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.

Rock is a material that is affected by wear, and the curvature of the asperities on a rock joint surface increases with the degree of wear after shearing. Based on the Greenwood and Williamson (GW) model, a new model considering the change of asperity curvature is proposed to explain the wear behaviour of rock joints. First, the shear stiffness formula for a joint surface is derived when the asperity curvature is constant, which shows that the shear stiffness increases with increase of asperity curvature. According to the Mohr–Coulomb criterion, the yield position of a single asperity under normal force and tangential friction force is discussed. Then, the critical normal force for a single asperity at a specific friction coefficient is obtained, which shows that the normal force corresponds to the curvature radius of the asperity. A rough surface model with multi-level curvature radius is proposed. With increase of normal force, the higher-order asperities gradually fail and the curvature radius become larger. A specific pressure value excites a specific radius of curvature, and the larger the pressure, the larger the radius of curvature. The relation between the normal force and the curvature radius is proposed and a shear stiffness formula considering the change of curvature radius of the asperity is derived. The proposed model is verified on the basis of the published experimental results. The calculation results of the proposed model can reflect the test results well: for a given joint surface, with increase in normal force the joint surface gradually becomes smooth; for different joint surfaces, with increase in roughness, the joint surface is more easily smoothed.

This paper uses a 3D model for stability assessment of Zhujiabaobao waste dump with ground cracks. The study data were gathered via reconnaissance, geomorphological analysis and laboratory experiment. A 3D finite extended element method model that can consider cracks was then used to calculate the factor of safety (FOS) of the waste dump via the strength reduction technique. The simulation shows the dump to have an FOS of 1.22 and both the position and depth of penetration of cracks in the waste dump have a crucial impact on the stability of the slope. Because the study area is located in a seismically active area, simulation and analysis of the dynamic response of the waste dump under different magnitudes of seismic waves (peak acceleration is 0.05, 0.15, 0.25 and 0.45g) were performed via an explicit dynamic model. The simulation shows that high steps in the slope are particularly responsive to earthquakes. The approach used here for analysing stability under static and dynamic loads is useful for hazard prevention and mitigation.

Groundwater recharge and runoff conditions are ascertained in the Suixiao coal-mining district using the hydrogen and oxygen isotopes and the trace elements in the unconsolidated pore aquifer of the Cenozoic group, the fissured sandstone aquifer of the Permian system, and the karst fissured limestone aquifer of the Carboniferous Taiyuan Formation and the Ordovician system, which are the main recharge aquifers during coal mining. The main water–rock interactions are pyrite oxidation, cation exchange and adsorption, and carbonate acidification, which are educed by principal component analysis of conventional ions. These results combined with geological conditions prove that hydraulic connection exists generally between the main recharge aquifers, and the groundwater circulation is controlled by faults in the sandstone and limestone aquifers. The water–rock interaction is very weak in the east of the district, which is proved to be a recharge area by Fisher discriminant analysis. This study provides the theoretical basis for the hydrochemistry exploration and the establishment of a water-inrush warning system in a concealed coalfield.

The need to characterize and distinguish geographically adjacent aggregate quarry sources prompted the SEM-EDS analysis of pyrite (FeS₂) within fill material taken from eight different quarry sources. This experiment was undertaken to investigate the possibility of geochemically separating these quarry sources based on the major element concentration of their pyrite. The results show that median values for Fe and S vary by up to 7.6 and 8.55 wt% respectively. By implementing statistical methods, including k-means clustering and principal component analysis, it is possible to geochemically discriminate three of the eight sources.

The Clay-with-flints Formation outcrops on the high chalk plateaux and interfluves of the chalk downs in southern England. Both current and historical definitions of the Clay-with-flints are detailed and important distinctions are identified with other deposits that appear identical but are formed in different ways. Historically pedological or geomorphological studies have been carried out on the deposit. Engineering studies are only carried out where the deposit is crossed by infrastructure. The physical and chemical processes acting on the deposit and the resulting effects on the physical properties are discussed.

A logistic regression model and a bivariate statistical analysis were used in this paper to evaluate the groundwater recharge susceptibility. The approach is based on the assessment of the relationship involving groundwater recharge and parameters that influence this hydrological process. Surface parameters and aquifer-related parameters were evaluated as thematic map layers using ArcGIS. Then, a weighted-rating method was adopted to categorize each parameter's map. To assess the role of each parameter in the aquifer recharge, a logistic regression model and a bivariate statistical analysis were applied to the Guenniche phreatic aquifer (Tunisia). Models are explored to establish a map showing the aquifer recharge susceptibility. The code Modflow was used to simulate the consequence of the recharge. The recharge amount was introduced in the model and was tested to verify the recharge effect on the hydraulic head for the two models. The obtained results reveal that the recharge as mapped in the bivariate statistical model has a minor impact on the hydraulic head. Results of the logistic regression model are more significant as the hydraulic head is widely affected. This model provides good results in mapping the spatial distribution of the aquifer recharge susceptibility.

In 2015, the M_w 7.8 Gorkha earthquake struck Nepal, triggering thousands of landslides across the central and eastern Himalayas. These landslides had many adverse effects, including causing widespread damage to low-grade transport routes (e.g. tracks, footpaths) in rural regions that depend on tourism for survival. Langtang Valley is a glacial–periglacial landscape located 60 km north of Kathmandu. It is one of the most popular trekking regions in Nepal and has been severely affected by Gorkha earthquake-triggered and monsoon-triggered landsliding. Here, qualitative and quantitative observations from fieldwork and remote sensing are used to describe the materials and geomorphology of the landslides across Langtang Valley, and to quantify the extent to which coseismic and monsoon-triggered landslides have affected Langtang's trekking infrastructure. The dominant bedrock materials involved within Langtang landslides are found to be a range of gneisses and intruded leucogranites. In total, 64 landslides are found to have intersected trekking paths across Langtang, with coseismic and monsoon-triggered landslides having an impact on c. 3 km and 0.8 km of path respectively. It is observed that the practice of reconstructing paths through unstable landslide deposits is leaving the trekking infrastructure across Langtang increasingly vulnerable to future failure.

