Analytical characterization of full, intermediate, and empty AAV capsids.

Manufacturing of recombinant adeno-associated virus (AAV) vectors produces three types of capsids: full, intermediate, and empty. While there are different opinions about the impact of intermediate and empty capsids on safety and efficacy of AAV products, they are generally considered impurities because they are not the intended fully intact vector product. The presence of these impurities could impact product efficacy due to potential competition with fully packaged AAVs for cellular transduction, as well as have potential implications to patient safety due to increased capsid load during dosing. To determine the impact of intermediate capsids on potency, an AAV preparation was separated into fractions enriched for full, intermediate, or empty capsids. Using a matrix of in vitro (infectivity, gene expression, biological activity) and in vivo potency assays to determine potency as a function of capsid content, our results indicate that while intermediate capsids contribute to the vector genome titer of the product and are equally as infectious as full capsids, they do not contribute to the potency of the AAV product. This study confirms the criticality of reducing and controlling the level of intermediate capsids to ensure a more efficacious AAV product.

Occurrence and characterization of rmtB-harbouring Salmonella and Escherichia coli isolates from a pig farm in the UK.

OBJECTIVES: To characterize and elucidate the spread of amikacin-resistant Enterobacteriaceae isolates from environmental samples on a pig farm in the UK, following the previous identification of index Salmonella isolates harbouring the rmtB gene, a 16S rRNA methylase. METHODS: Environmental samples were collected during two visits to a pig farm in the UK. Isolates were recovered using selective media (amikacin 128 mg/L) followed by real-time PCR and WGS to analyse rmtB-carrying Salmonella and Escherichia coli isolates. RESULTS: Salmonella and E. coli isolates harbouring the rmtB gene were detected at both farm visits. All Salmonella isolates were found to be monophasic S. enterica serovar Typhimurium variant Copenhagen of ST34. rmtB-harbouring E. coli isolates were found to be one of three STs: ST4089, ST1684 and ST34. Long-read sequencing identified the rmtB gene to be chromosomally located in Salmonella isolates and on IncFII-type plasmids in E. coli isolates. The results showed the rmtB gene to be flanked by IS26 elements and several resistance genes. CONCLUSIONS: We report on the occurrence of rmtB-harbouring Enterobacteriaceae on a pig farm in the UK. rmtB confers resistance to multiple aminoglycosides and this work highlights the need for surveillance to assess dissemination and risk.

Hydrogen Storage in Partially Exfoliated Magnesium Diboride Multilayers.

Metal boride nanostructures have shown significant promise for hydrogen storage applications. However, the synthesis of nanoscale metal boride particles is challenging because of their high surface energy, strong inter- and intraplanar bonding, and difficult-to-control surface termination. Here, it is demonstrated that mechanochemical exfoliation of magnesium diboride in zirconia produces 3-4 nm ultrathin MgB2 nanosheets (multilayers) in high yield. High-pressure hydrogenation of these multilayers at 70 MPa and 330 °C followed by dehydrogenation at 390 °C reveals a hydrogen capacity of 5.1 wt%, which is ≈50 times larger than the capacity of bulk MgB2 under the same conditions. This enhancement is attributed to the creation of defective sites by ball-milling and incomplete Mg surface coverage in MgB2 multilayers, which disrupts the stable boron-boron ring structure. The density functional theory calculations indicate that the balance of Mg on the MgB2 nanosheet surface changes as the material hydrogenates, as it is energetically favorable to trade a small number of Mg vacancies in Mg(BH4 )2 for greater Mg coverage on the MgB2 surface. The exfoliation and creation of ultrathin layers is a promising new direction for 2D metal boride/borohydride research with the potential to achieve high-capacity reversible hydrogen storage at more moderate pressures and temperatures.

Germinal center centroblasts transition to a centrocyte phenotype according to a timed program and depend on the dark zone for effective selection.

