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This study assesses college students' intent to intervene when presented with a hypothetical peer exhibiting depression in one of three scenarios: depression, sadness, and depression in quarantine during COVID-19. Using the Theory of Planned Behavior (TPB), variations in constructs associated with intent were examined by context (external triggers vs. no trigger), knowledge of, and experience with depression. One hundred and sixteen health sciences students read three vignettes and completed an enhanced TPB questionnaire. Intent to intervene was greater when the vignette target was experiencing depression with external stressors. Prior experience with depression and knowing someone with depression were associated with greater intent to connect the hypothetical peer to counseling resources regardless of vignette scenario. Due to increased mental health concerns resulting from the COVID-19 pandemic, efforts promoting awareness of mental illness in peers may benefit from increasing education about stressors and causes of depression that may not be observable.

A major limitation of pulmonary delivery is that drugs can exhibit suboptimal pharmacokinetic profiles resulting from rapid elimination from the pulmonary tissue. This can lead to systemic side effects and a short duration of action. A series of dibasic dipeptides attached to the poorly lung-retentive muscarinic M3 receptor antagonist piperidin-4-yl 2-hydroxy-2,2-diphenylacetate (1) through a pH-sensitive-linking group have been evaluated. Extensive optimization resulted in 1-(((R)-2-((S)-2,6-diaminohexanamido)-3,3-dimethylbutanoyl)oxy)ethyl 4-(2-hydroxy-2,2-diphenylacetoxy)piperidine-1-carboxylate (23), which combined very good in vitro stability and very high rat lung binding. Compound 23 progressed to pharmacokinetic studies in rats, where, at 24 h post dosing in the rat lung, the total lung concentration of 23 was 31.2 ?M. In addition, high levels of liberated drug 1 were still detected locally, demonstrating the benefit of this novel prodrug approach for increasing the apparent pharmacokinetic half-life of drugs in the lungs following pulmonary dosing.

Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny; whether macrophages need to sense a replicating virus to drive cytokine release; and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1–derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.

Efficient delivery of highly lipophilic drugs or prodrugs to the mesenteric lymph nodes (MLN) can be achieved following oral administration with lipids. However, it remains unclear which specific MLN can be targeted and to what extent. Moreover, the efficiency of drug delivery to the retroperitoneal lymph nodes (RPLN) has not been assessed. The aim of this study was to assess the distribution of a highly lipophilic model drug cannabidiol (CBD), known to undergo intestinal lymphatic transport following administration with lipids, into specific MLN and RPLN in rats at various time-points post dosing. In vivo studies showed that at 2 h following administration, significantly higher concentrations of CBD were present in the region second from the apex of the MLN chain. From 3 h following administration, concentrations in all MLN were similar. CBD was also found at substantial levels in RPLN. This study demonstrates that drug concentrations in specific MLN are different, at least at the peak of the absorption process. Moreover, in addition to the MLN, the RPLN may also be targeted by oral route of administration, which may have further implications for treatment of a range of diseases.

The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1β production. In contrast, HDAC7 initiates the pentose phosphate pathway (PPP) for NADPH and reactive oxygen species (ROS) production in response to the more proximal threat of nearby bacteria, as exemplified by studies on uropathogenic Escherichia coli (UPEC). HDAC7-mediated PPP engagement via 6-phosphogluconate dehydrogenase (6PGD) generates NADPH for antimicrobial ROS production, as well as D-ribulose-5-phosphate (RL5P) that both synergizes with ROS for UPEC killing and suppresses selective inflammatory responses. This dual functionality of the HDAC7-6PGD-RL5P axis prioritizes responses to proximal threats. Our findings thus reveal that the PPP metabolite RL5P has both antimicrobial and immunomodulatory activities and that engagement of enzymes in catabolic versus anabolic metabolic pathways triages responses to different types of danger for generation of inflammatory versus antimicrobial responses, respectively.

Hereditary angioedema (HAE) is a rare genetic disorder in which patients experience sudden onset of swelling in various locations of the body. HAE is associated with uncontrolled plasma kallikrein (PKa) enzyme activity and generation of the potent inflammatory mediator, bradykinin, resulting in episodic attacks of angioedema. Herein, we disclose the discovery and optimization of novel small molecule PKa inhibitors. Starting from molecules containing highly basic P1 groups, which typically bind to an aspartic acid residue (Asp189) in the serine protease S1 pocket, we identified novel P1 binding groups likely to have greater potential for oral-drug-like properties. The optimization of P4 and the central core together with the particularly favorable properties of 3-fluoro-4-methoxypyridine P1 led to the development of sebetralstat, a potent, selective, orally bioavailable PKa inhibitor in phase 3 for on-demand treatment of HAE attacks.