Leprosy is caused by Mycobacterium leprae, and it remains underdiagnosed in Burkina Faso. We investigated the use of fluorescent in situ hybridization (FISH) for detecting M. leprae in 27 skin samples (skin biopsy samples, slit skin samples, and skin lesion swabs) collected from 21 patients from Burkina Faso and three from Côte d’Ivoire who were suspected of having cutaneous leprosy. In all seven Ziehl-Neelsen-positive skin samples (four skin biopsy samples and three skin swabs collected from the same patient), FISH specifically identified M. leprae, including one FISH-positive skin biopsy sample that remained negative after testing with PCR targeting the rpoB gene and with the GenoType LepraeDR assay. Twenty other skin samples and three negative controls all remained negative for Ziehl-Neelsen staining, FISH, and rpoB PCR. These data indicate the usefulness of a microscopic examination of skin samples after FISH for first-line diagnosis of cutaneous leprosy. Accordingly, FISH represents a potentially useful point-of-care test for the diagnosis of cutaneous leprosy.

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.

Q fever, caused by Coxiella burnetii, is a worldwide zoonotic disease that may cause severe forms in humans and requires a specific and prolonged antibiotic treatment. Although current serological and molecular detection tools allow a reliable diagnosis of the disease, culture of C. burnetii strains is mandatory to assess their susceptibility to antibiotics and sequence their genome in order to optimize patient management and epidemiological studies. However, cultivating this fastidious microorganism is difficult and restricted to reference centers, as it requires biosafety level 3 laboratories and relies on cell culture performed by experienced technicians. In addition, the culture yield is low, which results in a small number of isolates being available. In this work, we developed a novel high-content screening (HCS) isolation strategy based on optimized high-throughput cell culture and automated microscopic detection of infected cells with specifically designed algorithms targeting cytopathic effects. This method was more efficient than the shell vial assay, at the level of time dependency, when applied to both frozen specimens (7 isolates recovered by HCS only, sensitivity 91% versus 78% for shell vial) and fresh samples (1 additional isolate using HCS, sensitivity 7% versus 5% for shell vial), for which most strains were recovered more rapidly with the new technique. In addition, detecting positive cultures by an automated microscope reduced the need for expertise and saved 24% of technician working time. Application of HCS to antibiotic susceptibility testing of 12 strains demonstrated that it was as efficient as the standard procedure that combines shell vial culture and quantitative PCR.

Quantitative bacterial culture of bronchoalveolar lavage fluids (BALF) is labor-intensive, and the delay involved in performing culture, definitive identification, and susceptibility testing often results in prolonged use of broad-spectrum antibiotics. The Unyvero lower respiratory tract (LRT) panel (Curetis, Holzgerlingen, Germany) allows the multiplexed rapid detection and identification of 20 potential etiologic agents of pneumonia within 5 h of collection. In addition, the assay includes detection of gene sequences that confer antimicrobial resistance. We retrospectively compared the performance of the molecular panel to routine quantitative bacterial culture methods on remnant BALF. Upon testing 175 BALF, we were able to analyze positive agreement of 181 targets from 129 samples, and 46 samples were negative. The positive percent agreement (PPA) among the microbial targets was 96.5%, and the negative percent agreement (NPA) was 99.6%. The targets with a PPA of <100% were Staphylococcus aureus (34/37 [91.9%]), Streptococcus pneumoniae (10/11 [90.9%]), and Enterobacter cloacae complex (2/4 [50%]). For the analyzable resistance targets, concordance with phenotypic susceptibility testing was 79% (14/18). This study found the Unyvero LRT panel largely concordant with culture results; however, no outcome or clinical impact studies were performed.

Klebsiella species are problematic pathogens in neonatal units and may cause outbreaks, for which the sources of transmission may be challenging to elucidate. We describe the use of whole-genome sequencing (WGS) to investigate environmental sources of transmission during an outbreak of extended-spectrum-β-lactamase (ESBL)-producing Klebsiella michiganensis colonizing neonates. Ceftriaxone-resistant Klebsiella spp. isolated from neonates (or their mothers) and the hospital environment were included. Short-read sequencing (Illumina) and long-read sequencing (MinION; Oxford Nanopore Technologies) were used to confirm species taxonomy, to identify antimicrobial resistance genes, and to determine phylogenetic relationships using single-nucleotide polymorphism profiling. A total of 21 organisms (10 patient-derived isolates and 11 environmental isolates) were sequenced. Standard laboratory methods identified the outbreak strain as an ESBL-producing Klebsiella oxytoca, but taxonomic assignment from WGS data suggested closer identity to Klebsiella michiganensis. Strains isolated from multiple detergent-dispensing bottles were either identical or closely related by single-nucleotide polymorphism comparison. Detergent bottles contaminated by K. michiganensis had been used for washing milk expression equipment. No new cases were identified once the detergent bottles were removed. Environmental reservoirs may be an important source in outbreaks of multidrug-resistant organisms. WGS, in conjunction with traditional epidemiological investigation, can be instrumental in revealing routes of transmission and guiding infection control responses.

