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.

Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte–EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation.

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 Carboniferous Bowland Shale Formation of the UK is a proven hydrocarbon source rock and currently a target for shale gas exploration. Most existing analysis details lithofacies and geochemical assessment of a small number of boreholes. Given a paucity of relevant borehole cores, surface samples provide a valuable contribution to the assessment of this unconventional gas source. This study reviews existing literature on the formation's hydrocarbon geochemistry and provides new lithological descriptions of seven lithofacies, XRD mineralogy and hydrocarbon-specific geochemical data for 32 outcrop localities within the Craven and Edale basins, respectively in the northern and southern parts of the resource area. Low oxygen indices suggest that the majority of samples are relatively unaltered (in terms of hydrocarbon geochemistry), and therefore suitable for the characterization of the shale organic character. Total organic carbon (TOC) ranges from 0.7 to 6.5 wt%, with highest values associated with maximum flooding surfaces. Mean T_max values of 447 and 441°C for the Edale and Craven basins, respectively, suggest that nearly all the samples were too immature to have generated appreciable amounts of dry gas. The oil saturation index is consistently below the >100 mg g^–1 TOC benchmark, suggesting that they are not prospective for shale oil.

Supplementary material: A table summarizing the location, geological description and age of all of the samples in this paper is available at https://doi.org/10.6084/m9.figshare.c.4444589

Detailed interpretation of a 3D seismic data volume reveals the detrimental effect that post-depositional tectonic deformation has had on buried Lower Carboniferous (Dinantian–Namurian) shales and its consequences for shale gas exploration in the SW part (Fylde area) of the Craven Basin in NW England. The structural styles primarily result from Devono-Carboniferous (syn-sedimentary) extension, post-rift subsidence and Variscan inversion, a renewed phase of Permo-Triassic extension, and Cenozoic uplift and basin exhumation. In contrast to the shallow dips and bedding continuity that characterizes productive shale gas plays in other basins (e.g. in the USA and Argentina), our mapping shows that the area is affected by deformation that results in the Bowland Shale Formation targets being folded and dissected into fault-bound compartments defined by SW–NE striking (Lower Carboniferous and Variscan) reverse faults and SSW–NNE to N–S striking (Permo-Triassic) normal faults. The fault networks and the misalignment between the elongate compartments they contain and the present-day minimum horizontal stress orientation limit the length over which long lateral boreholes can remain in a productive horizon, placing an important constraint on optimal well positioning, reducing the size of the shale gas resource and affecting well productivity. Our subsurface mapping using this high-fidelity dataset provides an accurate picture of the Upper Palaeozoic structure and demonstrates that faulting is denser and more complex than apparent from geological mapping of the surface outcrop. That structural complexity has direct and significant consequences for: the location of well pads; the lateral continuity of target shale gas horizons; the evaluation of the risk of inducing seismicity on seismically resolvable (large displacement) fault planes prior to drilling; and the likelihood of faults with small throws (below seismic resolution) being present.

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.

Accurate detection of influenza A virus (IAV) is crucial for patient management, infection control, and epidemiological surveillance. The World Health Organization and the Centers for Disease Control and Prevention have recommended using the M gene as the diagnostic gene target for reverse-transcription-PCR (RT-PCR). However, M gene RT-PCR has reduced sensitivity for recent IAV due to novel gene mutations. Here, we sought to identify novel diagnostic targets for the molecular detection of IAV using long-read third-generation sequencing. Direct nanopore sequencing from 18 nasopharyngeal specimens and one saliva specimen showed that the 5' and 3' ends of the PB2 gene and the entire NS gene were highly abundant. Primers selected for PB2 and NS genes were well matched with seasonal or avian IAV gene sequences. Our novel PB2 and NS gene real-time RT-PCR assays showed limits of detection similar to or lower than that of M gene RT-PCR and achieved 100% sensitivity and specificity in the detection of A(H1N1), A(H3N2), and A(H7N9) in nasopharyngeal and saliva specimens. For 10 patients with IAV detected by M gene RT-PCR conversion in sequentially collected specimens, NS and/or PB2 gene RT-PCR was positive in 2 (20%) of the initial specimens that were missed by M gene RT-PCR. In conclusion, we have shown that PB2 or NS gene RT-PCRs are suitable alternatives to the recommended M gene RT-PCR for diagnosis of IAV. Long-read nanopore sequencing facilitates the identification of novel diagnostic targets.

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.

The new decade of the 21^st century (2020) started with the emergence of a novel coronavirus known as SARS-CoV-2 that caused an epidemic of coronavirus disease (COVID-19) in Wuhan, China. It is the third highly pathogenic and transmissible coronavirus after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in humans. The source of origin, transmission to humans, and mechanisms associated with the pathogenicity of SARS-CoV-2 are not yet clear, however, its resemblance to SARS-CoV and several other bat coronaviruses was recently confirmed through genome sequencing-related studies. The development of therapeutic strategies is necessary in order to prevent further epidemics and cure infections. In this review, we summarize current information about the emergence, origin, diversity, and epidemiology of three pathogenic coronaviruses with a specific focus on the current outbreak in Wuhan, China. Furthermore, we discuss the clinical features and potential therapeutic options that may be effective against SARS-CoV-2.

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.