Germinal center (GC) B cells cycle between the dark zone (DZ) and light zone (LZ) during antibody affinity maturation. Whether this movement is necessary for GC function has not been tested. Here we show that CXCR4-deficient GC B cells, which are restricted to the LZ, are gradually outcompeted by WT cells indicating an essential role for DZ access. Remarkably, the transition between DZ centroblast and LZ centrocyte phenotypes occurred independently of positioning. However, CXCR4-deficient cells carried fewer mutations and were overrepresented in the CD73(+) memory compartment. These findings are consistent with a model where GC B cells change from DZ to LZ phenotype according to a timed cellular program but suggest that spatial separation of DZ cells facilitates more effective rounds of mutation and selection. Finally, we identify a network of DZ CXCL12-expressing reticular cells that likely support DZ functions.

Visualization of splenic marginal zone B-cell shuttling and follicular B-cell egress.

The splenic marginal zone is a unique microenvironment where resident immune cells are exposed to the open blood circulation. Even though it has an important role in responses against blood-borne antigens, lymphocyte migration in the marginal zone has not been intravitally visualized due to challenges associated with achieving adequate imaging depth in this abdominal organ. Here we develop a two-photon microscopy procedure to study marginal zone and follicular B-cell movement in the live mouse spleen. We show that marginal zone B cells are highly motile and exhibit long membrane extensions. Marginal zone B cells shuttle between the marginal zone and follicles with at least one-fifth of the cells exchanging between compartments per hour, a behaviour that explains their ability to deliver antigens rapidly from the open blood circulation to the secluded follicles. Follicular B cells also transit from follicles to the marginal zone, but unlike marginal zone B cells, they fail to undergo integrin-mediated adhesion, become caught in fluid flow and are carried into the red pulp. Follicular B-cell egress via the marginal zone is sphingosine-1-phosphate receptor-1 (S1PR1)-dependent. This study shows that marginal zone B cells migrate continually between marginal zone and follicles and establishes the marginal zone as a site of S1PR1-dependent B-cell exit from follicles. The results also show how adhesive differences of similar cells critically influence their behaviour in the same microenvironment.

Evaluation of Protein Kinase Inhibitors with PLK4 Cross-Over Potential in a Pre-Clinical Model of Cancer.

Polo-like kinase 4 (PLK4) is a cell cycle-regulated protein kinase (PK) recruited at the centrosome in dividing cells. Its overexpression triggers centrosome amplification, which is associated with genetic instability and carcinogenesis. In previous work, we established that PLK4 is overexpressed in pediatric embryonal brain tumors (EBT). We also demonstrated that PLK4 inhibition exerted a cytostatic effect in EBT cells. Here, we examined an array of PK inhibitors (CFI-400945, CFI-400437, centrinone, centrinone-B, R-1530, axitinib, KW-2449, and alisertib) for their potential crossover to PLK4 by comparative structural docking and activity inhibition in multiple established embryonal tumor cell lines (MON, BT-12, BT-16, DAOY, D283). Our analyses demonstrated that: (1) CFI-400437 had the greatest impact overall, but similar to CFI-400945, it is not optimal for brain exposure. Also, their phenotypic anti-cancer impact may, in part, be a consequence of the inhibition of Aurora kinases (AURKs). (2) Centrinone and centrinone B are the most selective PLK4 inhibitors but they are the least likely to penetrate the brain. (3) KW-2449, R-1530 and axitinib are the ones predicted to have moderate-to-good brain penetration. In conclusion, a new selective PLK4 inhibitor with favorable physiochemical properties for optimal brain exposure can be beneficial for the treatment of EBT.

Molecular epidemiology of isolates with multiple mcr plasmids from a pig farm in Great Britain: the effects of colistin withdrawal in the short and long term.