Screening for Chlamydia trachomatis and Neisseria gonorrhoeae at the pharyngeal, urogenital, and anorectal sites is recommended for men who have sex with men (MSM). Combining the three individual-site samples into a single pooled sample could result in significant cost savings, provided there is no significant sensitivity reduction. The aim of this study was to examine the sensitivity of pooled samples for detecting chlamydia and gonorrhea in asymptomatic MSM using a nucleic acid amplification test. Asymptomatic MSM who tested positive for chlamydia or gonorrhoea were invited to participate. Paired samples were obtained from participants prior to administration of treatment. To form the pooled sample, the anorectal swab was agitated in the urine specimen transport tube and then discarded. The pharyngeal swab and 2 ml of urine sample were then added to the tube. The difference in sensitivity between testing of pooled samples and individual-site testing was calculated against an expanded gold standard, where an individual is considered positive if either pooled-sample or individual-site testing returns a positive result. All samples were tested using the Aptima Combo 2 assay. A total of 162 MSM were enrolled in the study. Sensitivities of pooled-sample testing were 86% (94/109; 95% confidence interval [CI], 79 to 92%]) for chlamydia and 91% (73/80; 95% CI, 83 to 96%) for gonorrhea. The sensitivity reduction was significant for chlamydia (P = 0.02) but not for gonorrhea (P = 0.34). Pooling caused 22 infections (15 chlamydia and 7 gonorrhoea) to be missed, and the majority were single-site infections (19/22). Pooling urogenital and extragenital samples from asymptomatic MSM reduced the sensitivity of detection by approximately 10% for chlamydia but not for gonorrhea.

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.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections that can cause significant morbidity and mortality. CDI diagnosis involves laboratory testing in conjunction with clinical assessment. The objective of this study was to assess the performance of various C. difficile tests and to compare clinical characteristics, Xpert C. difficile/Epi (PCR) cycle threshold (C_T), and Singulex Clarity C. diff toxins A/B (Clarity) concentrations between groups with discordant test results. Unformed stool specimens from 200 hospitalized adults (100 PCR positive and 100 negative) were tested by cell cytotoxicity neutralization assay (CCNA), C. diff Quik Chek Complete (Quik Chek), Premier Toxins A and B, and Clarity. Clinical data, including CDI severity and CDI risk factors, were compared between discordant test results. Compared to CCNA, PCR had the highest sensitivity at 100% and Quik Chek had the highest specificity at 100%. Among clinical and laboratory data studied, prevalences of leukocytosis, prior antibiotic use, and hospitalizations were consistently higher across all subgroups in comparisons of toxin-positive to toxin-negative patients. Among PCR-positive samples, the median C_T was lower in toxin-positive samples than in toxin-negative samples; however, C_T ranges overlapped. Among Clarity-positive samples, the quantitative toxin concentration was significantly higher in toxin-positive samples than in toxin-negative samples as determined by CCNA and Quik Chek Toxin A and B. Laboratory tests for CDI vary in sensitivity and specificity. The quantitative toxin concentration may offer value in guiding CDI diagnosis and treatment. The presence of leukocytosis, prior antibiotic use, and previous hospitalizations may assist with CDI diagnosis, while other clinical parameters may not be consistently reliable.

Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis. However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 μg/ml) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two time points: R1 (mean, 10 days) and R2 (mean, 13 days) for each PZA concentration. A consensus reference test, composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. A total of 182 samples were evaluated. The sensitivity and specificity for 400 μg/ml ranged from 76.9 to 89.7 and from 93.0 to 97.9%, respectively, and for 800 μg/ml ranged from 71.8 to 82.1 and from 95.8 to 98.6%, respectively. Compared to MGIT-PZA, our test showed a similar turnaround time (medians of 10 and 12 days for PZA-sensitive and -resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line antituberculosis drugs. Further optimization of test conditions would be useful in order to increase its performance.

This minireview focuses on the microbiologic evaluation of patients with asymptomatic bacteriuria, as well as indications for antibiotic treatment. Asymptomatic bacteriuria is defined as two consecutive voided specimens (preferably within 2 weeks) with the same bacterial species, isolated in quantitative counts of ≥10⁵ CFU/ml in women, including pregnant women; a single voided urine specimen with one bacterial species isolated in a quantitative count ≥10⁵ CFU/ml in men; and a single catheterized urine specimen with one or more bacterial species isolated in a quantitative count of ≥10⁵ CFU/ml in either women or men (or ≥10² CFU/ml of a single bacterial species from a single catheterized urine specimen). Any urine specimen with ≥10⁴ CFU/ml group B Streptococcus is significant for asymptomatic bacteriuria in a pregnant woman. Asymptomatic bacteriuria occurs, irrespective of pyuria, in the absence of signs or symptoms of a urinary tract infection. The two groups with the best evidence of adverse outcomes in the setting of untreated asymptomatic bacteriuria include pregnant women and patients who undergo urologic procedures with risk of mucosal injury. Screening and treatment of asymptomatic bacteriuria is not recommended in the following patient populations: pediatric patients, healthy nonpregnant women, older patients in the inpatient or outpatient setting, diabetic patients, patients with an indwelling urethral catheter, patients with impaired voiding following spinal cord injury, patients undergoing nonurologic surgeries, and nonrenal solid-organ transplant recipients. Renal transplant recipients beyond 1 month posttransplant should not undergo screening and treatment for asymptomatic bacteriuria. There is insufficient evidence to recommend for or against screening of renal transplant recipients within 1 month, patients with high-risk neutropenia, or patients with indwelling catheters at the time of catheter removal. Unwarranted antibiotics place patients at increased risk of adverse effects (including Clostridioides difficile diarrhea) and contribute to antibiotic resistance. Methods to reduce unnecessary screening for and treatment of asymptomatic bacteriuria aid in antibiotic stewardship.