Dual amylin and calcitonin receptor agonists (DACRAs) are novel candidates for treatment of type 2 diabetes and obesity because of their beneficial effects on body weight, blood glucose, insulin sensitivity, and food preference, at least short-term. DACRAs activate the receptors for a prolonged time period, resulting in metabolic effects superior to those of amylin. Because of the prolonged receptor activation, different dosing intervals and, hence, less frequent receptor activation might change the efficacy of DACRA treatment in terms of weight loss and food preference. In this study, we compared daily dosing to dosing every other day with the aim of understanding the optimal balance between efficacy and tolerability. Obese and lean male Sprague-Dawley rats were treated with the DACRA KBP-088, applying two different dosing intervals (1.5 nmol/kg once daily and 3 nmol/kg every other day) to assess the effect on body weight, food intake, glucose tolerance, and food preference when given the choice between chow (13% fat) and a high-fat diet (60% fat). Treatment with KBP-088 induced significant weight loss, reduction in adiposity, improvement in glucose control, and altered food preference toward food that is less calorie-dense. KBP-088 dosed every other day (3 nmol/kg) was superior to KBP-088 once daily (1.5 nmol/kg) in terms of weight loss and improvement of food preference. The beneficial effects were evident in both lean and obese rats. Hence, dosing KBP-088 every other day positively affects overall efficacy on metabolic parameters regardless of the lean/obese state, suggesting that less-frequent dosing with KBP-088 could be feasible.

PF06821497 has been identified as an orally available small-molecule enhancer of zeste homolog 2 inhibitor. The objectives of the present study were to characterize pharmacokinetic-pharmacodynamic-disease relationships of PF06821497 in xenograft mouse models with diffuse large B-cell lymphoma (Karpas422). An indirect-response model reasonably fit dose-dependent pharmacodynamic responses [histone H3 on lysine 27 (H3K27) me3 inhibition] with an unbound EC₅₀ of 76 nM, whereas a signal-transduction model sufficiently fit dose-dependent disease responses (tumor growth inhibition) with an unbound tumor stasis concentration (T_sc) of 168 nM. Thus, effective concentration for 70% of maximal effect (EC₇₀) for H3K27me3 inhibition was roughly comparable to T_sc, suggesting that 70% H3K27me3 inhibition could be required for tumor stasis. Consistently, an integrated pharmacokinetic-pharmacodynamic-disease model adequately describing tumor growth inhibition also suggested that ~70% H3K27me3 inhibition was associated with tumor stasis. Based on these results, we would propose that an EC₇₀ estimate for H3K27me3 inhibition corresponding to tumor stasis could be considered a minimum target efficacious concentration of PF06821497 in cancer patients.

Behavioral flexibility is important in a changing environment. Previous research suggests that systems consolidation, a long-term poststorage process that alters memory traces, may reduce behavioral flexibility. However, exactly how systems consolidation affects flexibility is unknown. Here, we tested how systems consolidation affects: (1) flexibility in response to value changes and (2) flexibility in response to changes in the optimal sequence of actions. Mice were trained to obtain food rewards in a Y-maze by switching nose pokes between three arms. During initial training, all arms were rewarded and mice simply had to switch arms in order to maximize rewards. Then, after either a 1 or 28 d delay, we either devalued one arm, or we reinforced a specific sequence of pokes. We found that after a 1 d delay mice adapted relatively easily to the changes. In contrast, mice given a 28 d delay struggled to adapt, especially for changes to the optimal sequence of actions. Immediate early gene imaging suggested that the 28 d mice were less reliant on their hippocampus and more reliant on their medial prefrontal cortex. These data suggest that systems consolidation reduces behavioral flexibility, particularly for changes to the optimal sequence of actions.

PF-05089771 is an aryl sulfonamide Nav1.7 channel blocker that binds to the inactivated state of Nav1.7 channels with high affinity but binds only weakly to channels in the resting state. Such aryl sulfonamide Nav1.7 channel blockers bind to the extracellular surface of the S1-S4 voltage-sensor segment of homologous Domain 4, whose movement is associated with inactivation. This binding site is different from that of classic sodium channel inhibitors like lidocaine, which also bind with higher affinity to the inactivated state than the resting state but bind at a site within the pore of the channel. The common dependence on gating state with distinct binding sites raises the possibility that inhibition by aryl sulfonamides and by classic local anesthetics might show an interaction mediated by their mutual state dependence. We tested this possibility by examining the state-dependent inhibition by PF-05089771 and lidocaine of human Nav1.7 channels expressed in human embryonic kidney 293 cells. At –80 mV, where a small fraction of channels are in an inactivated state under drug-free conditions, inhibition by PF-05089771 was both enhanced and speeded in the presence of lidocaine. The results suggest that lidocaine binding to the channel enhances PF-05089771 inhibition by altering the equilibrium between resting states (with D4S4 in the inner position) and inactivated states (with D4S4 in the outer position). The gating state–mediated interaction between the compounds illustrates a principle applicable to many state-dependent agents.

The incidence of abnormal cerebrospinal fluid (CSF) flow dynamics in children with central nervous system (CNS) tumors before intraventricular therapy has not been described. Methods: We performed a single-institution, retrospective review of patients with primary or metastatic CNS tumors treated between 2003 and 2018 (15 y). Patients underwent ¹¹¹In-diethylenetriaminepentaacetic acid injection into the CSF intraventricular space followed by nuclear medicine imaging at 90 min, 4 h, 24 h, and 48 h (if required). CSF flow was classified as normal, delayed, asymmetric, or obstructed. Results: In total, 278 CSF flow studies were performed on 224 patients, 202 of whom (90%) were less than 18 y old. Of these, 116 patients (52%) had metastatic CNS neuroblastoma, 57 (25%) had medulloblastoma, and 51 (23%) had other histologic types of CNS tumors. Of the 278 studies, 237 (85%) were normal, 9 (3%) required neurosurgical intervention, 25 (9%) were delayed, and 7 (3%) were asymmetric. Conclusion: Abnormal CSF flow and the necessity for neurosurgical intervention must be considered when attempting to ensure appropriate intraventricular therapy in the pediatric population.