High-intensity wildland fires can produce extreme flaming and smoke emissions that develop into a fire-cloud chimney, reaching into the upper troposphere or lower stratosphere. Termed pyrocumulonimbus, these storms are both conventional and counterintuitive. They have been observed to produce lightning, hail, downdraft wind hazards, and tornadoes as expected with severe convective storms, but counterintuitively, they are not associated with significant precipitation. Pyrocumulonimbus storms have been noticed outside wildfire expert circles following Australia’s Black Summer in 2019/20, and have since repeatedly made headlines in the United States. However, much is unknown about their behavior, energetics, history, and impact on the Earth/atmosphere system. We address several questions and science challenges related to these unknowns. Our worldwide record of pyrocumulonimbus events from 2013 to 2021 shows that the phenomenon is neither new nor rare. Despite high occurrences in 2019 and 2021, these data do not support identification of a trend. Future studies require an expansive record of pyrocumulonimbus occurrence globally and regionally, both historically and continuously forward in time.

Evolutionary responses to sudden changes in the environment can, in theory, be rapid if they involve small shifts in allele frequencies at many loci. Such adaptation has proven hard to characterise in wild populations. We overcome these problems, in quantifying the genetic response of European ash trees (Fraxinus excelsior) to the strong selective challenge imposed by the invasive alien fungal pathogenHymenoscyphus fraxineus, by exploiting a previous study that had estimated effect sizes for many single nucleotide polymorphism (SNP) loci associated with resistance to the fungus in large field trials. We ask if the selective response, in a new natural setting of a multigenerational wild ash woodland, involves allele frequency changes at the 10,000 loci which provided the best genomic prediction of resistance in the field trials. We conducted whole genome resequencing of each tree and calculated its genetic merit as a Genomic Estimated Breeding Value (GEBV), using the previous estimates of SNP effect sizes. The GEBV of trees established after the start of the epidemic were significantly higher than those of related adults from the pre-epidemic generation, with the size of the change in the alleles’ frequency corresponding to their effect sizes. To produce a GEBV shift of this magnitude, would require truncation selection eliminating at least 13% of the juvenile population. Thus, we document shifts in allele frequency at very many loci producing a heritable micro-evolutionary adaptive change over a single generation. Adaptation could be further accelerated by a breeding programme informed by genomic selection.Significance statementWe demonstrate contemporary natural selection as European ash trees are exposed to the novel fungal epidemic ofHymenoscyphus fraxineus. We detect adaptive shifts in allele frequencies at thousands of loci. This mode of rapid evolution in a highly polygenic trait has been theorised since R. A. Fisher first proposed it, but has been hard to demonstrate in the wild. The approach we have applied could be widely used, where genomic prediction is possible in natural populations and there is a clear change in selective regime. The results for European ash trees indicate a degree of natural evolutionary rescue, after the fungus arrives. An effective practical application would be to accelerate this response by human-directed genomic selection.

Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air–liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.

Spruce budworm (Choristoneura fumiferana (Clemens)) defoliation has been shown to increase the likelihood of large forest fires in central Canada. However, the time frame of heightened risk based on the duration of spruce budworm defoliation has not yet been quantified. In this article, we document the extent of stand breakdown and surface fuel accumulation after a period of spruce budworm defoliation that occurred between 1972 and 1976. Data on stand characteristics were derived from previous studies at three different locations in the boreal mixedwood forests of central Canada: Aubinadong (B.J. Stocks. 1987. For. Chron. 63: 8–14), Gogama, and Gowganda in Ontario. Stand breakdown was measured using a series of transects set in plots 7 years following aerially mapped defoliation (1977–1983). Results show that during the 4 years following 5 years of defoliation, crown breakage, a typical symptom of defoliation, increased by nearly 200%, and surface fuel increased by 145% from predisturbance levels. The high correlation between crown breakage and surface fuels linked defoliation to fuel buildup. We begin to solve the challenge of measuring fuel structure over the expansive scale of spruce budworm outbreaks by quantifying the relationship among stand breakdown, time since the end of defoliation, and the duration of defoliation so that the expected fuel structure can be modelled from annual defoliation surveys.

TLR‐inducible zinc toxicity is an antimicrobial mechanism utilized by macrophages, however knowledge of molecular mechanisms mediating this response is limited. Here, we show that E. coli exposed to zinc stress within primary human macrophages reside in membrane‐bound vesicular compartments. Since SLC30A zinc exporters can deliver zinc into the lumen of vesicles, we examined LPS‐regulated mRNA expression of Slc30a/SLC30A family members in primary mouse and human macrophages. A number of these transporters were dynamically regulated in both cell populations. In human monocyte‐derived macrophages, LPS strongly up‐regulated SLC30A1 mRNA and protein expression. In contrast, SLC30A1 was not LPS‐inducible in macrophage‐like PMA‐differentiated THP‐1 cells. We therefore ectopically expressed SLC30A1 in these cells, finding that this was sufficient to promote zinc‐containing vesicle formation. The response was similar to that observed following LPS stimulation. Ectopically expressed SLC30A1 localized to both the plasma membrane and intracellular zinc‐containing vesicles within LPS‐stimulated THP‐1 cells. Inducible overexpression of SLC30A1 in THP‐1 cells infected with the Escherichia coli K‐12 strain MG1655 augmented the zinc stress response of intracellular bacteria and promoted clearance. Furthermore, in THP‐1 cells infected with an MG1655 zinc stress reporter strain, all bacteria contained within SLC30A1‐positive compartments were subjected to zinc stress. Thus, SLC30A1 marks zinc‐containing compartments associated with TLR‐inducible zinc toxicity in human macrophages, and its ectopic over‐expression is sufficient to initiate this antimicrobial pathway in these cells. Finally, SLC30A1 silencing did not compromise E. coli clearance by primary human macrophages, suggesting that other zinc exporters may also contribute to the zinc toxicity response., Graphical Abstract The zinc transporter SLC30A1 is LPS‐inducible in human macrophages and can deliver a zinc toxicity response against intracellular Escherichia coli.

