrieval rather than enhancing the original fear learning. These results suggest memories for acute stress are susceptible to disruption, which could have clinical implications.

Annelies Wouters, Jan-Pieter Ploem, Sabine A. S. Langie, Tom Artois, Aziz Aboobaker, and Karen Smeets

Pluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian Schmidtea mediterranea. Exposure to the genotoxic compound methyl methanesulfonate (MMS) revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, responses differed between anterior and posterior fragments after amputation. Stem cell proliferation and differentiation proceeded successfully in the amputated heads, leading to regeneration of missing tissues. Stem cells in the amputated tails showed decreased proliferation and differentiation capacity. As a result, tails could not regenerate. Interference with the body-axis-associated component β-catenin-1 increased regenerative success in tail fragments by stimulating proliferation at an early time point. Our results suggest that differences in the Wnt signalling gradient along the body axis modulate stem cell responses to MMS.

Kristi McElmurry, Jessica E. Stone, Donghan Ma, Phillip Lamoureux, Yueyun Zhang, Michelle Steidemann, Lucas Fix, Fang Huang, Kyle E. Miller, and Daniel M. Suter

Previously, we have shown that bulk microtubule (MT) movement correlates with neurite elongation, and blocking either dynein activity or MT assembly inhibits both processes. However, whether the contributions of MT dynamics and dynein activity to neurite elongation are separate or interdependent is unclear. Here, we investigated the underlying mechanism by testing the roles of dynein and MT assembly in neurite elongation of Aplysia and chick neurites using time-lapse imaging, fluorescent speckle microscopy, super-resolution imaging and biophysical analysis. Pharmacologically inhibiting either dynein activity or MT assembly reduced neurite elongation rates as well as bulk and individual MT anterograde translocation. Simultaneously suppressing both processes did not have additive effects, suggesting a shared mechanism of action. Single-molecule switching nanoscopy revealed that inhibition of MT assembly decreased the association of dynein with MTs. Finally, inhibiting MT assembly prevented the rise in tension induced by dynein inhibition. Taken together, our results suggest that MT assembly is required for dynein-driven MT translocation and neurite outgrowth.

Background

Advances in information communications technology (ICT) provide opportunities for enhanced diabetes care. Knowledge of the more acceptable communication modalities in patients of different ages will help to inform the direction of future innovations.

Factors contributing to therapeutic inertia related to patients’ medication experiences include concerns about side effects and out-of-pocket costs, stigmatization for having diabetes, confusion about frequent changes in evidence-based guidelines, low health literacy, and social determinants of health. A variety of solutions to this multifactorial problem may be necessary, including integrating pharmacists into interprofessional care teams, using medication refill synchronization programs, maximizing time with patients to discuss fears and concerns, being cognizant of language used to discuss diabetes-related topics, and avoiding stigmatizing patients. Managing diabetes successfully is a team effort, and the full commitment of all team members (including patients) is required to achieve desired outcomes through an individualized approach.

Diabetes educators can be challenged by therapeutic inertia, as has been documented with other health care providers. There are many contributing factors related to the educators themselves, their patients, and the health care system in which they operate. To address this potentially significant barrier to quality patient care, diabetes educators can adopt numerous strategies to maximize their impact and address the factors contributing to therapeutic inertia in their practices.

Background and objectives

The effects of active case finding (ACF) models that mobilise community networks for early identification and treatment of tuberculosis (TB) remain unknown. We investigated and compared the effect of community-based ACF using a seed-and-recruit model with one-off roving ACF and passive case finding (PCF) on the time to treatment initiation and identification of bacteriologically confirmed TB.