From a behavioral perspective, therapeutic inertia can happen when obstacles to changing a diabetes treatment plan outweigh perceived benefits. There is a complex interaction of important treatment-related obstacles for people with diabetes (PWD), their treating health care professional (HCP), and the clinical setting in which they interact. Tipping the scales toward more effective action involve strategies that increase perceptions of the benefits of treatment intensification while addressing important obstacles so that treatment changes are seen by both PWD and HCPs as worthwhile and achievable.

ABSTRACT

Members of family Coronaviridae cause a variety of diseases in birds and mammals. Porcine hemagglutinating encephalomyelitis virus (PHEV), a lesser-researched coronavirus, can infect naive pigs of any age, but clinical disease is observed in pigs ≤4 weeks of age. No commercial PHEV vaccines are available, and neonatal protection from PHEV-associated disease is presumably dependent on lactogenic immunity. Although subclinical PHEV infections are thought to be common, PHEV ecology in commercial swine herds is unknown. To begin to address this gap in knowledge, a serum IgG antibody enzyme-linked immunosorbent assay (ELISA) based on the S1 protein was developed and evaluated on known-status samples and then used to estimate PHEV seroprevalence in U.S. sow herds. Assessment of the diagnostic performance of the PHEV S1 ELISA using serum samples (n = 924) collected from 7-week-old pigs (n = 84; 12 pigs per group) inoculated with PHEV, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, or porcine deltacoronavirus showed that a sample-to-positive cutoff value of ≥0.6 was both sensitive and specific, i.e., all PHEV-inoculated pigs were seropositive from days postinoculation 10 to 42, and no cross-reactivity was observed in samples from other groups. The PHEV S1 ELISA was then used to estimate PHEV seroprevalence in U.S. sow herds (19 states) using 2,756 serum samples from breeding females (>28 weeks old) on commercial farms (n = 104) with no history of PHEV-associated disease. The overall seroprevalence was 53.35% (confidence interval [CI], ±1.86%) and herd seroprevalence was 96.15% (CI, ±3.70%).

IMPORTANCE There is a paucity of information concerning the ecology of porcine hemagglutinating encephalomyelitis virus (PHEV) in commercial swine herds. This study provided evidence that PHEV infection is endemic and highly prevalent in U.S. swine herds. These results raised questions for future studies regarding the impact of endemic PHEV on swine health and the mechanisms by which this virus circulates in endemically infected populations. Regardless, the availability of the validated PHEV S1 enzyme-linked immunosorbent assay (ELISA) provides the means for swine producers to detect and monitor PHEV infections, confirm prior exposure to the virus, and to evaluate the immune status of breeding herds.

ABSTRACT

Aspergillus flavus, a ubiquitous and saprophytic fungus, is the second most common cause of aspergillosis worldwide. Several mechanisms contribute to the establishment of the fungal infection. Extracellular vesicles (EVs) have been described as "virulence factor delivery bags" in several fungal species, demonstrating a crucial role during the infection. In this study, we evaluated production of A. flavus EVs and their immunomodulatory functions. We verified that A. flavus EVs induce macrophages to produce inflammatory mediators, such as nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1β. Furthermore, the A. flavus EVs enhance phagocytosis and killing by macrophages and induce M1 macrophage polarization in vitro. In addition, a prior inoculation of A. flavus EVs in Galleria mellonella larvae resulted in a protective effect against the fungal infection. Our findings suggest that A. flavus EVs are biologically active and affect the interaction between A. flavus and host immune cells, priming the innate immune system to eliminate the fungal infection. Collectively, our results suggest that A. flavus EVs play a crucial role in aspergillosis.

IMPORTANCE Immunocompromised patients are susceptible to several fungal infections. The genus Aspergillus can cause increased morbidity and mortality. Developing new therapies is essential to understand the fungal biology mechanisms. Fungal EVs carry important virulence factors, thus playing pivotal roles in fungal pathophysiology. No study to date has reported EV production by Aspergillus flavus, a fungus considered to be the second most common cause of aspergillosis and relevant food contaminator found worldwide. In this study, we produced A. flavus EVs and evaluated the in vitro immunomodulatory effects of EVs on bone marrow-derived macrophages (BMDMs) and in vivo effects in a Galleria mellonella model.

The bioprocessing industry uses recombinant mammalian cell lines to generate therapeutic biologic drugs. To ensure consistent product quality of the therapeutic proteins, it is imperative to have a controlled production process. Regulatory agencies and the biotechnology industry consider cell line "clonal origin" an important aspect of maintaining process control. Demonstration of clonal origin of the cell substrate, or production cell line, has received considerable attention in the past few years, and the industry has improved methods and devised standards to increase the probability and/or assurance of clonal derivation. However, older production cell lines developed before the implementation of these methods, herein referred to as "legacy cell lines," may not meet current regulatory expectations for demonstration of clonal derivation. In this article, the members of the IQ Consortium Working Group on Clonality present our position that the demonstration of process consistency and product comparability of critical quality attributes throughout the development life cycle should be sufficient to approve a license application without additional genetic analysis to support clonal origin, even for legacy cell lines that may not meet current day clonal derivation standards. With this commentary, we discuss advantages and limitations of genetic testing methods to support clonal derivation of legacy cell lines and wish to promote a mutual understanding with the regulatory authorities regarding their optional use during early drug development, subsequent to Investigational New Drug (IND) application and before demonstration of product and process consistency at Biologics License Applications (BLA) submission.