Background: The environment, including farms, might act as a reservoir for mobile colistin resistance (mcr) genes, which has led to calls for reduction of usage in livestock of colistin, an antibiotic of last resort for humans. Objectives: To establish the molecular epidemiology of mcr Enterobacteriaceae from faeces of two cohorts of pigs, where one group had initially been treated with colistin and the other not, over a 5 month period following stoppage of colistin usage on a farm in Great Britain; faecal samples were also taken at ∼20 months. Methods: mcr-1 Enterobacteriaceae were isolated from positive faeces and was WGS performed; conjugation was performed on selected Escherichia coli and colistin MICs were determined. Results: E. coli of diverse ST harbouring mcr-1 and multiple resistance genes were isolated over 5 months from both cohorts. Two STs, from treated cohorts, contained both mcr-1 and mcr-3 plasmids, with some isolates also harbouring multiple copies of mcr-1 on different plasmids. The mcr-1 plasmids grouped into four Inc types (X4, pO111, I2 and HI2), with mcr-3 found in IncP. Multiple copies of mcr plasmids did not have a noticeable effect on colistin MIC, but they could be transferred simultaneously to a Salmonella host in vitro. Neither mcr-1 nor mcr-3 was detected in samples collected ∼20 months after colistin cessation. Conclusions: We report for the first known time on the presence in Great Britain of mcr-3 from MDR Enterobacteriaceae, which might concurrently harbour multiple copies of mcr-1 on different plasmids. However, control measures, including stoppage of colistin, can successfully mitigate long-term on-farm persistence.

Occurrence and characterization of mcr-1-harbouring Escherichia coli isolated from pigs in Great Britain from 2013 to 2015.

Objectives: To determine the occurrence of mcr-1 -harbouring Escherichia coli in archived pig material originating in Great Britain (GB) from 2013 to 2015 and characterize mcr-1 plasmids. Methods: Enrichment and selective culture of 387 archived porcine caecal contents and recovery from archive of 1109 E. coli isolates to identify colistin-resistant bacteria by testing for the presence of mcr-1 by PCR and RT-PCR. mcr-1 -harbouring E. coli were characterized by WGS and compared with other available mcr-1 WGS. Results: Using selective isolation following enrichment, the occurrence of mcr-1 E. coli in caeca from healthy pigs at slaughter from unique farms in GB was 0.6% (95% CI 0%-1.5%) in 2015. mcr-1 E. coli were also detected in isolates from two porcine veterinary diagnostic submissions in 2015. All isolates prior to 2015 were negative. WGS analysis of the four mcr-1 -positive E. coli indicated no other antimicrobial resistance (AMR) genes were linked to mcr-1 -plasmid-bearing contigs, despite all harbouring multiple AMR genes. The sequence similarity between mcr-1 -plasmid-bearing contigs identified and those found in GB, Chinese and South African human isolates and Danish, French and Estonian livestock-associated isolates was 90%-99%. Conclusions: mcr-1- harbouring plasmids were diverse, implying transposable elements are involved in mcr-1 transmission in GB. The low number of mcr-1 -positive E. coli isolates identified suggested mcr-1 is currently uncommon in E. coli from pigs within GB. The high sequence similarity between mcr-1 plasmid draft genomes identified in pig E. coli and plasmids found in human and livestock-associated isolates globally requires further investigation to understand the full implications.

Colistin resistance in Salmonella and Escherichia coli isolates from a pig farm in Great Britain.

OBJECTIVES: The objective of this study was to characterize colistin-resistant bacteria isolated from pigs on a farm in Great Britain following identification of a plasmid-borne colistin resistance mechanism in Escherichia coli from China. METHODS: Phenotypic antimicrobial susceptibility testing was undertaken by broth dilution and WGS was performed to detect the presence of genes encoding resistance and virulence. Transferable colistin resistance was investigated by conjugation. RESULTS: Two E. coli and one Salmonella Typhimurium variant Copenhagen were shown to be MDR, including resistance to colistin, with one E. coli and the Salmonella carrying the mcr-1 gene; all three harboured chromosomal mutations in genes conferring colistin resistance and both E. coli harboured ß-lactamase resistance. The Salmonella mcr-1 plasmid was highly similar to pHNSHP45, from China, while the E. coli mcr-1 plasmid only had the ISApII and mcr-1 genes in common. The frequency of mcr-1 plasmid transfer by conjugation to recipient Enterobacteriaceae from Salmonella was low, lying between 10(-7) and 10(-9) cfu/recipient cfu. We were unable to demonstrate mcr-1 plasmid transfer from the E. coli. Plasmid profiling indicated transfer of multiple plasmids from the Salmonella resulting in some MDR transconjugants. CONCLUSIONS: Identification of the mcr-1 gene in Enterobacteriaceae from pigs confirms its presence in livestock in Great Britain. The results suggest dissemination of resistance through different horizontally transferable elements. The in vitro transfer of multiple plasmids carrying colistin and other resistances from the Salmonella isolate underlines the potential for wider dissemination and recombination.