Human herpesvirus 6 (HHV-6) is an important cause of meningitis and meningoencephalitis. As testing for HHV-6 in cerebrospinal fluid (CSF) is more readily available using the FilmArray Meningitis/Encephalitis panel (FA-ME; BioFire Diagnostics, Salt Lake City, UT), we aimed to determine the clinical significance of detecting HHV-6 in order to identify true infections and to ensure appropriate antiviral initiation. Chart review on 25 patients positive for HHV-6 by FA-ME was performed to determine clinical presentation, comorbidity, treatment, and outcome. The presence of chromosomally integrated HHV-6 (ciHHV-6) DNA was also investigated. Of 1,005 children tested by FA-ME, HHV-6 was detected in 25 (2.5%). Five patients were diagnosed with either HHV-6 meningitis or meningoencephalitis based on HHV-6 detection in CSF, clinical presentation, and radiographic findings. Detection of HHV-6 by FA-ME led to discontinuation of acyclovir within 12.0 h in all 12 patients empirically treated with acyclovir. Six of the 12 patients were started on ganciclovir therapy within 6.8 h; 4 of these were treated specifically for HHV-6 infection, whereas therapy was discontinued in the remaining 2 patients. CSF parameters were not generally predictive of HHV-6 positivity. The presence of ciHHV-6 was confirmed in 3 of 18 patients who could be tested. Five of the 25 patients included in the study were diagnosed with HHV-6 meningitis/meningoencephalitis. FA-ME results led to discontinuation of empirical antiviral treatment in 12 patients and appropriate initiation of ganciclovir in 4 patients. In our institution, detection of HHV-6 using FA-ME led to faster establishment of disease etiology and optimization of antimicrobial therapy.

On 31 December 2019, the World Health Organization was informed of a cluster of cases of pneumonia of unknown etiology in Wuhan, China. Subsequent investigations identified a novel coronavirus, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from the affected patients. Highly sensitive and specific laboratory diagnostics are important for controlling the rapidly evolving SARS-CoV-2-associated coronavirus disease 2019 (COVID-19) epidemic. In this study, we developed and compared the performance of three novel real-time reverse transcription-PCR (RT-PCR) assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported RdRp-P2 assay, which is used in >30 European laboratories. Among the three novel assays, the COVID-19-RdRp/Hel assay had the lowest limit of detection in vitro (1.8 50% tissue culture infective doses [TCID₅₀]/ml with genomic RNA and 11.2 RNA copies/reaction with in vitro RNA transcripts). Among 273 specimens from 15 patients with laboratory-confirmed COVID-19 in Hong Kong, 77 (28.2%) were positive by both the COVID-19-RdRp/Hel and RdRp-P2 assays. The COVID-19-RdRp/Hel assay was positive for an additional 42 RdRp-P2-negative specimens (119/273 [43.6%] versus 77/273 [28.2%]; P < 0.001), including 29/120 (24.2%) respiratory tract specimens and 13/153 (8.5%) non-respiratory tract specimens. The mean viral load of these specimens was 3.21 x 10⁴ RNA copies/ml (range, 2.21 x 10² to 4.71 x 10⁵ RNA copies/ml). The COVID-19-RdRp/Hel assay did not cross-react with other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical specimens, whereas the RdRp-P2 assay cross-reacted with SARS-CoV in cell culture. The highly sensitive and specific COVID-19-RdRp/Hel assay may help to improve the laboratory diagnosis of COVID-19.

The QIAstat-Dx Respiratory Panel (QIAstat-Dx RP) is a multiplex in vitro diagnostic test for the qualitative detection of 20 pathogens directly from nasopharyngeal swab (NPS) specimens. The assay is performed using a simple sample-to-answer platform with results available in approximately 69 min. The pathogens identified are adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, human metapneumovirus A and B, influenza A, influenza A H1, influenza A H3, influenza A H1N1/2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, rhinovirus/enterovirus, respiratory syncytial virus A and B, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae. This multicenter evaluation provides data obtained from 1,994 prospectively collected and 310 retrospectively collected (archived) NPS specimens with performance compared to that of the BioFire FilmArray Respiratory Panel, version 1.7. The overall percent agreement between QIAstat-Dx RP and the comparator testing was 99.5%. In the prospective cohort, the QIAstat-Dx RP demonstrated a positive percent agreement of 94.0% or greater for the detection of all but four analytes: coronaviruses 229E, NL63, and OC43 and rhinovirus/enterovirus. The test also demonstrated a negative percent agreement of ≥97.9% for all analytes. The QIAstat-Dx RP is a robust and accurate assay for rapid, comprehensive testing for respiratory pathogens.

The IR Biotyper is a new automated typing system based on Fourier-transform infrared (FT-IR) spectroscopy that gives results within 4 h. We aimed (i) to use the IR Biotyper to retrospectively analyze an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) in a neonatal intensive care unit and to compare results to BOX-PCR and whole-genome sequencing (WGS) results as the gold standard and (ii) to assess how the cutoff values used to define clusters affect the discriminatory power of the IR Biotyper. The sample consisted of 18 isolates from 14 patients. Specimens were analyzed in the IR Biotyper using the default analysis settings, and spectra were analyzed using OPUS 7.5 software. The software contains a feature that automatically proposes a cutoff value to define clusters; the cutoff value defines up to which distance the spectra are considered to be in the same cluster. Based on FT-IR, the outbreak represented 1 dominant clone, 1 secondary clone, and several unrelated clones. FT-IR results, using the cutoff value generated by the accompanying software after 4 replicates, were concordant with WGS for all but 1 isolate. BOX-PCR was underdiscriminatory compared to the other two methods. Using the cutoff value generated after 12 replicates, the results of FT-IR and WGS were completely concordant. The IR Biotyper can achieve the same typeability and discriminatory power as genome-based methods. However, to attain this high performance requires either previous, strain-dependent knowledge about the optimal technical parameters to be used or validation by a second method.

