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Reintroduction has become an increasingly important conservation tool in Australia, yet the effects of stress on species during reintroduction programs have received little attention. The use of enzyme immunoassays to measure faecal glucocorticoid metabolites (FGM) is a useful non-invasive technique to monitor adrenal activity but requires validation before they can be reliably used. As part of a large reintroduction project, the goals of this study were to 1) monitor FGM in 53 western quolls (Dasyurus geoffroii) following capture from the wild and transfer to a holding facility and use this stressor to biologically validate an enzyme immunoassay; 2) determine if biological factors, such as sex, age, weight or source population affect baseline FGM levels; and 3) examine individual variation in the acute adrenal response of quolls to the capture and transfer associated with reintroductions. We successfully validated an assay that targets glucocorticoid metabolites with a 5α-3β,11β-diol structure and found that sex significantly influenced both baseline and peak FGM output in western quolls, whereas age, weight and source population did not. We also observed considerable variation among individuals in the magnitude and duration of their physiological response to capture and transfer. Using the methods described here, FGM analysis may provide further information about the adrenal activity of the western quoll and improve future conservation efforts for this threatened species.

Competition among microbes is a key determinant of successful host colonization and persistence. For Streptococcus pneumoniae, lower than predicted rates of co-colonizing strains suggests a competitive advantage for resident bacteria over newcomers. In light of evolutionary theory, we hypothesised that S. pneumoniae use “owner / intruder” asymmetries to settle contests, leading to the disproportionate success of the initial resident “owner”, regardless of the genetic identity of the “intruder”. We investigated the determinants of within-host competitive success utilizing S. pneumoniae colonization of the infant mouse upper respiratory tract. Within six hours, colonization by the resident inhibited colonization by an isogenic challenger. The competitive advantage of the resident was dependent on quorum sensing via the competence (Com) regulon and downstream choline binding protein D (CbpD) and the competence-induced bacteriocins (CibAB) implicated in fratricide. CbpD and CibAB are highly conserved across pneumococcal lineages, indicating evolutionary advantages for asymmetric competitive strategies within the species. Mathematical modelling supported a significant role for quorum sensing via the Com regulon in competition, even for strains with different competitive advantages. Our study suggests that asymmetric owner/intruder competitive strategies do not require complex cognition and are used by a major human pathogen to determine “ownership” of human hosts.

The human IL22RA2 gene co-produces three protein isoforms in dendritic cells [IL-22 binding protein isoform-1 (IL-22BPi1), IL-22BPi2, and IL-22BPi3]. Two of these, IL-22BPi2 and IL-22BPi3, are capable of neutralizing the biological activity of IL-22. The function of IL-22BPi1, which differs from IL-22BPi2 through an in-frame 32-amino acid insertion provided by an alternatively spliced exon, remains unknown. Using transfected human cell lines, we demonstrate that IL-22BPi1 is secreted detectably, but at much lower levels than IL-22BPi2, and unlike IL-22BPi2 and IL-22BPi3, is largely retained in the endoplasmic reticulum (ER). As opposed to IL-22BPi2 and IL-22BPi3, IL-22BPi1 is incapable of neutralizing or binding to IL-22 measured in bioassay or assembly-induced IL-22 co-folding assay. We performed interactome analysis to disclose the mechanism underlying the poor secretion of IL-22BPi1 and identified GRP78, GRP94, GRP170, and calnexin as main interactors. Structure-function analysis revealed that, like IL-22BPi2, IL-22BPi1 binds to the substrate-binding domain of GRP78 as well as to the middle domain of GRP94. Ectopic expression of wild-type GRP78 enhanced, and ATPase-defective GRP94 mutant decreased, secretion of both IL-22BPi1 and IL-22BPi2, while neither of both affected IL-22BPi3 secretion. Thus, IL-22BPi1 and IL-22BPi2 are bona fide clients of the ER chaperones GRP78 and GRP94. However, only IL-22BPi1 activates an unfolded protein response (UPR) resulting in increased protein levels of GRP78 and GRP94. Cloning of the IL22RA2 alternatively spliced exon into an unrelated cytokine, IL-2, bestowed similar characteristics on the resulting protein. We also found that CD14(++)/CD16(+) intermediate monocytes produced a higher level of IL22RA2 mRNA than classical and non-classical monocytes, but this difference disappeared in immature dendritic cells (moDC) derived thereof. Upon silencing of IL22RA2 expression in moDC, GRP78 levels were significantly reduced, suggesting that native IL22RA2 expression naturally contributes to upregulating GRP78 levels in these cells. The IL22RA2 alternatively spliced exon was reported to be recruited through a single mutation in the proto-splice site of a Long Terminal Repeat retrotransposon sequence in the ape lineage. Our work suggests that positive selection of IL-22BPi1 was not driven by IL-22 antagonism as in the case of IL-22BPi2 and IL-22BPi3, but by capacity for induction of an UPR response. This work was supported by the following grants: Grupos de Investigacion (IT512-10, PPG17/44) and MINECO (SAF2012-32118, SAF2016-74891R).