Hydrology (streamflow and stage) and water quality (suspended sediment, total nitrogen [N], ammonia [NH3], total Kjeldahl N, nitrate plus nitrite [NO3 + NO2], and total phosphorus [TP]) were monitored in two small agricultural drainages in northwestern Mississippi to document changes in water quality that coincided with the implementation of best management practices (BMPs) in upstream drainages. Using an event-based data set and bootstrapping techniques, we tested for difference and equivalence in median event concentration and differences in concentration-streamflow (C-Q) relationships between an early and late period at each site, where most of the major BMP implementation occurred during the early period. Results for Bee Lake Tributary were inconclusive. Using 95% confidence intervals, none of the constituents were statistically different or equivalent for median event concentrations between the periods, indicating a lack of evidence to determine whether water quality had changed or stayed the same, and only TP had a significantly higher C-Q intercept during the late period. At Lake Washington Tributary, more than half the constituents had a significantly different median, slope, or intercept between periods, indicating a 35% or more decrease in event concentration following a period of intense BMP implementation. These mixed results could be due to a variety of differences between the sites including the type and timing of BMP implementation, production practices, and crop types. We also used the monitoring data to generate synthetic data and performed a simulation-based power analysis to explore the ability to detect change under 25 scenarios of sampled event counts and hypothetical percentage changes. The simulation-based power analysis indicated that natural variability in event concentration and flow hindered our ability to detect change. The results from this study can be used to guide future decisions pertaining to monitoring efforts in small agricultural drainage basins in the Mississippi Alluvial Plain.

Societal Impact Statement The current ash dieback epidemic in Europe caused by Hymenoscyphus fraxineus poses a key question to policy makers: whether or not to commit time and resources to the initiation of a breeding programme for the development of more resistant ash, as a long‐term policy of adaptation to the epidemic. Here we review current evidence on the potential viability of such a programme, from a biological perspective. We conclude that a breeding programme for ash aimed at resistance to current strains of H. fraxineus in the British Isles is biologically feasible. Summary To evaluate the viability and feasibility of a future breeding programme to produce trees resistant to an emerging pest or pathogen, it is helpful to ask the following questions: How much variation in resistance exists in tree populations? To what extent is this resistance heritable? How many genetic loci are involved? What level of resistance is found in other species of the same genus? Here, we survey current knowledge of these issues in relation to the degree of resistance of European ash (Fraxinus excelsior) to H. fraxineus, the fungus causing ash dieback (ADB). Several studies have found a low frequency of heritable resistance in F. excelsior populations, which seems to be determined by many genetic loci. This suggests that a breeding programme is viable and that natural selection may also increase the mean resistance of populations over time. More research is needed on the genetic basis of resistance to ADB to understand how quickly natural selection can operate in woodlands and what acceleration may be possible in breeding programmes, including via use of genetic markers. Hybrid breeding programmes may also be a possibility, as some ash species appear to be more resistant to ADB than is F. excelsior, but more research is needed on this issue. We do not yet know if it will be possible to breed F. excelsior to have resistance to both ADB and the emerging threat of emerald ash borer. We recommend short‐term mitigation measures for the ADB epidemic and future research directions.

Although natural sesame oil has been shown to facilitate the lymphatic delivery and oral bioavailability of the highly lipophilic drug cannabidiol (CBD), considerable variability remains an unresolved challenge. Vegetable oils differ substantially in composition, which could lead to differences in promotion of intestinal lymphatic transport of lipophilic drugs. Therefore, the differences in composition of sesame, sunflower, peanut, soybean, olive and coconut oils and their corresponding role as vehicles in promoting CBD lymphatic targeting and bioavailability were investigated in this study. The comparative analysis suggests that the fatty acids profile of vegetable oils is overall similar to the fatty acids profile in the corresponding chylomicrons in rat lymph. However, arachidonic acid (C20:4), was introduced to chylomicrons from endogenous nondietary sources. Overall, fatty acid composition of natural vegetable oils vehicles affected the intestinal lymphatic transport and bioavailability of CBD following oral administration in this work. Olive oil led to the highest concentration of CBD in the lymphatic system and in the systemic circulation in comparison to the other natural vegetable oils following oral administration in rats.