How efficiently do corn- and soybean-based cropping systems use water? A systems modeling analysis.

Agricultural systems are being challenged to decrease water use and increase production while climate becomes more variable and the world's population grows. Low water use efficiency is traditionally characterized by high water use relative to low grain production and usually occurs under dry conditions. However, when a cropping system fails to take advantage of available water during wet conditions, this is also an inefficiency and is often detrimental to the environment. Here, we provide a systems-level definition of water use efficiency (sWUE) that addresses both production and environmental quality goals through incorporating all major system water losses (evapotranspiration, drainage, and runoff). We extensively calibrated and tested the Agricultural Production Systems sIMulator (APSIM) using 6 years of continuous crop and soil measurements in corn- and soybean-based cropping systems in central Iowa, USA. We then used the model to determine water use, loss, and grain production in each system and calculated sWUE in years that experienced drought, flood, or historically average precipitation. Systems water use efficiency was found to be greatest during years with average precipitation. Simulation analysis using 28 years of historical precipitation data, plus the same dataset with ± 15% variation in daily precipitation, showed that in this region, 430 mm of seasonal (planting to harvesting) rainfall resulted in the optimum sWUE for corn, and 317 mm for soybean. Above these precipitation levels, the corn and soybean yields did not increase further, but the water loss from the system via runoff and drainage increased substantially, leading to a high likelihood of soil, nutrient, and pesticide movement from the field to waterways. As the Midwestern United States is predicted to experience more frequent drought and flood, inefficiency of cropping systems water use will also increase. This work provides a framework to concurrently evaluate production and environmental performance of cropping systems.

Subsurface Drainage Nitrate and Total Reactive Phosphorus Losses in Bioenergy-Based Prairies and Corn Systems.

We compare subsurface-drainage NO-N and total reactive phosphorus (TRP) concentrations and yields of select bioenergy cropping systems and their rotational phases. Cropping systems evaluated were grain-harvested corn-soybean rotations, grain- and stover-harvested continuous corn systems with and without a cover crop, and annually harvested reconstructed prairies with and without the addition of N fertilizer in an Iowa field. Drainage was monitored when soils were unfrozen during 2010 through 2013. The corn-soybean rotations without residue removal and continuous corn with residue removal produced similar mean annual flow-weighted NO-N concentrations, ranging from 6 to 18.5 mg N L during the 4-yr study. In contrast, continuous corn with residue removal and with a cover crop had significantly lower NO-N concentrations of 5.6 mg N L when mean annual flow-weighted values were averaged across the 4 yr. Prairies systems with or without N fertilization produced significantly lower concentrations below <1 mg NO-N L than all the row crop systems throughout the study. Mean annual flow-weighted TRP concentrations and annual yields were generally low, with values <0.04 mg TRP L and <0.14 kg TRP ha, and were not significantly affected by any cropping systems or their rotational phases. Bioenergy-based prairies with or without N fertilization and continuous corn with stover removal and a cover crop have the potential to supply bioenergy feedstocks while minimizing NO-N losses to drainage waters. However, subsurface drainage TRP concentrations and yields in bioenergy systems will need further evaluation in areas prone to higher levels of P losses.

A two-dimensional model for the study of interpersonal attraction.

We describe a model for understanding interpersonal attraction in which attraction can be understood as a product of the initial evaluations we make about others. The model posits that targets are evaluated on two basic dimensions, capacity and willingness, such that affective and behavioral attraction result from evaluations of (a) a target's capacity to facilitate the perceiver's goals/needs and (b) a target's potential willingness to facilitate those goals/needs. The plausibility of the two-dimensional model of attraction is evaluated vis-à-vis the extant literature on various attraction phenomena including the reciprocity of liking effect, pratfall effect, matching hypothesis, arousal effects, and similarity effect. We conclude that considerable evidence across a wide range of phenomena supports the idea that interpersonal attraction is principally determined by inferences about the target's capacity and willingness.

Clarifying the links between grandiose narcissism and parenting.

