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Plants contain rapidly evolving specialized metabolic enzymes to support the synthesis of a myriad of functionally diverse natural products. In the case of coumarin biosynthesis, a BAHD acyltransferase-family enzyme COSY was recently discovered in Arabidopsis that catalyzes coumarin formation fromo-hydroxylatedtrans-hydroxycinnamoyl-CoA substrates. COSY is the first and only BAHD enzyme known to date that catalyzes an intramolecular acyl transfer reaction. Here we combine structural, biochemical, and computational approaches to investigate the mechanistic basis for the unique coumarin synthase activity of COSY. Comparative analyses of crystal structures ofArabidopsis thalianaCOSY relative to other BAHD proteins reveal that COSY possesses an unconventional active-site configuration adapted to its specialized activity. Through deuterium exchange experiments, we discover a unique proton exchange mechanism at the α-carbon of theo-hydroxylatedtrans-hydroxycinnamoyl-CoA substrates during the catalytic cycle of COSY. Mutagenesis studies and quantum mechanical cluster modeling further support that this mechanism is key to COSY’s ability to lower the activation energy of thetrans-to-cisisomerization of the hydroxycinnamoyl-CoA substrates, a critical rate-limiting step leading to coumarin production. This study unveils the emergence of an unconventional catalytic mechanism mediated by a BAHD-family enzyme, and sheds light on the potential evolutionary origin of COSY and its recruitment to the evolutionarily new coumarin biosynthetic pathway in eudicots.

Two new lactone lipids, scoriosin (1) and its methyl ester (2), with a rare furylidene ring joined to a tetrahydrofurandione ring, were isolated from Scorias spongiosa, commonly referred to as sooty mold. The planar structure of these compounds was assigned by 1D and 2D NMR. The conformational analysis of these molecules was undertaken to evaluate the relative and absolute configuration through GIAO NMR chemical shift analysis and ECD calculation. In addition to the potent antimicrobial activities, compound 2 strongly potentiated the activity of amphotericin B against Cryptococcus neoformans, suggesting the potential utility of this compound in combination therapies for treating cryptococcal infections.

High-dimensional mass cytometry provides unparalleled insight into the cellular composition of the immune system. Here, we describe a mass-cytometry-based protocol to examine memory CD4

A detailed analysis of steady coating flow of a thin film of a viscous fluid on the outside of a uniformly rotating horizontal circular cylinder in the absence of surface-tension effects but in the presence of a non-uniform pressure distribution due to an irrotational airflow with circulation shows that the presence of the airflow can result in qualitatively different behaviour of the fluid film from that in classical coating flow. Full-film solutions corresponding to a continuous film of fluid covering the entire cylinder are possible only when the flux and mass of fluid do not exceed critical values, which are determined in terms of the non-dimensional parameters $F$ and $K$ representing the speed of the far-field airflow and the circulation of the airflow, respectively. The qualitative changes in the behaviour of the film thickness as $F$ and $K$ are varied are described. In particular, the film thickness can have as many as four stationary points and, in general, has neither top-to-bottom nor right-to-left symmetry. In addition, when the circulation of the airflow is in the same direction as the rotation of the cylinder the maximum mass of fluid that can be supported on the cylinder is always less than that in classical coating flow, whereas when the circulation is in the opposite direction the maximum mass of fluid can be greater than that in classical coating flow.

Ancient shoreline–shelf depositional systems are influenced by an unusually wide array of geological, biological and hydrodynamic processes, with sediment transport and deposition primarily determined by the interaction of river, wave (including storm) and tidal processes, and changes in relative sea level. Understanding the impact of these processes on shoreline–shelf morphodynamics and stratigraphic preservation remains challenging. Numerical modelling integrated with traditional facies analysis provides an increasingly viable approach, with the potential to quantify, and thereby improve understanding of, the impact of these complex coastal sedimentary processes. An integrated approach is presented here that focuses on palaeotidal modelling to investigate the controls on ancient tides and their influence on sedimentary deposition and preservation – one of the three cornerstones of the ternary process classification scheme of shoreline-shelf systems. Numerical tidal modelling methodology is reviewed and illustrated in three palaeotidal model case studies of different scales and focus. The results are synthesised in the context of shoreline–shelf processes, including a critique and modification of the process-based classification scheme. The emphasis on tidal processes reflects their global importance throughout Earth’s history. Ancient palaeotidal models are able to highlight and quantify the following four controls on tidal processes: (1) the physiography (shape and depth) of oceans (1000s km scale) determines the degree of tidal resonance; (2) the physiography of ocean connections to partly enclosed water bodies (100–1000s km scale) determines the regional-scale flux of tidal energy (inflow versus outflow); (3) the physiography of continental shelves influences shelf tidal resonance potential; and (4) tides in relatively local-scale embayments (typically 1–10s km scale) are influenced by the balance of tidal amplification due to funnelling, shoaling and resonance effects versus frictional damping. In deep time, palaeogeographic and palaeobathymetric uncertainty can be accounted for in palaeotidal models by performing sensitivity analyses to different scenarios, across this range of spatial scales. These tidal process controls are incorporated into an updated predictive decision tree for determining shoreline–shelf process regime in terms of the relative interaction of wave, fluvial and tidal processes. The predictive decision tree considers the effects of basin physiography, shelf width and shoreline morphology on wave, fluvial and tidal processes separately. Uncertainty and ambiguity in applying the widely used three-tier process classification scheme are reduced by using the decision tree in conjunction with a proposed two-tier classification of process regime that is limited to primary and secondary processes. This two-tier classification scheme is illustrated in the three case studies, showing how integration of numerical modelling with facies analysis of the preserved stratigraphic record improves confidence in prediction of tide-influenced shoreline-shelf process regimes. Wider application of this approach will further improve process-based classifications and predictions of modern and ancient shoreline–shelf systems.