Macrophages are key cellular contributors to COVID-19 pathogenesis. Whether SARS-CoV-2 can enter macrophages, replicate and release new viral progeny remains controversial. Similarly, whether macrophages need to sense replicating virus to drive cytokine release is also unclear. Macrophages are heterogeneous cells poised to respond to their local microenvironment, and accordingly, the SARS-CoV-2 entry receptor ACE2 is only present on a subset of macrophages at sites of human infection. Here, we use in vitro approaches to investigate how SARS-CoV-2 interacts with ACE2-negative and ACE2-positive human macrophages and determine how these macrophage populations sense and respond to SARS-CoV-2. We show that SARS-CoV-2 does not replicate within ACE2-negative human macrophages and does not induce pro-inflammatory cytokine expression. By contrast, ACE2 expression in human macrophages permits SARS-CoV-2 entry, replication, and virion release. ACE2-expressing macrophages sense replicating virus to trigger pro-inflammatory and anti-viral programs that limit virus release. These combined findings resolve several controversies regarding macrophage-SARS-CoV-2 interactions and identify a signaling circuit by which macrophages sense SARS-CoV-2 cell entry and respond by restricting viral replication.One sentence summaryLack of macrophage ACE2 expression precludes SARS-CoV-2 entry and sensing, while ACE2-expressing macrophages sense intramacrophage SARS-CoV-2 replication to induce rapid anti-viral responses that limit new virion release.

We previously reported relatively normal pulmonary function (2 years of age) and computed tomography (CT, 1 year of age) in cystic fibrosis (CF) newborn screened (NBS) infants. We now report follow up of these children to preschool age.67 NBS children with CF and 41 healthy controls underwent pulmonary function tests in infancy (∼3 months, 1 year and 2 years) and at preschool (3-6 years). Broncho-alveolar lavage (BAL) and CT were undertaken in those with CF at 1 year. Primary outcomes at preschool were lung clearance index (LCI) and forced expired volume (FEVAt preschool age children with CF had poorer lung function than controls, mean(95% CI) difference in LCI z-score: 1.47(0.96;1.97) and FEVLung function deteriorates after 2 years in NBS children with CF. Isolation of Pseudomonas aeruginosa before 6 months and minor abnormalities of infant lung function tests and CT in infancy are associated with higher preschool LCI.

Acute Oak Decline (AOD) is complex syndrome affecting Britain’s keystone native oak species, (Quercus robur L. and Q. petraea L. (Matt.) Liebl.), in some cases causing mortality within five years of symptom development. The most distinguishable symptom is weeping stem lesions, from which four species of bacteria have been isolated: Brenneria goodwinii, Gibbsiella quercinecans, Lonsdalea britannica and Rahnella victoriana. We do not yet know where else these bacteria exist, and little is known about the relationship of the wider oak leaf microbiome (phyllosphere) to acute oak decline. Here we investigate whether incidental evidence from a large oak genome re-sequencing dataset could be used to detect these bacteria in oak foliage, and whether bacterial incidence co-varied with AOD status or location. Oak leaves and buds were sampled from 421 trees at five sites in England. Whole genomic DNA from these samples was shot-gun sequenced with short reads. Non-oak reads were extracted from these data and queried to microbial databases. Reads uniquely matching AOD-associated bacterial genomes were found to be present on trees from all five sites and included trees with active lesions, trees with historic lesions and trees without AOD symptoms. The abundance of the AOD-associated bacteria did not differ between tree health categories but did differ among sites. We conclude that the AOD-associated bacteria may be members of the normal oak microbiome, whose presence on a tree is not sufficient to cause AOD symptoms.

Oral sesame oil-based formulation facilitates the delivery of poorly water-soluble drug cannabidiol (CBD) to the lymphatic system and blood circulation. However, this natural oil-based formulation also leads to considerable variability in absorption of CBD. In this work, the performance of lipid-based formulations with the addition of medium-chain triglyceride (MCT) or surfactants to the sesame oil vehicle has been tested in vitro and in vivo using CBD as a model drug. The in vitro lipolysis has shown that addition of the MCT leads to a higher distribution of CBD into the micellar phase. Further addition of surfactants to MCT-containing formulations did not improve distribution of the drug into the micellar phase. In vivo, formulations containing MCT led to lower or similar concentrations of CBD in serum, lymph and MLNs, but with reduced variability. MCT improves the emulsification and micellar solubilization of CBD, but surfactants did not facilitate further the rate and extent of lipolysis. Even though addition of MCT reduces the variability, the in vivo performance for the extent of both lymphatic transport and systemic bioavailability remains superior with a pure natural oil vehicle.