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.

Large-scale metagenomic and metatranscriptomic data analyses are often restricted by their gene-centric approach, limiting the ability to understand organismal and community biology. De novo assembly of large and mosaic eukaryotic genomes from complex meta-omics data remains a challenging task, especially in comparison with more straightforward bacterial and archaeal systems. Here, we use a transcriptome reconstruction method based on clustering co-abundant genes across a series of metagenomic samples. We investigated the co-abundance patterns of ~37 million eukaryotic unigenes across 365 metagenomic samples collected during the Tara Oceans expeditions to assess the diversity and functional profiles of marine plankton. We identified ~12,000 co-abundant gene groups (CAGs), encompassing ~7 million unigenes, including 924 metagenomics-based transcriptomes (MGTs, CAGs larger than 500 unigenes). We demonstrated the biological validity of the MGT collection by comparing individual MGTs with available references. We identified several key eukaryotic organisms involved in dimethylsulfoniopropionate (DMSP) biosynthesis and catabolism in different oceanic provinces, thus demonstrating the potential of the MGT collection to provide functional insights on eukaryotic plankton. We established the ability of the MGT approach to capture interspecies associations through the analysis of a nitrogen-fixing haptophyte-cyanobacterial symbiotic association. This MGT collection provides a valuable resource for analyses of eukaryotic plankton in the open ocean by giving access to the genomic content and functional potential of many ecologically relevant eukaryotic species.

Transcription of a chromatin template involves the concerted interaction of many different proteins and protein complexes. Analyses of specific factors showed that these interactions change during stress and upon developmental switches. However, how the binding of multiple factors at any given locus is coordinated has been technically challenging to investigate. Here we used Epi-Decoder in yeast to systematically decode, at one transcribed locus, the chromatin binding changes of hundreds of proteins in parallel upon perturbation of transcription. By taking advantage of improved Epi-Decoder libraries, we observed broad rewiring of local chromatin proteomes following chemical inhibition of RNA polymerase. Rapid reduction of RNA polymerase II binding was accompanied by reduced binding of many other core transcription proteins and gain of chromatin remodelers. In quiescent cells, where strong transcriptional repression is induced by physiological signals, eviction of the core transcriptional machinery was accompanied by the appearance of quiescent cell–specific repressors and rewiring of the interactions of protein-folding factors and metabolic enzymes. These results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of multiple chromatin proteins under varying conditions and cell states. The systematic and comprehensive delineation of dynamic local chromatin proteomes will greatly aid in uncovering protein–protein relationships and protein functions at the chromatin template.

Cellular heterogeneity in gene expression is driven by cellular processes, such as cell cycle and cell-type identity, and cellular environment such as spatial location. The cell cycle, in particular, is thought to be a key driver of cell-to-cell heterogeneity in gene expression, even in otherwise homogeneous cell populations. Recent advances in single-cell RNA-sequencing (scRNA-seq) facilitate detailed characterization of gene expression heterogeneity and can thus shed new light on the processes driving heterogeneity. Here, we combined fluorescence imaging with scRNA-seq to measure cell cycle phase and gene expression levels in human induced pluripotent stem cells (iPSCs). By using these data, we developed a novel approach to characterize cell cycle progression. Although standard methods assign cells to discrete cell cycle stages, our method goes beyond this and quantifies cell cycle progression on a continuum. We found that, on average, scRNA-seq data from only five genes predicted a cell's position on the cell cycle continuum to within 14% of the entire cycle and that using more genes did not improve this accuracy. Our data and predictor of cell cycle phase can directly help future studies to account for cell cycle–related heterogeneity in iPSCs. Our results and methods also provide a foundation for future work to characterize the effects of the cell cycle on expression heterogeneity in other cell types.

The human immune system relies on highly complex and diverse transcripts and the proteins they encode. These include transcripts encoding human leukocyte antigen (HLA) receptors as well as B cell and T cell receptors (BCR and TCR). Determining which alleles an individual possesses for each HLA gene (high-resolution HLA typing) is essential to establish donor–recipient compatibility in organ and bone marrow transplantations. In turn, the repertoires of millions of unique BCR and TCR transcripts in each individual carry a vast amount of health-relevant information. Both short-read RNA-seq-based HLA typing and BCR/TCR repertoire sequencing (AIRR-seq) currently rely on our incomplete knowledge of the genetic diversity at HLA and BCR/TCR loci. Here, we generated over 10,000,000 full-length cDNA sequences at a median accuracy of 97.9% using our nanopore sequencing-based Rolling Circle Amplification to Concatemeric Consensus (R2C2) protocol. We used this data set to (1) show that deep and accurate full-length cDNA sequencing can be used to provide isoform-level transcriptome analysis for more than 9000 loci, (2) generate accurate sequences of HLA alleles, and (3) extract detailed AIRR data for the analysis of the adaptive immune system. The HLA and AIRR analysis approaches we introduce here are untargeted and therefore do not require prior knowledge of the composition or genetic diversity of HLA and BCR/TCR loci.