During ontogeny, macrophages develop from CD115+precursors, including erythro-myeloid progenitors (EMP). EMP arise in the embryonic yolk sac, the primary site of early haematopoiesis. In adults, CD115+bone marrow-derived monocytes represent essential macrophage precursors. Herein, we identify a CD115-macrophage precursor within the adult bone marrow that is unrelated to the classical monocyte lineage but rather shares transcriptomic and functional characteristics of embryonic EMP. These EMPROR (forErythroMyeloidPrecursor) cells are capable of efficiently generating macrophages in disease settings. During early development, EMPROR cells were largely absent from the yolk sac but were instead found at the embryonic-maternal interface in the uterine wall. Unexpectedly, the latter site contains robust haematopoietic activity and harbours defined embryonic haematopoietic progenitor cells, including classical CD115+EMP. Our data suggest the existence of an alternative pathway of macrophage generation in the adult. Further, we uncover a hitherto unknown site of earliest blood cell development.

Tissue-resident memory T cells (TRM) provide immune defence against local infection and can inhibit cancer progression. However, it is unclear to what extent chronic inflammation impacts TRMactivation and how the immune pressure exerted by TRMaffects developing tumours in humans. We performed deep profiling of lung cancers arising in never-smokers (NS) and ever-smokers (ES), finding evidence of enhanced TRMimmunosurveillance in ES lung. Only tumours arising in ES patients underwent clonal immune escape, even when evaluating cancers with similar tumour mutational burden to NS patients, suggesting that the timing of immune pressure exerted by TRMis a critical factor in the evolution of tumour immune evasion. Tumours grown in T cell quiescent NS lungs displayed little evidence of immune evasion and had fewer neoantigens with low diversity, paradoxically making them amenable to treatment with agonist of the costimulatory molecule, ICOS. These data demonstrate local environmental insults enhance TRMimmunosurveillance of human tissue, shape the evolution of tumour immunogenicity and that this interplay informs effective immunotherapeutic modalities.

High‐dimensional cytometry represents an exciting new era of immunology research, enabling the discovery of new cells and prediction of patient responses to therapy. A plethora of analysis and visualization tools and programs are now available for both new and experienced users; however, the transition from low‐ to high‐dimensional cytometry requires a change in the way users think about experimental design and data analysis. Data from high‐dimensional cytometry experiments are often underutilized, because of both the size of the data and the number of possible combinations of markers, as well as to a lack of understanding of the processes required to generate meaningful data. In this article, we explain the concepts behind designing high‐dimensional cytometry experiments and provide considerations for new and experienced users to design and carry out high‐dimensional experiments to maximize quality data collection., High‐dimensional cytometry represents an exciting new era of immunology research; however, the transition from low‐ to high‐dimensional cytometry requires a change in the way users think about experimental design and data analysis. We explain the concepts behind designing high‐dimensional cytometry experiments and provide considerations for new and experienced users to design and carry out high‐dimensional experiments. ​

Objectives: The immunologic events that build up to the fatal neurological stage of experimental cerebral malaria (ECM) are incompletely understood. Here, we dissect immune cell behaviour occurring in the central nervous system (CNS) when Plasmodium berghei ANKA (PbA)-infected mice show only minor clinical signs. Methods: A 2-photon intravital microscopy (2P-IVM) brain imaging model was used to study the spatiotemporal context of early immunological events in situ during ECM. Results: Early in the disease course, antigen-specific CD8+ T cells came in contact and arrested on the endothelium of post-capillary venules. CD8+ T cells typically adhered adjacent to, or were in the near vicinity of, perivascular macrophages (PVMs) that line post-capillary venules. Closer examination revealed that CD8+ T cells crawled along the inner vessel wall towards PVMs that lay on the abluminal side of large post-capillary venules. 'Activity hotspots' in large post-capillary venules were characterised by T-cell localisation, activated morphology and clustering of PVM, increased abutting of post-capillary venules by PVM and augmented monocyte accumulation. In the later stages of infection, when mice exhibited neurological signs, intravascular CD8+ T cells increased in number and changed their behaviour, actively crawling along the endothelium and displaying frequent, short-term interactions with the inner vessel wall at hotspots. Conclusion: Our study suggests an active interaction between PVM and CD8+ T cells occurs across the blood-brain barrier (BBB) in early ECM, which may be the initiating event in the inflammatory cascade leading to BBB alteration and neuropathology.

Unsteady two-dimensional coating flow of a thin film of a viscous fluid on the outside of a uniformly rotating horizontal circular cylinder in the presence of a steady two-dimensional irrotational airflow with circulation is considered. The analysis of this problem by Newell and Viljoen [Phys. Fluids 31(3), 034106 (2019)], who sought to generalize the work of Hinch and Kelmanson [Proc. R. Soc. London, Ser. A 459(2033), 1193–1213 (2003)] to include the effect of the airflow, is revisited. In contrast with the claim of Newell and Viljoen that the flow is conditionally unstable (in the sense that the solution for the film thickness grows without bound for certain values of the physical parameters), it is shown that, in fact, the film remains unconditionally stable in the presence of the airflow.

Objectives The immunologic events that build up to the fatal neurological stage of experimental cerebral malaria (ECM) are incompletely understood. Here, we dissect immune cell behaviour occurring in the central nervous system (CNS) when Plasmodium berghei ANKA (PbA)‐infected mice show only minor clinical signs. Methods A 2‐photon intravital microscopy (2P‐IVM) brain imaging model was used to study the spatiotemporal context of early immunological events in situ during ECM. Results Early in the disease course, antigen‐specific CD8+ T cells came in contact and arrested on the endothelium of post‐capillary venules. CD8+ T cells typically adhered adjacent to, or were in the near vicinity of, perivascular macrophages (PVMs) that line post‐capillary venules. Closer examination revealed that CD8+ T cells crawled along the inner vessel wall towards PVMs that lay on the abluminal side of large post‐capillary venules. ‘Activity hotspots’ in large post‐capillary venules were characterised by T‐cell localisation, activated morphology and clustering of PVM, increased abutting of post‐capillary venules by PVM and augmented monocyte accumulation. In the later stages of infection, when mice exhibited neurological signs, intravascular CD8+ T cells increased in number and changed their behaviour, actively crawling along the endothelium and displaying frequent, short‐term interactions with the inner vessel wall at hotspots. Conclusion Our study suggests an active interaction between PVM and CD8+ T cells occurs across the blood–brain barrier (BBB) in early ECM, which may be the initiating event in the inflammatory cascade leading to BBB alteration and neuropathology., Our study suggests that perivascular macrophages and CD8+ T cells interact across the blood–brain barrier (BBB) in the early stages of experimental cerebral malaria, which may be the initiating event in the inflammatory cascade leading to BBB alteration and neuropathology.