The substantial discrepancy between the strong effects of functional foods and various drugs, especially traditional Chinese medicines (TCMs), and the poor bioavailability of these substances remains a perplexing problem. Understanding the gut microbiota, which acts as an effective bioreactor in the human intestinal tract, provides an opportunity for the redefinition of bioavailability. Here, we discuss four different pathways associated with the role of the gut microbiota in the transformation of parent compounds to beneficial or detrimental small molecules, which can enter the body’s circulatory system and be available to target cells, tissues, and organs. We further describe and propose effective strategies for improving bioavailability and alleviating side effects with the help of the gut microbiota. This review also broadens our perspectives for the discovery of new medicinal components.

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.

BackgroundSelf-harm is a serious risk factor for suicide, a major public health concern, and a significant burden on the NHS. Rates of self-harm presentation in primary care are rising and GPs interact with patients both before and after they have self-harmed. There is significant public and political interest in reducing rates of self-harm, but there has been no robust synthesis of the existing literature on the role of GPs in the management of patients who self-harm.AimThis study aimed to explore the role of the GP in the management of patients with self-harm behaviour.Design and settingA systematic review and narrative synthesis of primary care literature.MethodThis systematic review was conducted and is reported in line with PRISMA guidance. Electronic databases systematically searched were MEDLINE, PsycINFO, EMBASE, CINAHL, Web of Science, and AMED. Two independent reviewers conducted study screening and selection, data extraction, and quality appraisal of all included studies. Thematic analysis was conducted.ResultsFrom 6976 unique citations, 12 studies met eligibility criteria and were included. These 12 studies, published from 1997–2016, of 789 GPs/family medicine physicians from Europe, the US, and Australia were of good methodological quality. Five themes were identified for facilitating GP management of self-harm: GP training, improved communication, service provision, clinical guidelines, and young people. Four barriers for GP management of self-harm were identified: assessment, service provision, local, and systemic factors.ConclusionGPs recognise self-harm as a serious risk factor for suicide, but some feel unprepared for managing self-harm. The role of the GP is multidimensional and includes frontline assessment and treatment, referral to specialist care, and the provision of ongoing support.

Merkel cell carcinoma (MCC) is often caused by persistent expression of Merkel cell polyomavirus (MCPyV) T-antigen (T-Ag). These non-self proteins comprise about 400 amino acids (AA). Clinical responses to immune checkpoint inhibitors, seen in about half of patients, may relate to T-Ag–specific T cells. Strategies to increase CD8⁺ T-cell number, breadth, or function could augment checkpoint inhibition, but vaccines to augment immunity must avoid delivery of oncogenic T-antigen domains. We probed MCC tumor-infiltrating lymphocytes (TIL) with an artificial antigen-presenting cell (aAPC) system and confirmed T-Ag recognition with synthetic peptides, HLA-peptide tetramers, and dendritic cells (DC). TILs from 9 of 12 (75%) subjects contained CD8⁺ T cells recognizing 1–8 MCPyV epitopes per person. Analysis of 16 MCPyV CD8⁺ TIL epitopes and prior TIL data indicated that 97% of patients with MCPyV⁺ MCC had HLA alleles with the genetic potential that restrict CD8⁺ T-cell responses to MCPyV T-Ag. The LT AA 70–110 region was epitope rich, whereas the oncogenic domains of T-Ag were not commonly recognized. Specific recognition of T-Ag–expressing DCs was documented. Recovery of MCPyV oncoprotein–specific CD8⁺ TILs from most tumors indicated that antigen indifference was unlikely to be a major cause of checkpoint inhibition failure. The myriad of epitopes restricted by diverse HLA alleles indicates that vaccination can be a rational component of immunotherapy if tumor immune suppression can be overcome, and the oncogenic regions of T-Ag can be modified without impacting immunogenicity.

Burkholderia sp. strain SG-MS1 and Pseudomonas sp. strain SG-MS2 have previously been found to mineralize (+)-pinoresinol through a common catabolic pathway. Here, we used comparative genomics, proteomics, protein semipurification, and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 that carries out the initial hydroxylation of (+)-pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene, and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a K_m of 1.17 μM for (+)-pinoresinol. The enzyme is overexpressed in strain SG-MS2 upon exposure to (+)-pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1-kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7-kb cluster in SG-MS1. The amino acid identities of four of the other proteins within the SG-MS2 cluster suggest they catalyze conversion of hydroxylated pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways, but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome genes.

IMPORTANCE (+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the β-β' linkages in lignin. We report a gene cluster, in one strain each of Pseudomonas and Burkholderia, that is involved in the oxidative catabolism of (+)-pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work casts new light on the biology of (+)-pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans.

Background:

The negative effect of adverse childhood experiences (ACEs) on physical and mental health has led to calls for routine screening for ACEs in primary care settings. We aimed to examine the association between maternal ACEs and children’s behaviour problems (externalizing and internalizing) at age 5 in the context of other known predictors.

Genetic variants are the primary driver of congenital heart disease (CHD) pathogenesis. However, our ability to identify causative variants is limited. To identify causal CHD genes that are associated with specific molecular functions, the study used prior knowledge to filter de novo variants from 2,881 probands with sporadic severe CHD. This approach enabled the authors to identify an association between left ventricular outflow tract obstruction lesions and genes associated with the WAVE2 complex and regulation of small GTPase-mediated signal transduction. Using CRISPR zebrafish knockdowns, the study confirmed that WAVE2 complex proteins brk1, nckap1, and wasf2 and the regulators of small GTPase signaling cul3a and racgap1 are critical to cardiac development.