The regulation of transposable element (TE) activity by small RNAs is a ubiquitous feature of germlines. However, despite the obvious benefits to the host in terms of ensuring the production of viable gametes and maintaining the integrity of the genomes they carry, it remains controversial whether TE regulation evolves adaptively. We examined the emergence and evolutionary dynamics of repressor alleles after P-elements invaded the Drosophila melanogaster genome in the mid-twentieth century. In many animals including Drosophila, repressor alleles are produced by transpositional insertions into piRNA clusters, genomic regions encoding the Piwi-interacting RNAs (piRNAs) that regulate TEs. We discovered that ~94% of recently collected isofemale lines in the Drosophila melanogaster Genetic Reference Panel (DGRP) contain at least one P-element insertion in a piRNA cluster, indicating that repressor alleles are produced by de novo insertion at an exceptional rate. Furthermore, in our sample of approximately 200 genomes, we uncovered no fewer than 80 unique P-element insertion alleles in at least 15 different piRNA clusters. Finally, we observe no footprint of positive selection on P-element insertions in piRNA clusters, suggesting that the rapid evolution of piRNA-mediated repression in D. melanogaster was driven primarily by mutation. Our results reveal for the first time how the unique genetic architecture of piRNA production, in which numerous piRNA clusters can encode regulatory small RNAs upon transpositional insertion, facilitates the nonadaptive rapid evolution of repression.

Genome-wide association studies have implicated thousands of noncoding variants across common human phenotypes. However, they cannot directly inform the cellular context in which disease-associated variants act. Here, we use open chromatin profiles from discrete mouse cell populations to address this challenge. We applied stratified linkage disequilibrium score regression and evaluated heritability enrichment in 64 genome-wide association studies, emphasizing schizophrenia. We provide evidence that mouse-derived human open chromatin profiles can serve as powerful proxies for difficult to obtain human cell populations, facilitating the illumination of common disease heritability enrichment across an array of human phenotypes. We demonstrate that signatures from discrete subpopulations of cortical excitatory and inhibitory neurons are significantly enriched for schizophrenia heritability with maximal enrichment in cortical layer V excitatory neurons. We also show that differences between schizophrenia and bipolar disorder are concentrated in excitatory neurons in cortical layers II-III, IV, and V, as well as the dentate gyrus. Finally, we leverage these data to fine-map variants in 177 schizophrenia loci nominating variants in 104/177. We integrate these data with transcription factor binding site, chromatin interaction, and validated enhancer data, placing variants in the cellular context where they may modulate risk.

Cohesin is a ring-shaped multiprotein complex that is crucial for 3D genome organization and transcriptional regulation during differentiation and development. It also confers sister chromatid cohesion and facilitates DNA damage repair. Besides its core subunits SMC3, SMC1A, and RAD21, cohesin in somatic cells contains one of two orthologous STAG subunits, STAG1 or STAG2. How these variable subunits affect the function of the cohesin complex is still unclear. STAG1- and STAG2-cohesin were initially proposed to organize cohesion at telomeres and centromeres, respectively. Here, we uncover redundant and specific roles of STAG1 and STAG2 in gene regulation and chromatin looping using HCT116 cells with an auxin-inducible degron (AID) tag fused to either STAG1 or STAG2. Following rapid depletion of either subunit, we perform high-resolution Hi-C, gene expression, and sequential ChIP studies to show that STAG1 and STAG2 do not co-occupy individual binding sites and have distinct ways by which they affect looping and gene expression. These findings are further supported by single-molecule localizations via direct stochastic optical reconstruction microscopy (dSTORM) super-resolution imaging. Since somatic and congenital mutations of the STAG subunits are associated with cancer (STAG2) and intellectual disability syndromes with congenital abnormalities (STAG1 and STAG2), we verified STAG1-/STAG2-dependencies using human neural stem cells, hence highlighting their importance in particular disease contexts.

High-constitutive activity of the DNA damage response protein checkpoint kinase 1 (CHK1) has been shown in glioblastoma (GBM) cell lines and in tissue sections. However, whether constitutive activation and overexpression of CHK1 in GBM plays a functional role in tumorigenesis or has prognostic significance is not known. We interrogated multiple glioma patient cohorts for expression levels of CHK1 and the oncogene cancerous inhibitor of protein phosphatase 2A (CIP2A), a known target of high-CHK1 activity, and examined the relationship between these two proteins in GBM. Expression levels of CHK1 and CIP2A were independent predictors for reduced overall survival across multiple glioma patient cohorts. Using siRNA and pharmacologic inhibitors we evaluated the impact of their depletion using both in vitro and in vivo models and sought a mechanistic explanation for high CIP2A in the presence of high-CHK1 levels in GBM and show that; (i) CHK1 and pSTAT3 positively regulate CIP2A gene expression; (ii) pSTAT3 and CIP2A form a recursively wired transcriptional circuit; and (iii) perturbing CIP2A expression induces GBM cell senescence and retards tumor growth in vitro and in vivo. Taken together, we have identified an oncogenic transcriptional circuit in GBM that can be destabilized by targeting CIP2A.

Pancreatic cancer is one of the most lethal human malignancies, partly because of its propensity for metastasis. However, the mechanisms of metastasis in pancreatic cancer remain unclear. Oxidized low-density lipoprotein receptor 1 (OLR1), a lectin-like scavenger receptor that recognizes several ligands, such as oxidized low-density lipoprotein, was previously reported in cardiovascular and metabolic diseases. The role and mechanism of OLR1 in pancreatic cancer is unclear. In this study, we found that OLR1 expression was significantly higher in pancreatic cancer tissues than that in adjacent normal tissues and closely associated with reduced overall survival. OLR1 promoted proliferation and metastasis of pancreatic cancer cells in vitro and in vivo. Mechanistically, OLR1 increased HMGA2 transcription by upregulating c-Myc expression to promote the metastasis of pancreatic cancer cells. In addition, patients with pancreatic cancer with high expression of OLR1–c-Myc–HMGA2 axis showed worse prognosis compared with patients with low expression of OLR1–c-Myc–HMGA2 axis.