When nanoparticles interact with human blood, a multitude of plasma components adsorb onto the surface of the nanoparticles, forming a biomolecular corona. Corona composition is known to be influenced by the chemical composition of nanoparticles. In contrast, the possible effects of variations in the human blood proteome between healthy individuals on the formation of the corona and its subsequent interactions with immune cells in blood are unknown. Herein, we prepared and examined a matrix of 11 particles (including organic and inorganic particles of three sizes and five surface chemistries) and plasma samples from 23 healthy donors to form donor-specific biomolecular coronas (personalized coronas) and investigated the impact of the personalized coronas on particle interactions with immune cells in human blood. Among the particles examined, poly(ethylene glycol) (PEG)-coated mesoporous silica (MS) particles, irrespective of particle size (800, 450, or 100 nm in diameter), displayed the widest range (up to 60-fold difference) of donor-dependent variance in immune cell association. In contrast, PEG particles (after MS core removal) of 860, 518, or 133 nm in diameter displayed consistent stealth behavior (negligible cell association), irrespective of plasma donor. For comparison, clinically relevant PEGylated doxorubicin-encapsulated liposomes (Doxil) (74 nm in diameter) showed significant variance in association with monocytes and B cells across all plasma donors studied. An in-depth proteomic analysis of each biomolecular corona studied was performed, and the results were compared against the nanoparticle-blood cell association results, with individual variance in the proteome driving differential association with specific immune cell types. We identified key immunoglobulin and complement proteins that explicitly enriched or depleted within the corona and which strongly correlated with the cell association pattern observed across the 23 donors. This study demonstrates how plasma variance in healthy individuals significantly influences the blood immune cell interactions of nanoparticles.

Ancient shoreline–shelf depositional systems are influenced by an unusually wide array of geological, biological and hydrodynamic processes, with sediment transport and deposition primarily determined by the interaction of river, wave (including storm) and tidal processes, and changes in relative sea level. Understanding the impact of these processes on shoreline–shelf morphodynamics and stratigraphic preservation remains challenging. Numerical modelling integrated with traditional facies analysis provides an increasingly viable approach, with the potential to quantify, and thereby improve understanding of, the impact of these complex coastal sedimentary processes. An integrated approach is presented here that focuses on palaeotidal modelling to investigate the controls on ancient tides and their influence on sedimentary deposition and preservation – one of the three cornerstones of the ternary process classification scheme of shoreline-shelf systems. Numerical tidal modelling methodology is reviewed and illustrated in three palaeotidal model case studies of different scales and focus. The results are synthesised in the context of shoreline–shelf processes, including a critique and modification of the process-based classification scheme.The emphasis on tidal processes reflects their global importance throughout Earth’s history. Ancient palaeotidal models are able to highlight and quantify the following four controls on tidal processes: (1) the physiography (shape and depth) of oceans (1000s km scale) determines the degree of tidal resonance; (2) the physiography of ocean connections to partly enclosed water bodies (100–1000s km scale) determines the regional-scale flux of tidal energy (inflow versus outflow); (3) the physiography of continental shelves influences shelf tidal resonance potential; and (4) tides in relatively local-scale embayments (typically 1–10s km scale) are influenced by the balance of tidal amplification due to funnelling, shoaling and resonance effects versus frictional damping. In deep time, palaeogeographic and palaeobathymetric uncertainty can be accounted for in palaeotidal models by performing sensitivity analyses to different scenarios, across this range of spatial scales.These tidal process controls are incorporated into an updated predictive decision tree for determining shoreline–shelf process regime in terms of the relative interaction of wave, fluvial and tidal processes. The predictive decision tree considers the effects of basin physiography, shelf width and shoreline morphology on wave, fluvial and tidal processes separately. Uncertainty and ambiguity in applying the widely used three-tier process classification scheme are reduced by using the decision tree in conjunction with a proposed two-tier classification of process regime that is limited to primary and secondary processes. This two-tier classification scheme is illustrated in the three case studies, showing how integration of numerical modelling with facies analysis of the preserved stratigraphic record improves confidence in prediction of tide-influenced shoreline-shelf process regimes. Wider application of this approach will further improve process-based classifications and predictions of modern and ancient shoreline–shelf systems.

Particle‐based pulmonary delivery has great potential for delivering inhalable therapeutics for local or systemic applications. The design of particles with enhanced aerodynamic properties can improve lung distribution and deposition, and hence the efficacy of encapsulated inhaled drugs. This study describes the nanoengineering and nebulization of metal–phenolic capsules as pulmonary carriers of small molecule drugs and macromolecular drugs in lung cell lines, a human lung model, and mice. Tuning the aerodynamic diameter by increasing the capsule shell thickness (from ≈100 to 200 nm in increments of ≈50 nm) through repeated film deposition on a sacrificial template allows precise control of capsule deposition in a human lung model, corresponding to a shift from the alveolar region to the bronchi as aerodynamic diameter increases. The capsules are biocompatible and biodegradable, as assessed following intratracheal administration in mice, showing >85% of the capsules in the lung after 20 h, but 90% of capsules remaining nonassociated with cells. The amenability to nebulization, capacity for loading, tunable aerodynamic properties, high biocompatibility, and biodegradability make these capsules attractive for controlled pulmonary delivery., The aerodynamic diameters of metal–phenolic capsules are nanoengineered by increasing their shell thickness, which facilitates tailored capsule deposition in a mechanical lung model. The engineered capsules are promising for pulmonary delivery owing to their robustness for nebulization, biocompatibility, biodegradability, and their capacity for cargo loading and surface functionalization.