Conservation of long‐distance migratory species poses unique challenges. Migratory connectivity, that is, the extent to which groupings of individuals at breeding sites are maintained in wintering areas, is frequently used to evaluate population structure and assess use of key habitat areas. However, for species with complex or variable annual cycle movements, this traditional bimodal framework of migratory connectivity may be overly simplistic. Like many other waterfowl, sea ducks often travel to specific pre‐ and post‐breeding sites outside their nesting and wintering areas to prepare for migration by feeding extensively and, in some cases, molting their flight feathers. These additional migrations may play a key role in population structure, but are not included in traditional models of migratory connectivity. Network analysis, which applies graph theory to assess linkages between discrete locations or entities, offers a powerful tool for quantitatively assessing the contributions of different sites used throughout the annual cycle to complex spatial networks. We collected satellite telemetry data on annual cycle movements of 672 individual sea ducks of five species from throughout eastern North America and the Great Lakes. From these data, we constructed a multi‐species network model of migratory patterns and site use over the course of breeding, molting, wintering, and migratory staging. Our results highlight inter‐ and intra‐specific differences in the patterns and complexity of annual cycle movement patterns, including the central importance of staging and molting sites in James Bay, the St. Lawrence River, and southern New England to multi‐species annual cycle habitat linkages, and highlight the value of Long‐tailed Ducks (Calengula haemalis) as an umbrella species to represent the movement patterns of multiple sea duck species. We also discuss potential applications of network migration models to conservation prioritization, identification of population units, and integrating different data streams.

We monitored faecal glucocorticoid metabolites (FGM) in 53 western quolls following capture. Using this stressor, we validated an assay for monitoring FGM in quolls. On average, FGM peak 24 hours after capture. Females exhibit higher baseline and peak FGM levels than males. Individuals vary in their physiological response to capture., Reintroduction has become an increasingly important conservation tool in Australia, yet the effects of stress on species during reintroduction programs have received little attention. The use of enzyme immunoassays to measure faecal glucocorticoid metabolites (FGM) is a useful non-invasive technique to monitor adrenal activity but requires validation before they can be reliably used. As part of a large reintroduction project, the goals of this study were to 1) monitor FGM in 53 western quolls (Dasyurus geoffroii) following capture from the wild and transfer to a holding facility and use this stressor to biologically validate an enzyme immunoassay; 2) determine if biological factors, such as sex, age, weight or source population affect baseline FGM levels; and 3) examine individual variation in the acute adrenal response of quolls to the capture and transfer associated with reintroductions. We successfully validated an assay that targets glucocorticoid metabolites with a 5α-3β,11β-diol structure and found that sex significantly influenced both baseline and peak FGM output in western quolls, whereas age, weight and source population did not. We also observed considerable variation among individuals in the magnitude and duration of their physiological response to capture and transfer. Using the methods described here, FGM analysis may provide further information about the adrenal activity of the western quoll and improve future conservation efforts for this threatened species.