Nosocomial pneumonia (NP), including ventilator-associated pneumonia (VAP), is increasingly associated with multidrug-resistant Gram-negative pathogens. This study describes the in vitro activity of ceftazidime-avibactam, ceftazidime, and relevant comparator agents against bacterial pathogens isolated from patients with NP, including VAP, enrolled in a ceftazidime-avibactam phase 3 trial. Gram-positive pathogens were included if coisolated with a Gram-negative pathogen. In vitro susceptibility was determined at a central laboratory using Clinical and Laboratory Standards Institute broth microdilution methods. Of 817 randomized patients, 457 (55.9%) had ≥1 Gram-negative bacterial pathogen(s) isolated at baseline, and 149 (18.2%) had ≥1 Gram-positive pathogen(s) coisolated. The most common isolated pathogens were Klebsiella pneumoniae (18.8%), Pseudomonas aeruginosa (15.8%), and Staphylococcus aureus (11.5%). Ceftazidime-avibactam was highly active in vitro against 370 isolates of Enterobacteriaceae, with 98.6% susceptible (MIC₉₀, 0.5 μg/ml) compared with 73.2% susceptible for ceftazidime (MIC₉₀, >64 μg/ml). The percent susceptibility values for ceftazidime-avibactam and ceftazidime against 129 P. aeruginosa isolates were 88.4% and 72.9% (MIC₉₀ values of 16 μg/ml and 64 μg/ml), respectively. Among ceftazidime-nonsusceptible Gram-negative isolates, ceftazidime-avibactam percent susceptibility values were 94.9% for 99 Enterobacteriaceae and 60.0% for 35 P. aeruginosa. MIC₉₀ values for linezolid and vancomycin (permitted per protocol for Gram-positive coverage) were within their respective MIC susceptibility breakpoints against the Gram-positive pathogens isolated. This analysis demonstrates that ceftazidime-avibactam was active in vitro against the majority of Enterobacteriaceae and P. aeruginosa isolates from patients with NP, including VAP, in a phase 3 trial. (This study has been registered at ClinicalTrials.gov under identifier NCT01808092.)

Infections with nontuberculous mycobacteria (NTM) have a poor prognosis in patients with underlying respiratory diseases. Clofazimine (CFZ) showed both experimental and clinical promising results against clinically relevant NTM. However, there are no data on CFZ in combination with the current recommended treatment; therefore, we aimed to study its in vivo activity in an aerosol mouse model of Mycobacterium avium. In an aerosol infection BALB/c mouse model using M. avium strain Chester, we treated 58 mice with four combinations of rifampin (RIF) at 10 mg/kg, CFZ at 25 mg/kg, and clarithromycin (CLR) and ethambutol (EMB) at 100 mg/kg. Treatment efficacy was assessed on the basis of lung CFU counts after 2 (M2) and 4 (M4) months of treatment. At M2, CLR-RIF-EMB was slightly but significantly more efficient than CFZ-RIF-EMB (3.02 ± 0.12 versus 3.55 ± 0.28, respectively, P < 0.01), whereas CLR-CFZ-EMB and CLR-CFZ-RIF-EMB dramatically decreased lung CFU counts by 4.32 and 4.47 log₁₀, respectively, compared to untreated group. At M4, CLR-RIF-EMB was significantly more efficient than CFZ-RIF-EMB (2 ± 0.53 versus 2.66 ± 0.22, respectively, P = 0.01). The addition of CLZ to CLR dramatically decreased the lung CFU count, with CFU counts 5.41 and 5.79 log₁₀ lower in the CLR-CFZ-EMB and CLR-CFZ-RIF-EMB groups, respectively, than in the untreated group. The addition of CFZ to CLR seems to improve the efficacy of CLR as early as M2 and was confirmed at M4. CFZ, in addition to RIF and EMB, on the other hand, is less effective than CLR-RIF-EMB. These results need to be confirmed by similar studies along with CFZ potential for shortening treatment.

The comparative efficacy of ceftazidime-avibactam and meropenem-vaborbactam for treatment of carbapenem-resistant Enterobacteriaceae (CRE) infections remains unknown. This was a multicenter, retrospective cohort study of adults with CRE infections who received ceftazidime-avibactam or meropenem-vaborbactam for ≥72 hours from February 2015 to October 2018. Patients with a localized urinary tract infection and repeat study drug exposures after the first episode were excluded. The primary endpoint was clinical success compared between treatment groups. Secondary endpoints included 30- and 90-day mortality, adverse events (AE), 90-day CRE infection recurrence, and development of resistance in patients with recurrent infection. A post hoc subgroup analysis was completed comparing patients who received ceftazidime-avibactam monotherapy, ceftazidime-avibactam combination therapy, and meropenem-vaborbactam monotherapy. A total of 131 patients were included (ceftazidime-avibactam, n = 105; meropenem-vaborbactam, n = 26), 40% of whom had bacteremia. No significant difference in clinical success was observed between groups (62% versus 69%; P = 0.49). Patients in the ceftazidime-avibactam arm received combination therapy more often than patients in the meropenem-vaborbactam arm (61% versus 15%; P < 0.01). No difference in 30- and 90-day mortality resulted, and rates of AE were similar between groups. In patients with recurrent infection, development of resistance occurred in three patients that received ceftazidime-avibactam monotherapy and in no patients in the meropenem-vaborbactam arm. Clinical success was similar between patients receiving ceftazidime-avibactam and meropenem-vaborbactam for treatment of CRE infections, despite ceftazidime-avibactam being used more often as a combination therapy. Development of resistance was more common with ceftazidime-avibactam monotherapy.