Pneumococcal meningitis is a highly lethal form of bacterial meningitis that occurs following brain infection by the Gram-positive cocci Streptococcus pneumoniae. Not only does it cause acute mortality, but pneumococcal meningitis also accounts for the highest proportion of survivors living with neurological sequelae, including behavioral disorders, cognitive deficits, hearing loss, motor impairment and epilepsy. More than 90 distinct pneumococcal serotypes have been identified worldwide based on their capsular compositions and serological responses. Serotype replacement continually poses great challenge to costly vaccination programs in developed countries (Koelman et al., 2020), this has therefore emphasized the need to develop new treatment strategies in addition to improving vaccine coverage. Various immunomodulatory agents, such as complement system inhibitors and matrix metalloproteinase inhibitors, have been shown to improve disease severity and mortality when tested in animal models (Bewersdorf et al., 2018). Nevertheless, given the evidence of long-term cognitive deficits and behavioral problems in patients who were clinically well recovered from pneumococcal meningitis, it remains unclear how the protection at the early inflammatory stage may translate into long-term functional recovery. With this in mind, we have established an integrated approach to investigate the interplay between acute host inflammatory response and ensuing neurological deficits in a mouse model of pneumococcal meningitis in animals that survive the lethal disease due to antibiotic ceftriaxone treatment. This has enabled the identification of a nexus between the toll-like receptors (TLRs) 2 and 4, interferon-gamma (IFN-γ) and the enzyme indoleamine 2,3-dioxygenase-1 (IDO-1) that contributes to enduring neurological impairments. Here, we will highlight the findings of our systematic studies in the hope of opening avenues for future research relevant to both meningitis as well as other neurological diseases.

This chapter was inadvertently published with the acknowledgement section leaving out the following sentence: "This work received funding from South Australian Government PRIF program Project "International Cluster on Nanosafety" of Nicolas H. Voelcker and Enzo Lombi." This correction has been updated in the chapter.

Streptococcus pneumoniae is a major meningitis-causing pathogen globally, bringing about significant morbidity and mortality, as well as long-term neurological sequelae in almost half of the survivors. Subsequent to nasopharyngeal colonisation and systemic invasion, translocation across the blood‒brain barrier (BBB) by S. pneumoniae is a crucial early step in the pathogenesis of meningitis. The BBB, which normally protects the central nervous system (CNS) from deleterious molecules within the circulation, becomes dysfunctional in S. pneumoniae invasion due to the effects of pneumococcal toxins and a heightened host inflammatory environment of cytokines, chemokines and reactive oxygen species intracranially. The bacteria‒host interplay within the CNS likely determines not only the degree of BBB pathological changes, but also host survival and the extent of neurological damage. This review explores the relationship between S. pneumoniae bacteria and the host inflammatory response, with an emphasis on the BBB and its roles in CNS protection, as well as both the acute and long-term pathogenesis of meningitis.

Significance Here, we identify two noncatalytic chalcone isomerase-fold proteins, which are critical for high-efficiency prenylchalcone production in Humulus lupulus. Our results provide insights into their evolutionary development from the ancestral noncatalytic fatty acid-binding chalcone isomerase-fold proteins to specialized auxiliary proteins supporting flavonoid biosynthesis in plants, and open up the possibility of producing high-value plant prenylchalcones using heterologous systems., Xanthohumol (XN) and demethylxanthohumol (DMX) are specialized prenylated chalconoids with multiple pharmaceutical applications that accumulate to high levels in the glandular trichomes of hops (Humulus lupulus L.). Although all structural enzymes in the XN pathway have been functionally identified, biochemical mechanisms underlying highly efficient production of XN have not been fully resolved. In this study, we characterized two noncatalytic chalcone isomerase (CHI)-like proteins (designated as HlCHIL1 and HlCHIL2) using engineered yeast harboring all genes required for DMX production. HlCHIL2 increased DMX production by 2.3-fold, whereas HlCHIL1 significantly decreased DMX production by 30%. We show that CHIL2 is part of an active DMX biosynthetic metabolon in hop glandular trichomes that encompasses a chalcone synthase (CHS) and a membrane-bound prenyltransferase, and that type IV CHI-fold proteins of representative land plants contain conserved function to bind with CHS and enhance its activity. Binding assays and structural docking uncover a function of HlCHIL1 to bind DMX and naringenin chalcone to stabilize the ring-open configuration of these chalconoids. This study reveals the role of two HlCHILs in DMX biosynthesis in hops, and provides insight into their evolutionary development from the ancestral fatty acid-binding CHI-fold proteins to specialized auxiliary proteins supporting flavonoid biosynthesis in plants.

How to study nanoparticle–cell interactions is the key question that puzzles researchers in the fields of nanomedicine as well as in nanotoxicology. In nanotoxicology, the amount of nanoparticles internalized by the cells or bound to the external surfaces of cells determines the toxic profile of those particles. In medical applications, cellular uptake and binding of medically effective nanoparticles decides their efficacy. Despite the importance of understanding the extent and mode of nanoparticle–cell interactions, these processes are underinvestigated, mainly due to the lack of suitable user-friendly methodologies. Here we discuss the advantages and limitations of currently available (and most advanced) microscopic, spectroscopic, and other bioanalytical methods that could be used to assess cell-nanoparticle interactions either qualitatively or quantitatively. Special emphasis is given to the methods that enable analysis and identification of nanoparticles at single-cell level, and allow intracellular localization and speciation analysis of nanoparticles. This article is categorized under: Nanotechnology Approaches to Biology > Cells at the Nanoscale Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials. Refereed/Peer-reviewed

Gamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection with Plasmodium species. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used the Plasmodium berghei ANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA for Irgm1 and Irgm3 was found in the brains and spleens of infected mice at times of peak IFN-γ production. Irgm3 −/− but not Irgm1 −/− mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8 + T cells within the brain. Although antigen-specific proliferation of transferred CD8 + T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred into Irgm3 −/− recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens of Irgm3 −/− mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology in Irgm3 −/− mice occurs due to impaired generation of CD8 + effector function. This defect is nonintrinsic to CD8 + T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.