CXCL17 is the most recently described chemokine. It is principally expressed by mucosal tissues, where it facilitates chemotaxis of monocytes, dendritic cells, and macrophages and has antimicrobial properties. CXCL17 is also implicated in the pathology of inflammatory disorders and progression of several cancers, as well as being highly upregulated during viral infections of the lung. However, the exact role of CXCL17 in health and disease is largely unknown, mainly due to a lack of known molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET) based assays, here we demonstrate that CXCL17 inhibits CXCR4-mediated signalling and ligand binding. Moreover, CXCL17 interacts with neuropillin-1, a VEGFR2 co-receptor. Additionally, we find CXCL17 only inhibits CXCR4 ligand binding in intact cells and demonstrate that this effect is mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. This indicates that CXCL17 inhibits CXCR4 by a unique mechanism of action that potentially requires the presence of a glycosaminoglycan containing accessory protein. Altogether, our results reveal that CXCL17 is an endogenous inhibitor of CXCR4 and represents an important discovery in our understanding of the (patho) physiological functions of CXCL17 and regulation of CXCR4 signalling.

The two G protein-coupled receptors (GPCRs) C-X-C chemokine receptor type 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are part of the class A chemokine GPCR family and represent important drug targets for human immunodeficiency virus (HIV) infection, cancer, and inflammation diseases. CXCR4 is one of only three chemokine receptors with a US Food and Drug Administration approved therapeutic agent, the small-molecule modulator AMD3100. In this review, known modulators of the two receptors are discussed in detail. Initially, the structural relationship between receptors and ligands is reviewed on the basis of common structural motifs and available crystal structures. To date, no atypical chemokine receptor has been crystallized, which makes ligand design and predictions for these receptors more difficult. Next, the selectivity, receptor activation, and the resulting ligand-induced signaling output of chemokines and other peptide ligands are reviewed. Binding of pepducins, a class of lipid-peptides whose basis is the internal loop of a GPCR, to CXCR4 is also discussed. Finally, small-molecule modulators of CXCR4 and ACKR3 are reviewed. These modulators have led to the development of radio- and fluorescently labeled tool compounds, enabling the visualization of ligand binding and receptor characterization both in vitro and in vivo. SIGNIFICANCE STATEMENT To investigate the pharmacological modulation of CXCR4 and ACKR3, significant effort has been focused on the discovery and development of a range of ligands, including small-molecule modulators, pepducins, and synthetic peptides. Imaging tools, such as fluorescent probes, also play a pivotal role in the field of drug discovery. This review aims to provide an overview of the aforementioned modulators that facilitate the study of CXCR4 and ACKR3 receptors.

Significance Uropathogenic Escherichia coli (UPEC) is responsible for most urinary tract infections and is also a frequent cause of sepsis, thus necessitating an understanding of UPEC-mediated subversion of innate immunity. The role of zinc in the innate immune response against UPEC infection, and whether this pathogen counters this response, has not been examined. Here we demonstrate, both in vitro and in vivo, that UPEC both evades and resists innate immune-mediated zinc toxicity to persist and disseminate within the host. Moreover, we have defined the set of UPEC genes conferring zinc resistance and have developed highly selective E. coli reporter systems to track zinc toxicity. These innovative approaches substantially enhance our understanding of immune-mediated metal ion toxicity and bacterial pathogenesis., Toll-like receptor (TLR)-inducible zinc toxicity is a recently described macrophage antimicrobial response used against bacterial pathogens. Here we investigated deployment of this pathway against uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections. Primary human macrophages subjected EC958, a representative strain of the globally disseminated multidrug-resistant UPEC ST131 clone, to zinc stress. We therefore used transposon-directed insertion site sequencing to identify the complete set of UPEC genes conferring protection against zinc toxicity. Surprisingly, zinc-susceptible EC958 mutants were not compromised for intramacrophage survival, whereas corresponding mutants in the nonpathogenic E. coli K-12 strain MG1655 displayed significantly reduced intracellular bacterial loads within human macrophages. To investigate whether the intramacrophage zinc stress response of EC958 reflected the response of only a subpopulation of bacteria, we generated and validated reporter systems as highly specific sensors of zinc stress. Using these tools we show that, in contrast to MG1655, the majority of intramacrophage EC958 evades the zinc toxicity response, enabling survival within these cells. In addition, EC958 has a higher tolerance to zinc than MG1655, with this likely being important for survival of the minor subset of UPEC cells exposed to innate immune-mediated zinc stress. Indeed, analysis of zinc stress reporter strains and zinc-sensitive mutants in an intraperitoneal challenge model in mice revealed that EC958 employs both evasion and resistance against zinc toxicity, enabling its dissemination to the liver and spleen. We thus demonstrate that a pathogen of global significance uses multiple mechanisms to effectively subvert innate immune-mediated zinc poisoning for systemic spread.