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung condition, causing progressive decline in lung function leading to premature death. Acute exacerbations in COPD patients are predominantly associated with respiratory viruses. Ribavirin is a generic broad-spectrum antiviral agent that could be used for treatment of viral respiratory infections in COPD. Using the Particle Replication In Nonwetting Templates (PRINT) technology, which produces dry-powder particles of uniform shape and size, two new inhaled formulations of ribavirin (ribavirin-PRINT-CFI and ribavirin-PRINT-IP) were developed for efficient delivery to the lung and to minimize bystander exposure. Ribavirin-PRINT-CFI was well tolerated in healthy participants after single dosing and ribavirin-PRINT-IP was well tolerated in healthy and COPD participants after single and repeat dosing. Ribavirin-PRINT-CFI was replaced with ribavirin-PRINT-IP since the latter formulation was found to have improved physicochemical properties and it had a higher ratio of active drug to excipient per unit dose. Ribavirin concentrations were measured in lung epithelial lining fluid in both healthy and COPD participants and achieved target concentrations. Both formulations were rapidly absorbed with approximately dose proportional pharmacokinetics in plasma. Exposure to bystanders was negligible based on both the plasma and airborne ribavirin concentrations with the ribavirin-PRINT-IP formulation. Thus, ribavirin-PRINT-IP allowed for an efficient and convenient delivery of ribavirin to the lungs while minimizing systemic exposure. Further clinical investigations would be required to demonstrate ribavirin-PRINT-IP antiviral characteristics and impact on COPD viral-induced exacerbations. (The clinical trials discussed in this study have been registered at ClinicalTrials.gov under identifiers NCT03243760 and NCT03235726.)

Current treatments for Acanthamoeba keratitis rely on a combination of chlorhexidine gluconate, propamidine isethionate, and polyhexamethylene biguanide. These disinfectants are nonspecific and inherently toxic, which limits their effectiveness. Furthermore, in 10% of cases, recurrent infection ensues due to the difficulty in killing both trophozoites and double-walled cysts. Therefore, development of efficient, safe, and target-specific drugs which are capable of preventing recurrent Acanthamoeba infection is a critical unmet need for averting blindness. Since both trophozoites and cysts contain specific sets of membrane sterols, we hypothesized that antifungal drugs targeting sterol 14-demethylase (CYP51), known as conazoles, would have deleterious effects on A. castellanii trophozoites and cysts. To test this hypothesis, we first performed a systematic screen of the FDA-approved conazoles against A. castellanii trophozoites using a bioluminescence-based viability assay adapted and optimized for Acanthamoeba. The most potent drugs were then evaluated against cysts. Isavuconazole and posaconazole demonstrated low nanomolar potency against trophozoites of three clinical strains of A. castellanii. Furthermore, isavuconazole killed trophozoites within 24 h and suppressed excystment of preformed Acanthamoeba cysts into trophozoites. The rapid action of isavuconazole was also evident from the morphological changes at nanomolar drug concentrations causing rounding of trophozoites within 24 h of exposure. Given that isavuconazole has an excellent safety profile, is well tolerated in humans, and blocks A. castellanii excystation, this opens an opportunity for the cost-effective repurposing of isavuconazole for the treatment of primary and recurring Acanthamoeba keratitis.

Etravirine (ETR) is a nonnucleoside reverse transcriptase inhibitor (NNRTI) used in treatment-experienced individuals. Genotypic resistance test-interpretation systems can predict ETR resistance; however, genotype-based algorithms are derived primarily from HIV-1 subtype B and may not accurately predict resistance in non-B subtypes. The frequency of ETR resistance among recombinant subtype C HIV-1 and the accuracy of genotypic interpretation systems were investigated. HIV-1_LAI containing full-length RT from HIV-1 subtype C-positive individuals experiencing virologic failure (>10,000 copies/ml and >1 NNRTI resistance-associated mutation) were phenotyped for ETR susceptibility. Fold change (FC) was calculated against a composite 50% effective concentration (EC₅₀) from treatment-naive individuals and three classifications were assigned: (i) <2.9-FC, susceptible; (ii) ≥2.9- to 10-FC, partially resistant; and (iii) >10-FC, fully resistant. The Stanford HIVdb-v8.4 was used for genotype predictions merging the susceptible/potential low-level and low-level/intermediate groups for 3 x 3 comparison. Fifty-four of a hundred samples had reduced ETR susceptibility (≥2.9-FC). The FC correlated with HIVdb-v8.4 (Spearman’s rho = 0.62; P < 0.0001); however, 44% of samples were partially (1 resistance classification difference) and 4% completely discordant (2 resistance classification differences). Of the 34 samples with an FC of >10, 26 were HIVdb-v8.4 classified as low-intermediate resistant. Mutations L100I, Y181C, or M230L were present in 27/34 (79%) of samples with an FC of >10 but only in 2/46 (4%) of samples with an FC of <2.9. No other mutations were associated with ETR resistance. Viruses containing the mutation K65R were associated with reduced ETR susceptibility, but 65R reversions did not increase ETR susceptibility. Therefore, genotypic interpretation systems were found to misclassify ETR susceptibility in HIV-1 subtype C samples. Modifications to genotypic algorithms are needed to improve the prediction of ETR resistance for the HIV-1 subtype C.