Recent advances in cough research suggest a more widespread use of cough-provocation tests to demonstrate the hypersensitivity of the cough reflex arc. Cough-provocation tests with capsaicin or acidic aerosols have been used for decades in scientific studies. Several factors have hindered their use in everyday clinical work: i.e. lack of standardisation, the need for special equipment and the limited clinical importance of the response. Cough-provocation tests with hypertonic aerosols (CPTHAs) involve provocations with hypertonic saline, hypertonic histamine, mannitol and hyperpnoea. They probably act via different mechanisms than capsaicin and acidic aerosols. They are safe and well tolerated and the response is repeatable. CPTHAs can assess not only the sensitivity of the cough reflex arc but also the tendency of the airway smooth muscles to constrict (airway hyper-responsiveness). They can differentiate between subjects with asthma or chronic cough and healthy subjects. The responsiveness to CPTHAs correlates with the cough-related quality of life among asthmatic subjects. Furthermore, the responsiveness to them decreases during treatment of chronic cough. A severe response to CPTHAs may indicate poor long-term prognosis in chronic cough. The mannitol test has been stringently standardised, is easy to administer with simple equipment, and has regulatory approval for the assessment of airway hyper-responsiveness. Manual counting of coughs during a mannitol challenge would allow the measurement of the function of the cough reflex arc as a part of clinical routine.

ABSTRACT

The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease (cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, β-lactamase (βlac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop (⁷⁶⁷DKE⁷⁶⁹) connecting α15 and α16 (cis-loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K⁷⁶⁸ was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis-loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis-loop in LC translocation, independently of pore formation.

IMPORTANCE How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, β-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (βlac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K⁷⁶⁸ in LC translocation. K⁷⁶⁸ was located between α15 and α16 (termed the cis-loop). Cellular assays showed that K⁷⁶⁸ did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K⁷⁶⁸ is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.

Among the emerging techniques to detect the real footprint of buried ore deposits is isotope tracing. Novel and automated preparation systems such as continuous flow isotope ratio mass spectrometry, off-axis integrated cavity output spectroscopy for isotopic compositions of selected molecules, multi-collector inductively coupled-plasma mass spectrometry (ICP-MS), triple quadrupole ICP-MS, laser ablation ICP-MS, and a multitude of inline preparation systems have facilitated the use of isotopes as tracers in mineral exploration, as costs for isotope analyses have decreased and the time required for the analyses has improved. In addition, the isotope systems being used have expanded beyond the traditional light stable and Pb isotopes to include a multitude of elements that behave differently during processes that promote the mobilization of elements during both primary and secondary dispersion. Isotopes are also being used to understand barren areas that lack a critical process to form an ore deposit and to reveal precise redox mechanisms. The goal is to be able to use isotopes to reflect a definitive process that occurs in association with the deposit and not in barren systems, and then to relate these to something that is easier to measure, namely elemental concentrations. As new generations of exploration and environmental scientists are becoming more comfortable with the application of isotopes to effectively trace processes involved in geoscience, and new technologies for rapid and inexpensive analyses of isotopes are continually being developed, novel applications of isotope tracing are becoming more mainstream.

Thematic collection: This article is part of the Exploration 17 collection available at: https://www.lyellcollection.org/cc/exploration-17

In the past decade, significant research efforts have been devoted to mineral chemistry studies to assist porphyry exploration. These activities can be divided into two major fields of research: (1) porphyry indicator minerals (PIMs), which are used to identify the presence of, or potential for, porphyry-style mineralization based on the chemistry of magmatic minerals such as zircon, plagioclase and apatite, or resistate hydrothermal minerals such as magnetite; and (2) porphyry vectoring and fertility tools (PVFTs), which use the chemical compositions of hydrothermal minerals such as epidote, chlorite and alunite to predict the likely direction and distance to mineralized centres, and the potential metal endowment of a mineral district. This new generation of exploration tools has been enabled by advances in and increased access to laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), short-wave length infrared (SWIR), visible near-infrared (VNIR) and hyperspectral technologies. PIMs and PVFTs show considerable promise for exploration and are starting to be applied to the diversity of environments that host porphyry and epithermal deposits globally. Industry has consistently supported development of these tools, and in the case of PVFTs encouraged by several successful blind tests where deposit centres have successfully been predicted from distal propylitic settings. Industry adoption is steadily increasing but is restrained by a lack of the necessary analytical equipment and expertise in commercial laboratories, and also by the ongoing reliance on well-established geochemical exploration techniques (e.g. sediment, soil and rock chip sampling) that have aided the discovery of near-surface resources over many decades, but are now proving less effective in the search for deeply buried mineral resources and for those concealed under cover.