© 2021 Elsevier B.V. Lipid-based formulations play a significant role in oral delivery of lipophilic drugs. Previous studies have shown that natural sesame oil promotes the intestinal lymphatic transport and oral bioavailability of the highly lipophilic drug cannabidiol (CBD). However, both lymphatic transport and systemic bioavailability were also associated with considerable variability. The aim of this study was to test the hypothesis that pre-digested lipid formulations (oleic acid, linoleic acid, oleic acid with 2-oleoylglycerol, oleic acid with 2-oleoylglycerol and oleic acid with glycerol) could reduce variability and increase the extent of the intestinal lymphatic transport and oral bioavailability of CBD. The in vivo studies in rats showed that pre-digested or purified triglyceride did not improve the lymphatic transport and bioavailability of CBD in comparison to sesame oil. Moreover, the results suggest that both the absorption of lipids and the absorption of co-administered CBD were more efficient following administration of natural sesame oil vehicle compared with pre-digested lipids or purified trioleate. Although multiple small molecule constituents and unique fatty acid compositions could potentially contribute to a better performance of sesame oil in oral absorption of lipids or CBD, further investigation will be needed to identify the mechanisms involved.

RationaleYoung children (typically those

The two predominant oak species in Britain areQuercus robur(English or pedunculate oak) andQ. petraea(sessile oak). We sequenced the whole genomes of 386 oak trees from four British parkland sites and found over 50 million nuclear single nucleotide polymorphisms (SNPs), allowing us to identify 360Q. robur, tenQ. petraeaand 16 hybrid individuals using clustering methods. ComparingQ. roburandQ. petraeatrees from Attingham Park, we found that the nuclear genomes of the two species are largely undifferentiated but identified 81 coding regions exhibiting strong interspecific differentiation. The nuclear genomes of our 360Q. roburindividuals showed no clear differentiation among the four parkland sites. Scans for selective sweeps inQ. roburhighlighted regions containing genes with putative involvement in stress tolerance, one of which was moderately differentiated fromQ. petraea. Reconstructions of past effective population sizes suggested a long population size decline in bothQ. roburandQ. petraeaover the Pleistocene, but population growth after the last glacial maximum. We assembled the whole chloroplast genomes of 287Q. robur, 8Q. petraeaand 14 hybrid trees. In a phylogenetic network, these fell into five major haplotypes, which were shared among species but differed in frequency among parkland sites. We matched our chloroplast genome haplotypes to restriction enzyme fragment haplotypes identified in older studies that had surveyed ancient woodlands in Britain and much of Europe. This suggested that the parkland populations in our study derive from local seed sources.

ObjectivesThrombotic and microvascular complications are frequently seen in deceased COVID-19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation-induced endothelial activation remains highly contentious.MethodsHere, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial-endothelial cell barrier to show that primary human endothelial cells express very low levels the SARS-CoV-2 receptor ACE2 and the protease TMPRSS2.ResultsAccordingly, endothelial cells can only be infected when SARS-CoV-2 is present at very high concentrations. However, this is not a productive infection (i.e. no infectious virus is produced) and viral entry induces an inflammatory response. We also show that SARS-CoV-2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co-culture model endothelial cells are not infected with SARS-CoV-2. They do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM-1 expression and releasing pro-inflammatory cytokines.ConclusionsTaken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS-CoV-2 and that infection is only likely to occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS-CoV-2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells are still likely to play a key role in SARS-CoV-2 pathogenesis by sensing adjacent infection and mounting a pro-inflammatory response to SARS-CoV-2.

P. aeruginosa (PA) continues to pose a threat to global public health due to its high levels of antimicrobial resistance (AMR). The ongoing AMR crisis has led to an alarming shortage of effective treatments for resistant microbes, and hence there is a pressing demand for the development of novel antimicrobial interventions. The potential use of antivirulence therapeutics to tackle bacterial infections has attracted considerable attention over the past decades as they hamper the pathogenicity of target microbes with reduced selective pressure, minimizing the emergence of resistance. One such approach is to interfere with the PA pqs quorum sensing system which upon the interaction of PqsR, a Lys-R type transcriptional regulator, with its cognate signal molecules 4-hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), governs multiple virulence traits and host-microbe interactions. In this study, we report the hit identification and optimization of PqsR antagonists using virtual screening coupled with whole cell assay validation. The optimized hit compound 61 ((R)-2-(4-(3-(6-chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxypropoxy)phenyl)acetonitrile) was found to inhibit the expression of the PA P-pqsA promoter controlled by PqsR with an IC50 of 1 mu M. Using isothermal titration calorimetry, a K-d of 10 nM for the P-qsR ligand binding domain (PqsR(LBD)) was determined for 61. Furthermore, the crystal structure of 61 with PqsR(LBD) was attained with a resolution of 2.65 angstrom. Compound 61 significantly reduced levels of pyocyanin, PQS, and HHQ in PAO1-L, PA14 lab strains and PAK6085 clinical isolate. Furthermore, this compound potentiated the effect of ciprofloxacin in early stages of biofilm treatment and in Galleria mellonella infected with PA. Altogether, this data shows 61 as a potent PqsR inhibitor with potential for hit to lead optimization toward the identification of a PA QS inhibitor which can be advanced into preclinical development.