Dalbavancin offers a possible treatment option for infectious peritonitis associated with peritoneal dialysis (PD) due to its coverage of Gram-positive bacteria and pharmacokinetic properties. We aimed to evaluate the clinical pharmacokinetics (PK) and pharmacodynamics of dalbavancin in a prospective, randomized, open-label, crossover PK study of adult patients with end-stage renal disease ESRD who were receiving PD. Sampling occurred prior to a single 30-min infusion of dalbavancin at 1,500 mg and at 1, 2, 3, 4, and 6 h and 7 and 14 days postadministration. Concentration-time data were analyzed via noncompartmental analysis. Pharmacodynamic parameters against common infectious peritonitis-causing pathogens were evaluated. Ten patients were enrolled. Patients were a median of 55 years old and had a median weight of 78.2 kg, 50% were female, and 70% were Caucasian. The terminal plasma half-life of dalbavancin was 181.4 ± 35.5 h. The day 0 to day 14 dalbavancin mean area under the curve (AUC) was 40,573.2 ± 9,800.3 mg·h/liter. The terminal-phase half-life of dalbavancin within the peritoneal fluid was 4.309 x 10⁸ ± 1.140 x 10⁹ h. The day 0 to day 14 dalbavancin mean peritoneal fluid AUC was 2,125.0 ± 1,794.3 mg·h/liter. The target plasma AUC/MIC was attained with the intravenous dose in all 10 patients for all Staphylococcus and Streptococcus species at the recommended MIC breakpoints. The intraperitoneal arm of the study was stopped early, because the first 3 patients experienced moderate to severe pain and bloating within 1 h following the administration of dalbavancin. Dalbavancin at 1,500 mg administered intravenously can be utilized without dose adjustment in peritoneal dialysis patients and will likely achieve the necessary peritoneal fluid concentrations to treat peritonitis caused by typical Gram-positive pathogens.

The effects of multiple-dose administration of tenofovir disoproxil fumarate (TDF) on the pharmacokinetics of morinidazole (MOR) were compared in healthy subjects. MOR exposure was similar, with an area under the curve from 0 h to infinity (AUC_0-) treatment ratio for MOR+TDF/MOR of 1.01 (90% confidence interval, 0.97 to 1.06). No relevant differences were observed regarding plasma exposure of metabolites. Renal clearances of MOR and its metabolites were not affected by TDF. No unexpected safety or tolerability issues were observed.

In children requiring lopinavir coformulated with ritonavir in a 4:1 ratio (lopinavir-ritonavir-4:1) and rifampin, adding ritonavir to achieve a 4:4 ratio with lopinavir (LPV/r-4:4) overcomes the drug-drug interaction. Possible drug-drug interactions within this regimen may affect abacavir concentrations, but this has never been studied. Children weighing <15 kg needing rifampin and LPV/r-4:4 were enrolled in a pharmacokinetic study and underwent intensive pharmacokinetic sampling on 3 visits: (i) during the intensive and (ii) continuation phases of antituberculosis treatment with LPV/r-4:4 and (iii) 1 month after antituberculosis treatment completion on LPV/r-4:1. Pharmacometric modeling and simulation were used to compare exposures across weight bands with adult target exposures. Eighty-seven children with a median (interquartile range) age and weight of 19 (4 to 64) months and 8.7 (3.9 to 14.9) kg, respectively, were included in the abacavir analysis. Abacavir pharmacokinetics were best described by a two-compartment model with first-order elimination and transit compartment absorption. After allometric scaling adjusted for the effect of body size, maturation could be identified: clearance was predicted to be fully mature at about 2 years of age and to reach half of this mature value at about 2 months of age. Abacavir bioavailability decreased 36% during treatment with rifampin and LPV/r-4:4 but remained within the median adult recommended exposure, except for children in the 3- to 4.9-kg weight band, in which the exposures were higher. The observed predose morning trough concentrations were higher than the evening values. Though abacavir exposure significantly decreased during concomitant administration of rifampin and LPV/r-4:4, it remained within acceptable ranges. (This study is registered in ClinicalTrials.gov under identifier NCT02348177.)

Currently, the expansion of the novel human respiratory coronavirus (known as SARS-CoV-2 [severe acute respiratory syndrome coronavirus 2], COVID-2019 [coronavirus disease 2019], or 2019-nCoV [2019 novel coronavirus]) has stressed the need for therapeutic alternatives to alleviate and stop this new epidemic. The previous epidemics of infections by high-morbidity human coronaviruses, such as SARS-CoV in 2003 and the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, prompted the characterization of compounds that could be potentially active against the currently emerging novel coronavirus, SARS-CoV-2. The most promising compound is remdesivir (GS-5734), a nucleotide analog prodrug currently in clinical trials for treating Ebola virus infections. Remdesivir inhibited the replication of SARS-CoV and MERS-CoV in tissue cultures, and it displayed efficacy in nonhuman animal models. In addition, a combination of the human immunodeficiency virus type 1 (HIV-1) protease inhibitors lopinavir/ritonavir and interferon beta (LPV/RTV–IFN-β) was shown to be effective in patients infected with SARS-CoV. LPV/RTV–IFN-β also improved clinical parameters in marmosets and mice infected with MERS-CoV. Remarkably, the therapeutic efficacy of remdesivir appeared to be superior to that of LPV/RTV–IFN-β against MERS-CoV in a transgenic humanized mouse model. The relatively high mortality rates associated with these three novel human coronavirus infections, SARS-CoV, MERS-CoV, and SARS-CoV-2, have suggested that proinflammatory responses might play a role in the pathogenesis. It remains unknown whether the generated inflammatory state should be targeted. Therapeutics that target the coronavirus alone might not be able to reverse highly pathogenic infections. This minireview aims to provide a summary of therapeutic compounds that have shown potential in fighting SARS-CoV-2 infections.