Cerebral malaria (CM) is a severe complication of and a leading cause of death due toPlasmodium falciparuminfection. CM is likely the result of interrelated events, including mechanical obstruction due to parasite sequestration in the microvasculature, and upregulation of Th1 immune responses. In parallel, blood-brain-barrier (BBB) breakdown and damage or death of microglia, astrocytes, and neurons occurs. We found that a novel formulation of a liposome-encapsulated glucocorticosteroid,β-methasone hemisuccinate (nSSL-BMS), prevents experimental cerebral malaria (ECM) in a murine model and creates a survival time-window, enabling administration of an antiplasmodial drug before severe anemia develops. nSSL-BMS treatment leads to lower levels of cerebral inflammation, expressed by altered levels of corresponding cytokines and chemokines. The results indicate the role of integrated immune responses in ECM induction and show that the new steroidal nanodrug nSSL-BMS reverses the balance between the Th1 and Th2 responses in malaria-infected mice so that the proinflammatory processes leading to ECM are prevented. Overall, because of the immunopathological nature of CM, combined immunomodulator/antiplasmodial treatment should be considered for prevention/treatment of human CM and long-term cognitive damage.

Concurrent ectoparasite infections on fishes are commonly encountered in the wild and in aquaculture (Rohde 1984; Hoffman 1999), for example, infections of both Ichthyophthirius multifiliis Fouquet, 1876 (Hymenostomatida: Ichthyopththiriidae; Colorni 2008) and gill‐infecting monogenoids (Platyhelminthes: Monogenoidea; Whittington and Chisholm 2008). The decision to aggressively treat infections of either of these parasites can be prudent in aquaculture systems. Both taxa have direct life cycles and short generation times, which may result in high infection intensities that can debilitate or kill the fish host (Noga 2010). Various chemical therapies have been evaluated to prevent, reduce, or eliminate such infections. Typical treatments for killing monogenoids include: freshwater or saltwater baths, formalin, copper sulfate (CuSO4), hydrogen peroxide, mebendazole, trichlorphon, and praziquantel (Whittington and Chisholm 2008). Treatments for infections of I. multifiliis (Ich) include: temporary exposure to high temperature, formalin, malachite green, CuSO4, potassium permanganate, and sodium chloride (Colorni 2008). Despite the common occurrence of these parasites on fish, little species‐specific information exists for the vast majority of fish–parasite combinations across the diversity of culture systems and captive settings. Formalin is approved by the US Food and Drug Administration (US FDA) for use as a treatment for external monogenetic trematodes in fish at up to 250 mg/L for up to 1 h (US FWS/AFS 2008). The ambiguity of this recommendation suggests a wide therapeutic range with no interpretation of repeated applications. Likewise, little information exists on the safety or efficacy of repeated formalin treatments on fish, and CuSO4 has an indeterminate approval status but is widely accepted as having high efficacy. In October 2009, white bass, Morone chrysops (Rafinesque, 1820; Perciformes: Moronidae), in a flow‐through culture system were diagnosed with concurrent infections of the gill‐infecting monogenoid Onchocleidus mimus (Mueller, 1936; Monogenoidea: Ancyrocephalidae) and Ich. O. mimus has a direct life cycle with no intermediate host required and as the intensity of infection increases can become problematic to a population. This study reports an opportunistic evaluation of the efficacy of repeated applications of formalin and CuSO4 to reduce, control, or eradicate these infections in cultured white bass.

The complete extent to which the human genome is translated into polypeptides is of fundamental importance. We report a peptidomic strategy to detect short open reading frame (sORF)-encoded polypeptides (SEPs) in human cells. We identify 90 SEPs, 86 of which are previously uncharacterized, which is the largest number of human SEPs ever reported. SEP abundances range from 10-1,000 molecules per cell, identical to abundances of known proteins. SEPs arise from sORFs in noncoding RNAs as well as multicistronic mRNAs, and many SEPs initiate with non-AUG start codons, indicating that noncanonical translation may be more widespread in mammals than previously thought. In addition, coding sORFs are present in a small fraction (8 out of 1,866) of long intergenic noncoding RNAs. Together, these results provide strong evidence that the human proteome is more complex than previously appreciated.

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-γ) was strongly induced, and IFN-γ−/− mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-γ. Furthermore, production of IFN-γ was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-γ induction. The influence of IFN-γ gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-γ−/− mice, both monocyte recruitment and CCL2 production were less in infected IFN-γ−/− mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-γ−/− mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-γ−/− mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-γ contributes via multiple pathways to pathology during S. pneumoniae meningitis.