© 2020 The Author(s) Common carp (Cyprinus carpio) are one of the world's most destructive vertebrate pests. In Australia, they dominate many aquatic ecosystems causing a severe threat to aquatic plants, invertebrates, water quality, native fish and social amenity. The Australian Government is considering release of cyprinid herpesvirus-3 (CyHV-3) as a control measure and consequently a robust, continental-scale estimate of the carp population and biomass is essential to inform planning and risk management. Here, we pioneer a novel model-based approach to provide the first estimate of carp density (no/ha) and biomass density (kg/ha) at river reach/waterbody, basin and continental scales. We built a spatial layer of rivers and waterbodies, classified aquatic habitats and calculated the area of each throughout the range of carp in Australia. We then developed a database of fishery-independent electrofishing catch-per-unit-effort (CPUE) for habitat types, containing catch information for 574,145 carp caught at 4831 sites. Eastern Australia accounted for 96% of carp biomass and 92% of the total available wetted habitat area (16,686 km2) was occupied. To correct these data for variable detection efficiencies, we used existing electrofishing data and undertook additional field experiments to establish relationships between relative and absolute abundances. We then scaled-up site-based estimates to habitat types to generate continental estimates. The number of carp was estimated at 199.2 M (95%Crl: 106 M to 357.6 M) for an ‘average’ hydrological scenario and 357.5 M (95%Crl: 178.9 M to 685.1 M) for a ‘wet’ hydrological scenario. In eastern Australia, these numbers correspond with biomasses of 205,774 t (95%Crl: 117,532–356,482 t) (average scenario) and 368,357 t (95%Crl: 184,234–705,630 t) (wet scenario). At a continental scale the total biomass was estimated at 215,456 t for an ‘average’ hydrological scenario. Perennial lowland rivers had the highest CPUE and greatest biomass density (up to 826 kg/ha) and the modelled biomass exceeded a density-impact threshold of 80–100 kg/ha in 54% of wetlands and 97% of stream area in large lowland rivers. The continental-scale biomass estimates provide a baseline for focusing national conservation strategies to reduce carp populations below thresholds needed to restore aquatic ecosystems at a range of spatial scales.

Objectives Thrombotic and microvascular complications are frequently seen in deceased COVID‐19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation‐induced endothelial activation remains highly contentious. Methods Here, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial–endothelial cell barrier. Results We show that primary human endothelial cells express very low levels of the SARS‐CoV‐2 receptor ACE2 and the protease TMPRSS2, which blocks their capacity for productive viral infection, and limits their capacity to produce infectious virus. Accordingly, endothelial cells can only be infected when they overexpress ACE2, or are exposed to very high concentrations of SARS‐CoV‐2. We also show that SARS‐CoV‐2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co‐culture model endothelial cells are not infected with SARS‐CoV‐2. Endothelial cells do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM‐1 expression and releasing pro‐inflammatory cytokines. Conclusions Taken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS‐CoV‐2 and that infection may only occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS‐CoV‐2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells may still play a key role in SARS‐CoV‐2 pathogenesis by sensing adjacent infection and mounting a pro‐inflammatory response to SARS‐CoV‐2., We show that endothelial cells are not productively infected with SARS‐CoV‐2 but mount a pro‐inflammatory response to the virus.

The combined antiretroviral therapy (cART) can efficiently suppress HIV replication, but the cessation of cART usually results in viral rebound, mostly due to the presence of viral reservoirs. The mesenteric lymphatic system, including mesenteric lymph nodes (MLNs), is an important viral reservoir into which antiretroviral drugs poorly penetrate. In this work, we proposed a novel lipophilic ester prodrug approach, combined with oral lipid-based formulation, to efficiently deliver lopinavir (LPV) to the mesenteric lymph and MLNs. A series of prodrugs was designed using an in-silico model for prediction of affinity to chylomicrons (CMs), and then synthesized. The potential for mesenteric lymphatic targeting and bioconversion to LPV in physiologically relevant media was assessed in vitro and ex vivo. Subsequently, LPV and selected prodrug candidates were evaluated for their in vivo pharmacokinetics and biodistribution in rats. Oral co-administration of lipids alone could not facilitate the delivery of unmodified LPV to the mesenteric lymphatic system and resulted in undetectable levels of LPV in these tissues. However, a combination of the lipophilic prodrug approach with lipid-based formulation resulted in efficient targeting of LPV to HIV reservoirs in mesenteric lymph and MLNs. The maximum levels of LPV in mesenteric lymph were 1.6- and 16.9-fold higher than protein binding-adjusted IC90 (PA-IC90) of LPV for HIV-1 (140 ng/mL) following oral administration of simple alkyl ester prodrug and activated ester prodrug, respectively. Moreover, the concentrations of LPV in MLNs were 1.1- and 7.2-fold higher than PA-IC90 following administration of simple alkyl ester prodrug and activated ester prodrug, respectively. Furthermore, the bioavailability of LPV was also substantially increased following oral administration of activated ester prodrug compared to unmodified LPV. This approach, especially if can be translated to other antiretroviral drugs, has potential for reducing the size of HIV reservoirs within the mesenteric lymphatic system.