This study investigated the links between parenting and grandiose narcissism in hopes of clarifying recent empirical discrepancies. One-hundred forty-five participants completed the Narcissistic Personality Inventory and reported about their parents' support, coldness, monitoring, psychological control, and overvaluation. Psychological control was associated positively with narcissism, whereas monitoring and coldness were associated negatively. Overvaluation and parental support showed no reliable associations with narcissism. Analysis of the components of narcissism further elucidated these links. The results are interpreted in light of previous findings and as consistent with social learning and psychodynamic theories regarding the origins of narcissism.

Humic acid transport in saturated porous media: influence of flow velocity and influent concentration.

Understanding the transport of humic acids (HAs) in porous media can provide important and practical evidence needed for accurate prediction of organic/inorganic contaminant transport in different environmental media and interfaces. A series of column transport experiments was conducted to evaluate the transport of HA in different porous media at different flow velocities and influent HA concentrations. Low flow velocity and influent concentration were found to favor the adsorption and deposition of HA onto sand grains packed into columns and to give higher equilibrium distribution coefficients and deposition rate coefficients, which resulted in an increased fraction of HA being retained in columns. Consequently, retardation factors were increased and the transport of HA through the columns was delayed. These results suggest that the transport of HA in porous media is primarily controlled by the attachment of HA to the solid matrix. Accordingly, this attachment should be considered in studies of HA behavior in porous media.

Reducing emergency room utilization in end-stage COPD - feasible or fantasy?

The emergency room (ER) is a common point of care transition for patients with end-stage chronic obstructive pulmonary disease. Many of these patients present to the ER because of dyspnea, anxiety and caregiver burden that is precipitated by fragmented and reactive systems of care that fail to meet their needs. This article uses an illustrative case report to outline the challenges patients and caregivers face and presents the core elements of care required to improve quality of care and decrease reliance on the ER.

Lithium-induced alterations in soybean nodulation and nitrogen fixation through multifunctional mechanisms.

The increasing footprints of lithium (Li) in agroecosystems combined with limited recycling options have raised uncertain consequences for important crops. Nitrogen (N2)-fixation by legumes is an important biological response process, but the cause and effect of Li exposure on plant root-nodule symbiosis and biological N2-fixation (BNF) potential are still unclear. Soybean as a model plant was exposed to Li at low (25 mg kg-1), medium (50 mg kg-1), and high (100 mg kg-1) concentrations. We found that soybean growth and nodulation capacity had a concentration-dependent response to Li. Li at 100 mg kg-1 reduced the nodule numbers, weight, and BNF potential of soybean in comparison to the low and medium levels. Significant shift in soybean growth and BNF after exposure to Li were associated with alteration in the nodule metabolic pathways involved in nitrogen uptake and metabolism (urea, glutamine and glutamate). Importantly, poor soybean nodulation after high Li exposure was due in part to a decreased abundance of bacterium Ensifer in the nodule bacterial community. Also, the dominant N2-fixing bacterium Ensifer was significantly correlated with carbon and nitrogen metabolic pathways. The findings of our study offer mechanistic insights into the environmental and biological impacts of Li on soybean root-nodule symbiosis and N2-acquisition and provide a pathway to develop strategies to mitigate the challenges posed by Li in agroecosystems.

Genomic surveillance of extended-spectrum cephalosporin-resistant Escherichia coli isolated from poultry in the UK from 2016 to 2020.