MicroRNAs (miRNAs) are posttranscriptional regulatory molecules that have been implicated in the regulation of immune responses, but their role in the immune response to Plasmodium infection is unknown. We studied the expression of selected miRNAs following infection of CBA mice with Plasmodium berghei ANKA (PbA), which causes cerebral malaria (CM), or Plasmodium berghei K173 (PbK), which causes severe malaria but without cerebral complications, termed non-CM. The differential expression profiles of selected miRNAs (let-7i, miR-27a, miR-150, miR-126, miR-210, and miR-155) were analyzed in mouse brain and heart tissue by quantitative reverse transcription-PCR (qRT-PCR). We identified three miRNAs that were differentially expressed in the brain of PbA-infected CBA mice: let7i, miR-27a, and miR-150. In contrast, no miRNA changes were detected in the heart, an organ with no known pathology during acute malaria. To investigate the involvement of let-7i, miR-27a, and miR-150 in CM-resistant mice, we assessed the expression levels in gamma interferon knockout (IFN-γ −/− ) mice on a C57BL/6 genetic background. The expression of let-7i, miR-27a, and miR-150 was unchanged in both wild-type (WT) and IFN-γ −/− mice following infection. Overexpression of these three miRNAs during PbA, but not PbK, infection in WT mice may be critical for the triggering of the neurological syndrome via regulation of their potential downstream targets. Our data suggest that in the CBA mouse at least, miRNA may have a regulatory role in the pathogenesis of severe malaria.

We model tides and associated bed shear stress in the Early Jurassic Laurasian Seaway of northwest Europe. Sensitivity tests with different water depths highlight those regions that are least affected by paleobathymetric uncertainty. Results show that although the vast seaway was largely microtidal, the tides were still capable of affecting sediment transport more than 2000 km from the open ocean. Flow constriction associated with shallow platforms and straits produced elevated bed shear stresses (a direct proxy for the entrainment and transport of sediment) that were decoupled from tidal range and were capable of transporting sand. Areas of increased bed shear stress broadly correlate with published geological data. Varying the water depth in the basin provides an insight into the effects of an idealized transgression. Increasing the water level leads to a net reduction in the bed shear stress at the shelf edge, but promotes further penetration of tidal energy into the basin interior, although it is still focused into areas of flow constriction. Further drowning of the seaway eventually widens the straits to the point that they fail to constrict the flow and bed shear stress is reduced. Increasing water level counterintuitively leads to an increase in bed shear stress in a small number of locations.

Streptococcus pneumoniae is a major meningitis-causing pathogen globally, bringing about significant morbidity and mortality, as well as long-term neurological sequelae in almost half of the survivors. Subsequent to nasopharyngeal colonisation and systemic invasion, translocation across the blood‒brain barrier (BBB) by S. pneumoniae is a crucial early step in the pathogenesis of meningitis. The BBB, which normally protects the central nervous system (CNS) from deleterious molecules within the circulation, becomes dysfunctional in S. pneumoniae invasion due to the effects of pneumococcal toxins and a heightened host inflammatory environment of cytokines, chemokines and reactive oxygen species intracranially. The bacteria‒host interplay within the CNS likely determines not only the degree of BBB pathological changes, but also host survival and the extent of neurological damage. This review explores the relationship between S. pneumoniae bacteria and the host inflammatory response, with an emphasis on the BBB and its roles in CNS protection, as well as both the acute and long-term pathogenesis of meningitis.

Graphical abstract, Lung complications during malaria infection can range from coughs and impairments in gas transfer to the development of acute respiratory distress syndrome (ARDS). Infecting C57BL/6 mice with Plasmodium berghei K173 strain (PbK) resulted in pulmonary oedema, capillaries congested with leukocytes and infected red blood cells (iRBCs), and leukocyte infiltration into the lungs. This new model of malaria-associated lung pathology, without any accompanying cerebral complications, allows the investigation of mechanisms leading to the lung disease. The activity of the amiloride-sensitive epithelial sodium channel (ENaC) in alveolar epithelial cells is decreased by several respiratory tract pathogens and this is suggested to contribute to pulmonary oedema. We show that PbK, a pathogen that remains in the circulation, also decreased the activity and expression of ENaC, suggesting that infectious agents can have indirect effects on ENaC activity in lung epithelial cells. The reduced ENaC activity may contribute to the pulmonary oedema induced by PbK malaria.

Reduced survival of fish eggs is often a result of infestation with fungi Saprolegnia spp. However, timely chemical treatments often limit these infestations and increase survival. The effect of copper sulfate pentahydrate (CSP; 10 mg of CSP/L of water), diquat bromide (25 mg of diquat cation/L), formalin (433 mg/L), and hydrogen peroxide (H2O2; 250 mg/L) on percent hatch and fungal infestation in eggs of channel catfish Ictalurus punctatus was compared in two identical trials (five replicates for each chemical treatment per trial). The percent hatch in all chemical treatments was significantly better than percent hatch of the controls. The amount of fungal coverage on egg masses treated with CSP, formalin, and H2O2 was significantly less than that observed on the controls. Although not statistically different from the other treatments, H2O2 at 250 mg/L exhibited the highest average percent hatch (64%; control hatch = 34%), the lowest fungal coverage (1.5 cm; control coverage = 7.2 cm), and the l...

In a text where “[m]istakes are everywhere and errors abound,” 1 no error or mistake is so important in Finnegans Wake as humanity’s fall. The profundity of this error, a seemingly indelible stroke, is matched by the difficulty of its erasure, for nothing less than the death of a God could wipe it clean. The celebration of such a bestowal of divine grace upon an undeserving humanity is sung in the “Exultet” hymn of Holy Saturday in the Catholic Church, and the phrase “O felix culpa,” taken from it, forms an “energetic non-centre” of Finnegans Wake, according to Fritz Senn (164). More than just a phrase from the Mass or even a representation of the fall of humanity itself, “O felix culpa” also marks the influence of John Milton’s Paradise Lost upon the Wake and points to the murders of Chief Secretary Lord Frederick Cavendish and Under-Secretary Thomas Henry Burke in Phoenix Park, as well as to the exposure of Richard Pigott as a forger. After a brief survey of these references, I will show that the full importance of the felix culpa for Joyce is only encountered when the fall is understood as a fall into text (becoming a word of the flesh), that the fortuity of the fall is found in the act of writing itself, and that “felix culpa” marks the inter-penetration of divine and human creation in writing. I. In the Mass The “Exultet” hymn is the Praeconium to the Mass of Holy Saturday, Easter Eve. Holy Saturday forms the exact middle of the Easter vigil, between Christ’s crucifixion on Good Friday and his Easter resurrection. As such, it is a liminal moment between death and rebirth, not only in the life of Christ but in the life of the Christian congregation as well. Ildefonso Schuster’s annotation of the Sacramentary describes an ancient Easter tradition maintaining that the second coming of Christ was to occur on the anniversary of the night in which he rose from the tomb: “The faithful, therefore, assembled in the Church and kept watch in expectation of the parousia; but, when midnight had come and gone, and no one had appeared from heaven, they