Thematic collection: This article is part of the Exploration 17 collection available at: https://www.lyellcollection.org/cc/exploration-17

There have been several advances in inductively coupled plasma-mass spectrometer (ICP-MS) analytical technologies in the last decade. Collision/reaction cell ICP-MS and triple quadrupole ICP-MS techniques can produce lower detection limits for select elements that experience interferences with a standard quadrupole (e.g. Se and As). Triple quadrupole ICP-MS, in particular, can eliminate virtually all polyatomic or isobaric interferences for highly accurate measurements of some element isotopes systematics that are of great interest in mineral exploration, namely Pb/Pb. Laser ablation ICP-MS has become more popular as an effective analytical tool to measure mineral grain trace elements, which could assist in vectoring to mineralization or exploration drill targets. The ablation of a spot on a Li-borate fused glass disk paired with XRF analysis has also gained popularity as an alternative to total whole rock characterization packages that employ several separate digestions and analytical methods. While there have been several advancements in ICP-MS technologies in exploration geochemistry, they have not been widely accepted or implemented. This slow adaptation could be due to the extended recession in the mining industry between 2012 and 2017. It is also possible that standard ICP-MS data (i.e. no collision/reaction cell) is still fit for purpose. This stands in stark contrast to implementation of ICP-MS in the previous decade (1997–2007), which was transformational for the industry.

Consideration of all elements from large multi-element ICP-MS analytical suites for mineral exploration can be an extremely powerful tool in the exploration toolkit. The discovery of the White Gold District, Yukon, is a prime example of how the utilization of soil geochemical data, when plotted spatially, can vector to gold mineralization. The presence of Au + As + Sb soil anomalies were key to delineating mineralization, especially when accompanied by publicly available geological, geographical and geophysical data. Additionally, elements and element ratios not typically considered in Au exploration, including Ni and U, were utilized to determine the lithological and structural controls on mineralization. The availability of multi-element ICP-MS data was also useful in the discovery of the Cascadero Copper Taron Caesium deposit. Ore-grade Cs was discovered only because Cs was included in the multi-element ICP-MS exploration geochemistry suite. Before the availability of ICP-MS, it is unlikely that this deposit would have been discovered.

Thematic collection: This article is part of the Exploration 17 collection available at: https://www.lyellcollection.org/cc/exploration-17

Extractables and leachables (E&L) are identified and quantified so that their impact on patient safety can be established and assessed. The uncertainty in the impact assessment is affected by the uncertainty in the substance's experimentally determined identity and concentration. Thus, these experimentally determined quantities must be reported not only in terms of their absolute result but also in terms of the uncertainty in the result, which is based on the amount and rigor of the information on which the result is based. In this way, the impact assessment can be tempered to account for the uncertainty in its input data. To facilitate the assignment and reporting of uncertainty, classification hierarchies are proposed and discussed for both identification and quantitation. Both hierarchies establish levels or degrees of identification and quantitation based on the uncertainty of the result and contain descriptions of the quality and quantity of information required to achieve a certain level within the hierarchy. The minimal levels that must be achieved to support impact assessment are also established.

Although enteroviruses are associated with a wide variety of diseases and conditions, their mode of replication is well conserved. Their genome is carried as a single, positive-sense RNA strand. At the 5' end of the strand is an approximately 90-nucleotide self-complementary region called the 5' cloverleaf, or the oriL. This noncoding region serves as a platform upon which host and virus proteins, including the 3B, 3C, and 3D virus proteins, assemble in order to initiate replication of a negative-sense RNA strand. The negative strand in turn serves as a template for synthesis of multiple positive-sense RNA strands. Building on structural studies of individual RNA stem-loops, the structure of the intact 5' cloverleaf from rhinovirus has recently been determined via nuclear magnetic resonance/small-angle X-ray scattering (NMR/SAXS)-based methods, while structures have also been determined for enterovirus 3A, 3B, 3C, and 3D proteins. Analysis of these structures, together with structural and modeling studies of interactions between host and virus proteins and RNA, has begun to provide insight into the enterovirus replication mechanism and the potential to inhibit replication by blocking these interactions.