Treatments for cognitive deficits associated with central nervous system (CNS) disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system. The α7 nicotinic acetylcholine receptor (nAChR) has garnered substantial attention as a target for cognitive deficits based on receptor localization, robust preclinical effects, genetics implicating its involvement in cognitive disorders, and encouraging, albeit mixed, clinical data with α7 nAChR orthosteric agonists. Importantly, previous orthosteric agonists at this receptor suffered from off-target activity, receptor desensitization, and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility. To overcome the challenges with orthosteric agonists, we have identified a novel selective α7 positive allosteric modulator (PAM), BNC375. This compound is selective over related receptors and potentiates acetylcholine-evoked α7 currents with only marginal effect on the receptor desensitization kinetics. In addition, BNC375 enhances long-term potentiation of electrically evoked synaptic responses in rat hippocampal slices and in vivo. Systemic administration of BNC375 reverses scopolamine-induced cognitive deficits in rat novel object recognition and rhesus monkey object retrieval detour (ORD) task over a wide range of exposures, showing no evidence of an inverted U-shaped dose-effect curve. The compound also improves performance in the ORD task in aged African green monkeys. Moreover, ex vivo ¹³C-NMR analysis indicates that BNC375 treatment can enhance neurotransmitter release in rat medial prefrontal cortex. These findings suggest that α7 nAChR PAMs have multiple advantages over orthosteric α7 nAChR agonists for the treatment of cognitive dysfunction associated with CNS diseases.

Cinnamaldehyde (Cin), a bioactive cinnamon essential oil from traditional Chinese medicine herb Cinnamomum cassia, has been reported to have multipharmacological activities including anti-inflammation. However, its role and molecular mechanism of anti-inflammatory activity in musculoskeletal tissues remains unclear. Here, we first investigated the effects and molecular mechanisms of Cin in human synoviocyte cells. Then in vivo therapeutic effect of Cin on collagen-induced arthritis (CIA) also studied. Cell Counting Kit ‎CCK-8 assay was performed to evaluate the cell cytotoxicity. Proinflammatory cytokine expression was evaluated using quantitative polymerase chain reaction and ELISA. Protein expression was measured by western blotting. The in vivo effect of Cin (75 mg/kg per day) was evaluated in rats with CIA by gavage administration. Disease progression was assessed by clinical scoring, radiographic, and histologic examinations. Cin significantly inhibited interleukin (IL)-1β–induced IL-6, IL-8, and tumor necrosis factor-α release from human synoviocyte cells. The molecular analysis revealed that Cin impaired IL-6–induced activation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), and STAT3 signaling pathway by inhibiting the phosphorylation of JAK2, STAT1, and STAT3, without affecting NF-B pathway. Cin reduced collagen-induced swollen paw volume of arthritic rats. The anti-inflammation effects of Cin were associated with decreased severity of arthritis, joint swelling, and reduced bone erosion and destruction. Furthermore, serum IL-6 level was decreased when Cin administered therapeutically to CIA rats. Cin suppresses IL-1β–induced inflammation in synoviocytes through the JAK/STAT pathway and alleviated collagen-induced arthritis in rats. These data indicated that Cin might be a potential traditional Chinese medicine–derived, disease-modifying, antirheumatic herbal drug.

Extensive studies have shown that the ₁ receptor (₁R) interacts with and modulates the activity of multiple proteins with important biological functions. Recent crystal structures of ₁R as a homotrimer differ from a dimer-tetramer model postulated earlier. It remains inconclusive whether ligand binding regulates ₁R oligomerization. Here, novel nondenaturing gel methods and mutational analysis were used to examine ₁R oligomerization. In transfected cells, ₁R exhibited as multimers, dimers, and monomers. Overall, ₁R agonists decreased, whereas ₁R antagonists increased ₁R multimers, suggesting that agonists and antagonists differentially affect the stability of ₁R multimers. Endogenous ₁R in rat liver membranes also showed similar regulation of oligomerization as in cells. Mutations at key residues lining the trimerization interface (Arg119, Asp195, Phe191, Trp136, and Gly91) abolished multimerization without disrupting dimerization. Intriguingly, truncation of the N terminus reduced ₁R to apparent monomer. These results demonstrate that multiple domains play crucial roles in coordinating high-order quaternary organization of ₁R. The E102Q ₁R mutant implicated in juvenile amyotrophic lateral sclerosis formed dimers only, suggesting that dysregulation of ₁R multimeric assembly may impair its function. Interestingly, oligomerization of ₁R was pH-dependent and correlated with changes in [³H](+)-pentazocine binding affinity and B_max. Combined with mutational analysis, it is reasoned that ₁R multimers possess high-affinity and high-capacity [³H](+)-pentazocine binding, whereas monomers likely lack binding. These results suggest that ₁R may exist in interconvertible oligomeric states in a dynamic equilibrium. Further exploration of ligand-regulated ₁R multimerization may provide novel approaches to modulate the function of ₁R and its interacting proteins.

In vitro approaches for predicting drug-drug interactions (DDIs) caused by alterations in transporter protein regulation are not well established. However, reports of transporter regulation via nuclear receptor (NR) modulation by drugs are increasing. This study examined alterations in transporter protein levels in sandwich-cultured human hepatocytes (SCHH; n = 3 donors) measured by liquid chromatography–tandem mass spectrometry–based proteomic analysis after treatment with N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide (T0901317), the first described synthetic liver X receptor agonist. T0901317 treatment (10 μM, 48 hours) decreased the levels of organic cation transporter (OCT) 1 (0.22-, 0.43-, and 0.71-fold of control) and organic anion transporter (OAT) 2 (0.38-, 0.38-, and 0.53-fold of control) and increased multidrug resistance protein (MDR) 1 (1.37-, 1.48-, and 1.59-fold of control). The induction of NR downstream gene expression supports the hypothesis that T0901317 off-target effects on farnesoid X receptor and pregnane X receptor activation are responsible for the unexpected changes in OCT1, OAT2, and MDR1. Uptake of the OCT1 substrate metformin in SCHH was decreased by T0901317 treatment. Effects of decreased OCT1 levels on metformin were simulated using a physiologically-based pharmacokinetic (PBPK) model. Simulations showed a clear decrease in metformin hepatic exposure resulting in a decreased pharmacodynamic effect. This DDI would not be predicted by the modest changes in simulated metformin plasma concentrations. Altogether, the current study demonstrated that an approach combining SCHH, proteomic analysis, and PBPK modeling is useful for revealing tissue concentration–based DDIs caused by unexpected regulation of hepatic transporters by NR modulators.