Real-time continuous glucose monitoring (CGM) use may lead to behavioral modifications in food selection and physical activity, but there are limited data on the utility of CGM in facilitating lifestyle changes. This article describes an 18-item survey developed to explore whether patients currently using CGM believe the technology has caused them to change their behavior.

Background:

The Center to Reduce Cancer Health Disparities (CRCHD), NCI, implemented Screen to Save, NCI's Colorectal Cancer Outreach and Screening Initiative to promote awareness and knowledge of colorectal cancer in racial/ethnic and rural populations.

Adeno-associated virus (AAV) recombinants are currently the vector of choice for many gene therapy applications. As experimental therapies progress to clinical trials, the need to characterize recombinant adeno-associated viruses (rAAVs) accurately and reproducibly increases. Accurate determination of rAAV infectious titer is important for determining the activity of each vector lot and for ensuring lot-to-lot consistency. The following protocol developed in our laboratory uses a 96-well TCID₅₀ format and quantitative polymerase chain reaction (qPCR) detection for the determination of rAAV infectious titer.

This protocol is used to determine the concentration of DNase-resistant vector genomes (i.e., packaged in the capsid) in purified recombinant adeno-associated virus (rAAV) preparations. The protocol begins with treatment of the vector stock with DNase I to eliminate unencapsidated AAV DNA or contaminating plasmid DNA. This is followed by a heat treatment to heat-inactivate DNase I, to disrupt the viral capsid, and to release the packaged vector genomes for quantification by real-time polymerase chain reaction (PCR) using a set of standards (linearized plasmid used for vector production) containing known copy numbers. To accomplish high-throughput titration, the primer and probe sets used in real-time PCR are usually designed to target common elements present in most rAAV genomes, such as promoters and poly(A) signals. This strategy significantly reduces the number of PCRs, controls, and turnaround time. Several important controls should be included in the assay as follows: The first two controls should have a known copy number of the rAAV genome plasmid treated or not treated with DNase I. This control tests the effectiveness of DNase treatment. To control for potential cross-contamination between samples during the preparation process, a blank control containing nuclease-free water only should be processed and tested in parallel. A validation vector sample with a known titer should be included in every assay to monitor interassay variability. Finally, for the PCR run, a no-template control (NTC) is included to indicate cross-contamination during PCR setup.

This protocol describes a simple single-step column purification (SSCP) of rAAV2 by gravity flow based on its affinity to heparin, without ultracentrifugation.

Tobacco smoking is the primary risk factor for lung cancer, driven by the addictive nature of nicotine and the indisputable carcinogenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as well as other compounds. The integration of lung cancer chemoprevention with smoking cessation is one potential approach to reduce this risk and mitigate lung cancer mortality. Experimental data from our group suggest that kava, commonly consumed in the South Pacific Islands as a beverage to promote relaxation, may reduce lung cancer risk by enhancing NNK detoxification and reducing NNK-derived DNA damage. Building upon these observations, we conducted a pilot clinical trial to evaluate the effects of a 7-day course of kava on NNK metabolism in active smokers. The primary objective was to compare urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL plus its glucuronides, major metabolites of NNK) before and after kava administration as an indicator of NNK detoxification. Secondary objectives included determining kava's safety, its effects on DNA damage, tobacco use, and cortisol (a biomarker of stress). Kava increased urinary excretion of total NNAL and reduced urinary 3-methyladenine in participants, suggestive of its ability to reduce the carcinogenicity of NNK. Kava also reduced urinary total nicotine equivalents, indicative of its potential to facilitate tobacco cessation. Plasma cortisol and urinary total cortisol equivalents were reduced upon kava use, which may contribute to reductions in tobacco use. These results demonstrate the potential of kava intake to reduce lung cancer risk among smokers.

Colorectal cancer is a growing burden in adults less than 50 years old. In 2018, the American Cancer Society published a guideline update recommending a reduction in the colorectal cancer screening start age for average-risk individuals from 50 to 45. Implementing these recommendations would have important implications for public health. However, the approximate number of people impacted by this change, the average-risk population ages 45–49, is not well-described in the literature. Here, we provide methodology to conservatively estimate the average-risk and screening-eligible population in the United States, including those who would be impacted by a lowered colorectal cancer screening start age. Using multiple data sources, we estimated the current average-risk population by subtracting individuals with symptomatic colorectal cancer, with a family history of colorectal cancer, and with inflammatory bowel disease and hereditary nonpolyposis colorectal cancer from the total population. Within this population, we estimated the number of screening-eligible individuals by subtracting those with previous colorectal cancer screening (45- to 49-year-old) or up to date with colorectal cancer screening (50- to 74-year-old). The total average-risk population is estimated between 102.1 and 106.5 million people, of whom 43.4–45.2 million people are eligible for colorectal cancer screening. Lowering the screening age would add roughly 19 million people to the average-risk population and increase the current number of screening-eligible individuals on immediate implementation by over 60% (from 27 to 44 million). Estimating the population size impacted by lowering the recommended colorectal cancer screening start age enables more accurate decision-making for policymakers and epidemiologists focused on cancer prevention.

BACKGROUND AND PURPOSE:

Conventional nonadhesive liquid embolic agents currently are the criterion standard for endovascular embolization of cerebral AVMs. However, inadequate distal penetration into the nidus and unstable proximal plug formation are the major limitations of this approach and of the currently available embolic materials. The aim of this study was to evaluate the hypothetic efficacy of combining liquid embolic agents with different properties and viscosities for use in endovascular embolization of cerebral AVMs.