The capacity of Listeria monocytogenes to adapt to environmental changes is facilitated by a large number of regulatory proteins encoded by its genome. Among these proteins are the uncharacterized LysR-type transcriptional regulators (LTTRs). LTTRs can work as positive and/or negative transcription regulators at both local and global genetic levels. Previously, our group determined by comparative genome analysis that one member of the LTTRs (NCBI accession no. WP_003734782) was present in pathogenic strains but absent from nonpathogenic strains. The goal of the present study was to assess the importance of this transcription factor in the virulence of L. monocytogenes strain F2365 and to identify its regulons. An L. monocytogenes strain lacking lysR (the F2365lysR strain) displayed significant reductions in cell invasion of and adhesion to Caco-2 cells. In plaque assays, the deletion of lysR resulted in a 42.86% decrease in plaque number and a 13.48% decrease in average plaque size. Furthermore, the deletion of lysR also attenuated the virulence of L. monocytogenes in mice following oral and intraperitoneal inoculation. The analysis of transcriptomics revealed that the transcript levels of 139 genes were upregulated, while 113 genes were downregulated in the F2365lysR strain compared to levels in the wild-type bacteria. lysR-repressed genes included ABC transporters, important for starch and sucrose metabolism as well as glycerolipid metabolism, flagellar assembly, quorum sensing, and glycolysis/gluconeogenesis. Conversely, lysR activated the expression of genes related to fructose and mannose metabolism, cationic antimicrobial peptide (CAMP) resistance, and beta-lactam resistance. These data suggested that lysR contributed to L. monocytogenes virulence by broad impact on multiple pathways of gene expression.

IMPORTANCE Listeria monocytogenes is the causative agent of listeriosis, an infectious and fatal disease of animals and humans. In this study, we have shown that lysR contributes to Listeria pathogenesis and replication in cell lines. We also highlight the importance of lysR in regulating the transcription of genes involved in different pathways that might be essential for the growth and persistence of L. monocytogenes in the host or under nutrient limitation. Better understanding L. monocytogenes pathogenesis and the role of various virulence factors is necessary for further development of prevention and control strategies.

Despite the importance of the Bering Sea for subarctic oceanography and climate, relatively little is known of the foraminifera from the extensive Aleutian Basin. We report the occurrence of modern deep-water agglutinated foraminifera collected at seven sites cored during Integrated Ocean Drilling Program (IODP) Expedition 323 in the Bering Sea. Assemblages collected from core-top samples contained 32 genera and 50 species and are described and illustrated here for the first time. Commonly occurring species include typical deep-water Rhizammina, Reophax, Rhabdammina, Recurvoides and Nodulina. Assemblages from the northern sites also consist of accessory Cyclammina, Eggerelloides and Glaphyrammina, whilst those of the Bowers Ridge sites consist of other tubular genera and Martinottiella. Of the studied stations with the lowest dissolved oxygen concentrations, the potentially Bering Sea endemic Eggerelloides sp. 1 inhabits the northern slope, which has the highest primary productivity, and the potentially endemic Martinottiella sp. 3 inhabits Bowers Ridge, which has the lowest oxygen concentrations but relatively low annual productivity. Martinottiella sp. 3, with open pores on its test surface, has previously been reported in Pliocene to Recent material from Bowers Ridge. Despite relatively small sample sizes, ecological constraints may imply that the Bering Sea experienced high productivity and reduced oxygen at times since at least the Pliocene. We note the partially endemic nature of the agglutinated foraminiferal assemblages, which may at least in part be due to basin restriction, the geologically long time period of reduced oxygen, and high organic carbon flux. Our results indicate the importance of gathering further surface sample data from the Aleutian Basin.