It has been identified that arginine vasopressin (AVP), vasopressin receptor 2(V2R), and the aquaporin 2 (AQP2) signaling pathway in the inner ear play important roles in hearing and balance functions through regulating the endolymph equilibrium; however, the contributions of this signaling pathway to the development of motion sickness are unclear. The present study was designed to investigate whether the activation of the AVP-V2R-AQP2 signaling pathway in the inner ear is involved in the induction of motion sickness and whether mozavaptan, a V2R antagonist, could reduce motion sickness. We found that both rotatory stimulus and intraperitoneal AVP injection induced conditioned taste aversion (a confirmed behavioral index for motion sickness) in rats and activated the AVP-V2R-AQP2 signaling pathway with a responsive V2R downregulation in the inner ears, and AVP perfusion in cultured epithelial cells from rat endolymphatic sacs induced similar changes in this pathway signaling. Vestibular training, V2R antagonist mozavaptan, or PKA inhibitor H89 blunted these changes in the V2R-AQP2 pathway signaling while reducing rotatory stimulus– or DDAVP (a V2R agonist)-induced motion sickness in rats and dogs. Therefore, our results suggest that activation of the inner ear AVP-V2R-AQP2 signaling pathway is potentially involved in the development of motion sickness; thus, mozavaptan targeting AVP V2Rs in the inner ear may provide us with a new application option to reduce motion sickness.

Metastatic breast cancer is prevalent worldwide, and one of the most common sites of metastasis is long bones. Of patients with disease, the major symptom is pain, yet current medications fail to adequately result in analgesic efficacy and present major undesirable adverse effects. In our study, we investigate the potential of a novel monoacylglycerol lipase (MAGL) inhibitor, MJN110, in a murine model of cancer-induced bone pain. Literature has previously demonstrated that MAGL inhibitors function to increase the endogenous concentrations of 2-arachydonylglycerol, which then activates CB1 and CB2 receptors to inhibit inflammation and pain. We demonstrate that administration of MJN110 significantly and dose dependently alleviates spontaneous pain behavior during acute administration compared with vehicle control. In addition, MJN110 maintains its efficacy in a chronic-dosing paradigm over the course of 7 days without signs of receptor sensitization. In vitro analysis of MJN110 demonstrated a dose-dependent and significant decrease in cell viability and proliferation of 66.1 breast adenocarcinoma cells to a greater extent than KML29, an alternate MAGL inhibitor, or the CB2 agonist JWH015. Chronic administration of the compound did not appear to affect tumor burden, as evidenced by radiograph or histologic analysis. Together, these data support the application for MJN110 as a novel therapeutic for cancer-induced bone pain.

Despite the progress in the development of novel treatment modalities, a significant portion of patients with psoriasis remains undertreated relative to the severity of their disease. Recent evidence points to targeting the glucose transporter 1 and sugar metabolism as a novel therapeutic strategy for the treatment of psoriasis and other hyperproliferative skin diseases. In this review, we discuss glycoconjugation, an approach that facilitates the pharmacokinetics of cytotoxic molecules and ensures their preferential influx through glucose transporters. We propose pathways of glycoconjugate synthesis to increase effectiveness, cellular selectivity, and tolerability of widely used antipsoriatic drugs. The presented approach exploiting the heightened glucose requirement of proliferating keratinocytes bears the potential to revolutionize the management of psoriasis.

Glucagon-like peptide-2 (GLP-2) agonists have therapeutic potential in clinical indications in which the integrity or absorptive function of the intestinal mucosa is compromised, such as in short bowel syndrome (SBS). Native hGLP-2, a 33–amino acid peptide secreted from the small intestine, contributes to nutritional absorption but has a very short half-life because of enzymatic cleavage and renal clearance and thus is of limited therapeutic value. The GLP-2 analog teduglutide (Revestive/Gattex; Shire Inc.) has been approved for use in SBS since 2012 but has a once-daily injection regimen. Pharmacokinetic (PK) and pharmacodynamic studies confirm that apraglutide, a novel GLP-2 analog, has very low clearance, long elimination half-life, and high plasma protein binding compared with GLP-2 analogs teduglutide and glepaglutide. Apraglutide and teduglutide retain potency and selectivity at the GLP-2 receptor comparable to native hGLP-2, whereas glepaglutide was less potent and less selective. In rat intravenous PK studies, hGLP-2, teduglutide, glepaglutide, and apraglutide had clearances of 25, 9.9, 2.8, and 0.27 ml/kg per minute, respectively, and elimination half-lives of 6.4, 19, 16, and 159 minutes, respectively. The unique PK profile of apraglutide administered via intravenous and subcutaneous routes was confirmed in monkey and minipig and translated into significantly greater in vivo pharmacodynamic activity, measured as small intestinal growth in rats. Apraglutide showed greater intestinotrophic activity than the other peptides when administered at less-frequent dosing intervals because of its prolonged half-life. We postulate that apraglutide offers several advantages over existing GLP-2 analogs and is an excellent candidate for the treatment of gastrointestinal diseases, such as SBS.