A simple, sensitive and cost-effective HPLC-UV bioanalytical method for determination of lopinavir (LPV) in rat and human plasma was developed and validated. The plasma sample preparation procedure includes a combination of protein precipitation using cold acetonitrile and liquid-liquid extraction with n-hexane-ethyl acetate (7:3, v/v). A good chromatographic separation was achieved with a Phenomenex Gemini column (C18 , 150 mm × 2.0 mm, 5 µm) at 40°C with gradient elution, at 211 nm. Calibration curves were linear in the range 10-10,000 ng/mL, with a lower limit of quantification of 10 ng/mL using 100 µL of plasma. The accuracy and precision in all validation experiments were within the criteria range set by the guidelines of the Food and Drug Administration. This method was successfully applied to a preliminary pharmacokinetic study in rats following an intravenous bolus administration of LPV. Moreover, the method was subsequently fully validated for human plasma, allowing its use in therapeutic drug monitoring (TDM). In conclusion, this novel, simple and cost-efficient bioanalytical method for determination of LPV is useful for pharmacokinetic and drug delivery studies in rats, as well as TDM in human patients.

Innate immune cells such as macrophages and neutrophils initiate protective inflammatory responses and engage antimicrobial responses to provide frontline defence against invading pathogens. These cells can both restrict the availability of certain transition metals that are essential for microbial growth and direct toxic concentrations of metals towards pathogens as antimicrobial responses. Zinc is important for the structure and function of many proteins, however excess zinc can be cytotoxic. In recent years, several studies have revealed that innate immune cells can deliver toxic concentrations of zinc to intracellular pathogens. In this review, we discuss the importance of zinc status during infectious disease and the evidence for zinc intoxication as an innate immune antimicrobial response. Evidence for pathogen subversion of this response is also examined. The likely mechanisms, including the involvement of specific zinc transporters, that facilitate delivery of zinc by innate immune cells for metal ion poisoning of pathogens are also considered. Precise mechanisms by which excess levels of zinc can be toxic to microorganisms are then discussed, particularly in the context of synergy with other antimicrobial responses. Finally, we highlight key unanswered questions in this emerging field, which may offer new opportunities for exploiting innate immune responses for anti‐infective development.

Rising numbers of cases of multidrug- and extensively drug-resistant Pseudomonas aeruginosa over recent years have created an urgent need for novel therapeutic approaches to cure potentially fatal infections. One such approach is virulence attenuation where anti-virulence compounds, designed to reduce pathogenicity without affording bactericidal effects, are employed to treat infections. P. aeruginosa uses the pqs quorum sensing (QS) system, to coordinate the expression of a large number of virulence determinants as well as bacterial-host interactions and hence represents an excellent anti-virulence target. We report the synthesis and identification of a new series of thiazole-containing quinazolinones capable of inhibiting PqsR, the transcriptional regulator of the pqs QS system. The compounds demonstrated high potency (IC50, Graphical abstract Image 1, Highlights • Pseudomonas aeruginosa – a Gram-negative pathogenic bacterium. • Quorum sensing – a cell-to-cell communication strategy used by microbes to coordinate the production of various virulence factors. • Inhibitors of PqsR – small molecules that inhibit the signal of Pseudomonas aeruginosa Quinolone Signal Receptor. • X-ray crystal structure – used to demonstrate the 3-D structural orientation of the ligand in the protein structure.

For respiratory conditions, targeted drug delivery to the lungs could produce higher local concentrations with reduced risk of adverse events compared to systemic administration. Despite the increasing interest in pulmonary delivery, the pharmacokinetics (PK) of drugs following pulmonary administration remains to be elucidated. In this context, the application of modeling and simulation methodologies to characterize PK properties of compounds following pulmonary administration remains a scarcity. Pseudomonas aeruginosa (PA) lung infections are resistant to many of the current antibiotic therapies. Targeted treatments for pulmonary delivery could be particularly beneficial for these local conditions. In this study, we report the application of biopharmaceutical pharmacometrics (BPMX) for the analysis of PK data from three investigational antimicrobial agents following pulmonary administration of a suspension formulation. The observed drug concentration-time profiles in lungs and plasma of the compound series were combined for simultaneous analysis and modeling. The developed model describes the PK data, taking into account formulation properties, and provides a mechanism to predict dissolved drug concentrations in the lungs available for activity. The model was then used to evaluate formulation effects and the impact of variability on total and dissolved drug concentrations in lungs and plasma. The predictions suggest that these therapies for lung delivery should ideally be delivered in a sustained release formulation with high solubility for maximum local exposure in lungs for efficacy, with rapid systemic clearance in plasma for reduced risk of unwanted systemic adverse effects. This work shows the potential benefits of BPMX and the role it can play to support drug discovery and development in pulmonary delivery.