The free-living infective theront of Ichthyophthirius multifiliis historically has been thought to be the only stage susceptible to treatment. Here we introduce a technique to determine the toxicity of compounds to the newly released tomont, the encysted tomont and the developing tomites within the tomont that emerge as theronts. The toxicity of Wofasteril E400 (40% peracetic acid, PAA) to free-living forms of I. multifiliis was determined shortly after tomonts were physically removed from the surface of the fish and at 2.5 and 24 h after removal. Results indicate that 0.6 to 0.9 mg l(-1) PAA killed 39 to 82% of the newly released tomonts within 48 h when treated immediately. In a second experiment, tomonts were allowed to settle for 2.5 h after sampling from the skin and then treated for 12 h; concentrations > or =0.5 mg l(-1) PAA produced significantly fewer theronts than the controls. In a third experiment, encysted tomonts that were exposed to PAA 24 h after sampling from the skin and treated for 2 or 4 h produced a variable amount of theronts, but the concentrations tested (0.5 to 3.0 mg l(-1)) did not halt theront production. This research demonstrates that encysted I. multifiliis are less susceptible to chemical treatments.

Macrophages are central players in both lipid metabolism and innate immunity. Their determinant role in the pathogenesis of atherosclerosis is under the control of the ATP-binding cassette transporter (ABCA1), which by minimizing cellular lipid content, limits development of pro-inflammatory foam cells. Considering the differential contribution of monocyte subsets to the generation of vascular lesions we analyzed the immunophenotype of ABCA1-expressing cells in the myeloid lineage, by the combined use of flow cytometry and real-time quantitative RT-PCR. ABCA1 expression is limited to "non-inflammatory" Ly6C(lo) circulating monocytes and tissue-resident macrophages expressing markers of alternative activation. In ABCA1(-/-) peritoneal macrophages the transcriptional programs induced by LPS/IFN-gamma or IL-4 cytokines are altered and deviated phosphorylation patterns of STAT transcriptional regulators in response to stimuli are observed.

D. LLOYD, J.C. HARRIS, S. MAROULIS, A. MITCHELL, M.N. HUGHES, R.B. WADLEY AND M.R. EDWARDS. 2003. Aims: To investigate the antigiardial properties of the nitrosating agents: sodium nitrite, sodium nitroprusside andRoussin’s black salt.Methods and Results: Use of confocal laser scanning microscopy and flow cytometry indicated permeabilizationof the plasma membrane to the anionic fluorophore, DiBAC 4 (3) [bis(1,3-dibutylbarbituric acid) trimethine oxonol].Loss of plasma membrane electrochemical potential was accompanied by loss of regulated cellular volume control.Changes in ultrastructure revealed by electron microscopy and capacity for oxygen consumption, were alsoconsequences of nitrosative stress. Roussin’s black salt (RBS), active at micromolar concentrations was the mostpotent of the three agents tested.Conclusions: These multitargeted cytotoxic agents affected plasma membrane functions, inhibited cellularfunctions in Giardia intestinalis and led to loss of viability.Significance and Impact of the Study: Nitrosative damage, as an antigiardial strategy, may have implications fordevelopment of chemotherapy along with suggesting natural host defence mechanisms.Keywords: flow cytometry, nitric oxide, nitrite, nitroprusside, nitrosonium cations, plasma membrane potential.

Largemouth bass Micropterus salmoides are piscivorous fish raised on farms then sold live in Asian fish markets on the east and west coasts of the United States. In the winter of 1998, a major producer of feed-trained bass suffered a significant increase in fish mortality both during shipping and while the fish were still in ponds. No bacterial, viral, or significant parasitic pathogens were found at necropsy. Livers of affected fish were pale and translucent with 3–10 mm pink nodules on their surface and deeper in the parenchyma. Histological examination of these livers showed that the translucent regions of the liver contained few hepatocytes and were composed of tissue consistent with a chronic inflammatory response. Also present were eosinophils, islands of pancreatic and biliary cells, and granulomas that did not stain positively for mycobacteria. The pink nodules were areas of multifocal regeneration of normal hepatocytes. Sequential studies of bass in ponds revealed that the bass were progressively accumulating glycogen in their hepatocytes to an extent sufficient to explain the massive necrosis of that organ. In order to determine the effect of diets varying in available carbohydrate on fish growth, survival, and liver glycogen content, a 12-wk feeding trial was conducted in aquaria with juvenile largemouth bass. Nitrogen-free extract values indicated that an extruded trout diet, a steelhead trout diet, and a diet designed to contain 45% protein and 25% fat, contained 35, 27, and 21% carbohydrate, respectively. Weight gain was lowest in fish fed the extruded trout diet, while liver glycogen was significantly higher in fish fed diets with >27% carbohydrate than in fish fed the diet with 21% carbohydrate. The farmer switched to a diet similar to the 45–25 diet used in our trial. Subsequently, 16-mo-old fish examined in October 2000 had no hepatic nodules or necrosis, were significantly larger and less variable in size than previous crops, and the farmer experienced no significant losses in ponds or during shipping.