Introduction: Surveillance is vital for monitoring the increasing risk of antimicrobial resistance (AMR) in bacteria leading to failures in humans and animals to treat infections. In a One Health context, AMR bacteria from livestock and food can transfer through the food chain to humans, and vice versa, which can be characterized in detail through genomics. We investigated the critical aspects of AMR and the dynamics of AMR in poultry in the UK. Methods: In this study, we performed whole genome sequencing for genomic characterization of 761 extended-spectrum cephalosporinases (ESCs) harboring Escherichia coli isolated from poultry caeca and meat through EU harmonized monitoring of AMR in zoonotic and commensal bacteria from 2016 and 2018 and UK national monitoring in 2020. Results: The most common ESC in 2016 and 2018 was blaCTX-M-1; however, 2020 had a greater diversity of ESCs with blaCTX-M-55 dominant in chickens and blaCTX-M-15 more prevalent in turkeys. Co-resistance to sulphonamides, tetracycline, and trimethoprim was widespread, and there were several positive correlations between the sequence types (STs) and ESC genes. We identified certain AMR genotypes and STs that were frequent each year but not as successful in subsequent years, e.g., ST350 harboring blaCTX-M-1, sul2, and tetA-v4.Phylogenetic comparison of isolates prevalent in our panel with global ones from the same STs available in public databases showed that isolates from the UK generally clustered together, suggesting greater within-country than between-country transmission. Discussion: We conclude that future genomic surveillance of indicator organisms will be invaluable as it will enable detailed comparisons of AMR between and within neighboring countries, potentially identifying the most successful sequence types, plasmids, or emerging threats.

Surviving Long Enough to Die? An Analysis of Incomplete Assessments for Medical Assistance in Dying.

Background: Medical assistance in dying (MAiD) was legalized in Canada on June 17, 2016, yet many who request MAiD do not complete the assessment process and instead experience a natural death. This analysis of patients who made a formal request for MAiD aims to clarify timelines and factors associated with completion of the MAiD assessment process, and factors associated with completion or noncompletion of MAiD once eligible. Materials and Methods: This retrospective cohort study included all patients in Nova Scotia who requested MAiD between January 1, 2018 and December 31, 2018, were deceased at the time of analysis, did not withdraw their request, and were not formally deemed ineligible for the procedure (n = 218). Descriptive statistics, Kaplan-Meier curves, and logistic regression were used in data analysis. Results: Of 218 patients, 48 did not complete the MAiD assessment process. Of the 170 patients who completed the assessment process and were deemed eligible for MAiD, 79.4% (n = 135) completed the procedure. Those with an incomplete assessment had a median survival from request to death of 8.0 days (interquartile range [IQR] = 11.5), whereas for those deemed eligible, median survival from request to determination of MAiD eligibility was also 8.0 days (IQR = 16.0). Interpretation: Proximity to natural death and poor performance status at the time of MAiD request may drive incomplete MAiD assessments. The majority of patients deemed eligible for MAiD complete the procedure, and as such, patients who did not complete the MAiD assessment process may not have experienced their preferred mode of death.

Use of genomics to explore AMR persistence in an outdoor pig farm with low antimicrobial usage.

Food animals may be reservoirs of antimicrobial resistance (AMR) passing through the food chain, but little is known about AMR prevalence in bacteria when selective pressure from antimicrobials is low or absent. We monitored antimicrobial-resistant Escherichia coli over 1 year in a UK outdoor pig farm with low antimicrobial usage (AMU) compared to conventional pig farms in the United Kingdom. Short and selected long-read whole-genome sequencing (WGS) was performed to identify AMR genes, phylogeny and mobile elements in 385 E. coli isolates purified mainly from pig and some seagull faeces. Generally, low levels of antimicrobial-resistant E. coli were present, probably due to low AMU. Those present were likely to be multi-drug resistant (MDR) and belonging to particular Sequence Types (STs) such as ST744, ST88 or ST44, with shared clones (<14 Single Nucleotide Polymorphisms (SNPs) apart) isolated from different time points indicating epidemiological linkage within pigs of different ages, and between pig and the wild bird faeces. Although importance of horizontal transmission of AMR is well established, there was limited evidence of plasmid-mediated dissemination between different STs. Non-conjugable MDR plasmids or large AMR gene-bearing transposons were stably integrated within the chromosome and remained associated with particular STs/clones over the time period sampled. Heavy metal resistance genes were also detected within some genetic elements. This study highlights that although low levels of antimicrobial-resistant E. coli correlates with low AMU, a basal level of MDR E. coli can still persist on farm potentially due to transmission and recycling of particular clones within different pig groups. Environmental factors such as wild birds and heavy metal contaminants may also play important roles in the recycling and dissemination, and hence enabling persistence of MDR E. coli. All such factors need to be considered as any rise in AMU on low usage farms, could in future, result in a significant increase in their AMR burden.