ABSTRACTBackground:Some opportunities to routinely capture and improve respectful maternity care (RMC) during facility-based childbirth include quality improvement (QI) initiatives, community-based monitoring efforts through community score cards (CSC), and performance-based financing (PBF) initiatives. But there is limited guidance on which types of RMC indicators are best suited for inclusion in these initiatives. We sought to provide practical evidence-based recommendations on indicators that may be used for routine measurement of RMC in programs.Methods:We used a rapid review approach, which included (1) reviewing existing documents and publications to extract RMC indicators and identify which have or can be used in facility-based QI, CSCs, and PBF schemes; (2) surveying RMC and maternal health experts to rank indicators, and (3) analyzing survey data to select the most recommended indicators.Results:We identified 49 indicators spanning several domains of RMC and mistreatment including dignified/nondignified care, verbal and physical abuse, privacy/confidentiality, autonomy/loss of autonomy, supportive care/lack thereof, communication, stigma, discrimination, trust, facility environment/culture, responsiveness, and nonevidence-based care. Based on the analysis of the survey data, we recommend 33 indicators (between 2 and 6 indicators for each RMC domain) that may be suited for incorporation in both facility-based QI and CSC-related monitoring efforts.Conclusion:Integrating RMC indicators into QI and CSC initiatives, as well as in other maternal and neonatal health programs, could help improve RMC at the facility and community level. More research is needed into whether RMC can be integrated into PBF initiatives. Integration of RMC indicators into programs to improve quality of care and other health system outcomes will facilitate routine monitoring and accountability around experience of care. Measurement and improvement of women's experiences will increase maternal health service utilization and improve quality of care as a means of reducing maternal and neonatal morbidity and mortality.

ABSTRACTIntroduction:We evaluated a 2-way short message service (SMS) communication platform to improve continuation of pre-exposure prophylaxis (PrEP) for HIV prevention among Kenyan women who initiated PrEP within routine maternal child health (MCH) and family planning clinics.Methods:We adapted an existing SMS platform (Mobile WACh [mWACh]) to send PrEP-tailored, theory-based SMS and allow clients to communicate with a remote nurse. Women who did not have HIV and who were initiating PrEP at 2 MCH/family planning clinics in Kisumu County, Kenya, from February to October 2018, were offered enrollment into the mWACh-PrEP program; SMS communication was free. We evaluated acceptability, satisfaction, and implementation metrics. In a pre/postevaluation, we compared PrEP continuation at 1-month postinitiation among women who initiated PrEP in the period before (n=166) versus after mWACh-PrEP implementation, adjusting for baseline differences.Results:Of the 334 women who were screened for enrollment into the mWACh-PrEP program; 193 (58%) were eligible and of those, 190 (98%) accepted enrollment. Reasons for ineligibility (n=141) included no phone access (29%) and shared SIM cards (25%). Median age was 25 years (interquartile range=22–30), and 91% were MCH clients. Compared to women who initiated PrEP in the month before mWACh-PrEP implementation, women who enrolled in mWACh-PrEP were more likely to return for their first PrEP follow-up visit (40% vs. 53%; adjusted risk ratio [aRR]=1.26; 95% confidence interval [CI]= 1.06, 1.50; P=.008) and more likely to continue PrEP (22% vs. 43%; aRR=1.75; 95% CI=1.21, 2.55; P=.003). Among those who returned, 99% reported successful receipt of SMS through the mWACh-PrEP system and 94% reported that mWACh-PrEP helped them understand PrEP better. Concerns about PrEP use, how it works, and side effects accounted for the majority (80%) of issues raised by participants using SMS.Conclusions:Two-way SMS expanded support for PrEP and opportunities for dialogue beyond the clinic and enabled women to ask and receive answers in real time regarding PrEP, which facilitated its continued use.

OBJECTIVE

Nonalbuminuric diabetic kidney disease (DKD) has become the prevailing phenotype in patients with type 2 diabetes. However, it remains unclear whether its prognosis is poorer than that of other DKD phenotypes.

OBJECTIVE

Suboptimal adherence to insulin treatment is a main issue in adolescents with type 1 diabetes. However, to date, there are no available data on adherence to adjunct noninsulin medications in this population. Our aim was to assess adherence to ACE inhibitors and statins and explore potential determinants in adolescents with type 1 diabetes.

OBJECTIVE

With rising health care costs and finite health care resources, understanding the population needs of different type 2 diabetes mellitus (T2DM) patient subgroups is important. Sparse data exist for the application of population segmentation on health care needs among Asian T2DM patients. We aimed to segment T2DM patients into distinct classes and evaluate their differential health care use, diabetes-related complications, and mortality patterns.

OBJECTIVE

No study has reported global disability burden estimates for individual diabetes-related lower-extremity complications (DRLECs). The Global Burden of Disease (GBD) study presents a robust opportunity to address this gap.