Mast-cell degranulation is triggered by the bridging of Fc receptor-bound antigen-specific immunoglobulin IgE on the cell surface. In vitro experiments suggest that antibody affinity and nonspecific IgE may affect the mast-cell function, however, their importance in vivo is unclear. Investigations of the effects of these parameters on mast-cell sensitization were therefore carried out in a rat immunization model in which the IgE response is transient and peaks on days 10-15. Between these two timepoints, significant changes in the level of specific IgE were not observed, but the avidity of specific IgE increased (P < 0.05). Total serum IgE peaked on day 10 and slowly declined, with the relative proportion of specific to total IgE increasing from day 10-15 (P < 0.05). Despite similar levels of antigen-specific IgE, increasing avidity and an increased proportion of specific IgE between days 10 and 15, the biological activity of IgE in the serum peaks on day 10 and declines rapidly, dropping around seven-fold by day 15 (P < 0.001). Mechanisms that could explain this finding, such as differential expression of IgE isoforms and changes in the fine specificity of the IgE response, are discussed.

Therapeutic nanoparticles hold clinical promise for cancer treatment by avoiding limitations of conventional pharmaceuticals. Herein, a facile and rapid method is introduced to assemble poly(ethylene glycol) (PEG)-modified Pt prodrug nanocomplexes through metal-polyphenol complexation and combined with emulsification, which results in ≈100 nm diameter nanoparticles (PtP NPs) that exhibit high drug loading (0.15 fg Pt per nanoparticle) and low fouling properties. The PtP NPs are characterized for potential use as cancer therapeutics. Mass cytometry is used to quantify uptake of the nanoparticles and the drug concentration in individual cells in vitro. The PtP NPs have long circulation times, with an elimination half-life of ≈18 h in healthy mice. The in vivo antitumor activity of the PtP NPs is systematically investigated in a human prostate cancer xenograft mouse model. Mice treated with the PtP NPs demonstrate four times better inhibition of tumor growth than either free prodrug or cisplatin. This study presents a promising strategy to prepare therapeutic nanoparticles for biomedical applications.

During experimental cerebral malaria (ECM) mice develop a lethal neuropathological syndrome associated with microcirculatory dysfunction and intravascular leukocyte sequestration. The precise spatio-temporal context in which the intravascular immune response unfolds is incompletely understood. We developed a 2-photon intravital microscopy (2P-IVM)-based brain-imaging model to monitor the real-time behaviour of leukocytes directly within the brain vasculature during ECM. Ly6Chi monocytes, but not neutrophils, started to accumulate in the blood vessels of Plasmodium berghei ANKA (PbA)-infected MacGreen mice, in which myeloid cells express GFP, one to two days prior to the onset of the neurological signs (NS). A decrease in the rolling speed of monocytes, a measure of endothelial cell activation, was associated with progressive worsening of clinical symptoms. Adoptive transfer experiments with defined immune cell subsets in recombinase activating gene (RAG)-1-deficient mice showed that these changes were mediated by Plasmodium-specific CD8+ T lymphocytes. A critical number of CD8+ T effectors was required to induce disease and monocyte adherence to the vasculature. Depletion of monocytes at the onset of disease symptoms resulted in decreased lymphocyte accumulation, suggesting reciprocal effects of monocytes and T cells on their recruitment within the brain. Together, our studies define the real-time kinetics of leukocyte behaviour in the central nervous system during ECM, and reveal a significant role for Plasmodium-specific CD8+ T lymphocytes in regulating vascular pathology in this disease., Author Summary Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that takes a significant toll on human life. Blockage of the brain blood vessels contributes to the clinical signs of CM, however we know little about the precise pathological events that lead to this disease. To this end, studies in Plasmodium-infected mice, that also develop a similar fatal disease, have proven useful. These studies have revealed an important role for leukocytes not so much in protecting but rather promoting pathology in the brain. To better understand leukocyte behaviour during experimental CM, we established a brain-imaging model that allows us to ‘peek’ into the brain of living mice and watch immunological events as they unfold. We found that worsening of disease was accompanied by an accumulation of monocytes in the blood vessels. Monocyte accumulation was regulated by activated CD8+ T cells but only when present in critical numbers. Monocyte depletion resulted in reduced T cell trafficking to the brain, but this did not result in improved disease outcome. Our studies reveal the orchestration of leukocyte accumulation in real time during CM, and demonstrate that CD8+ T cells play a crucial role in promoting clinical signs in this disease.

Transendothelial migration of neutrophils in postcapillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we showed that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor α-hemolysin produced by S. aureus lyses perivascular macrophages, which leads to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin and indicate that S. aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy.

Sequestration of infected red blood cells (iRBC) within the cerebral and pulmonary microvasculature is a hallmark of human cerebral malaria (hCM). The interaction between iRBC and the endothelium in hCM has been studied extensively and is linked to the severity of malaria. Experimental CM (eCM) caused by Plasmodium berghei ANKA reproduces most features of hCM, although the sequestration of RBC infected by P. berghei ANKA (PbA-iRBC) has not been completely delineated. The role of PbA-iRBC sequestration in the severity of eCM is not well characterized. Using static and flow cytoadherence assays, we provide the first direct in vitro evidence for the binding of PbA-iRBC to murine brain and lung microvascular endothelial cells (MVEC). We found that basal PbA-iRBC cytoadherence to MVECs was significantly higher than that of normal red blood cells (NRBC) and of RBC infected with P. berghei K173 (PbK173-iRBC), a strain that causes noncerebral malaria (NCM). MVEC prestimulation with tumor necrosis factor (TNF) failed to promote any further significant increase in mixed-stage iRBC adherence. Interestingly, enrichment of the blood for mature parasites significantly increased PbA-iRBC binding to the MVECs prestimulated with TNF, while blockade of VCAM-1 reduced this adhesion. Our study provides evidence for the firm, flow-resistant binding to endothelial cells of iRBC from strain ANKA-infected mice, which develop CM, and for less binding of iRBC from strain K173-infected mice, which develop NCM. An understanding of P. berghei cytoadherence may help elucidate the importance of sequestration in the development of CM and aid the development of antibinding therapies to help reduce the burden of this syndrome.