The pneumococcal conjugate vaccine (PCV) strongly protects against vaccine serotypes, but the rapid expansion of non-vaccine serotype disease and the vaccine’s high expense has reduced its overall impact. We have developed Protein Glycan Coupling Technology (PGCT) as a flexible methodology for making low-cost polysaccharide/protein glycoconjugates recombinantly in Escherichia coli. We have used PGCT to make a recombinant PCV containing serotype 4 capsular polysaccharide linked to the Streptococcus pneumoniae proteins NanA, PiuA, and Sp0148. The introduction of the Campylobacter jejuni UDP-glucose 4-epimerase gene GalE (gne) into E. coli improved the yield of the resulting glycoprotein. PGCT glycoconjugate vaccination generated strong antibody responses in mice to both the capsule and the carrier protein antigens, with the PiuA/capsule glycoconjugate inducing similar anti-capsular antibody responses as the commercial PCV Prevnar-13. Antibody responses to PGCT glycoconjugates opsonised S. pneumoniae and Streptococcus mitis expressing the serotype 4 capsule and promoted neutrophil phagocytosis of S. pneumoniae to a similar level as antisera generated by vaccination with Prevnar-13. Vaccination with the PGCT glycoconjugates protected mice against meningitis and septicaemia with the same efficacy as vaccination with Prevnar-13. In addition, vaccination with the protein antigen components from PGCT glycoconjugates alone provided partial protection against septicaemia and colonisation. These data demonstrate that a vaccine made by PGCT is as effective as Prevnar-13, identifies PiuA as a carrier protein for glycoconjugate vaccines, and demonstrates that linking capsular antigen to S. pneumoniae protein antigens has additional protective benefits that could provide a degree of serotype-independent immunity., Pneumococcal vaccines: evaluation of a multivalent pneumococcal glycoconjugate vaccine The current pneumococcal conjugate vaccine induces protective immunity to infection, but this protection is limited to the dominant disease-causing strains included in the preparation. Emerging strains not included in the vaccine are not covered, which limits the vaccine’s protective effect in areas associated with alternative pneumococcal strains. Here Jeremy Brown, Brendan Wren and colleagues have optimised and applied Protein Glycan Coupling Technology to produce recombinant pneumococcal polysaccharide/protein glycoconjugates and evaluated these in a murine model of infection. Glycoconjugate vaccination resulted in antibody production specific to both capsule and the carrier proteins, which resulted in protection in murine models of meningitis and septicaemia equivalent to that of currently used vaccination strategies. This animal study highlights the potential of glycoconjugate vaccine approaches in the context of pneumococcal mediated disease.

Many important interactions between bacterial pathogens and their hosts are highly specific binding events that involve host or pathogen carbohydrate structures (glycans). Glycan interactions can mediate adhesion, invasion and immune evasion and can act as receptors for toxins. Several bacterial pathogens can also enzymatically alter host glycans to reveal binding targets, degrade the host cell glycans or alter the function of host glycoproteins. In recent years, high-throughput screening technologies, such as lectin, glycan and mucin microarrays, have transformed the field by identifying new bacterial–host glycointeractions, which are crucial for colonization, persistence and disease. In this Review, we discuss interactions involving both host and bacterial glycans that have a role in bacterial pathogenesis. We also highlight recent technological advances that have illuminated the glycoscience of microbial pathogenesis. In this Review, Jennings and colleagues discuss interactions involving host and bacterial glycans that have a role in bacterial pathogenesis. They also highlight recent technological advances that have illuminated the glycoscience of microbial pathogenesis.

Cholestasis‐induced accumulation of bile acids in the liver leads to farnesoid X receptor (FXR)‐mediated transcriptional down‐regulation of the bile acid importer Na+‐taurocholate cotransporting protein (NTCP) and to induction of endoplasmic reticulum (ER) stress. However, whether ER stress affects bile acid uptake is largely unknown. Here, we investigated the role of ER stress on the regulation and function of the bile acid transporter NTCP. ER stress was induced using thapsigargin or subtilase cytotoxin in human osteosarcoma (U2OS) and human hepatocellular carcinoma (HepG2) cells stably expressing NTCP. Cellular bile acid uptake was determined using radiolabeled taurocholate (TCA). NTCP plasma membrane expression was determined by cell surface biotinylation. Mice received a single injection of thapsigargin, and effects of ER stress on NTCP messenger RNA (mRNA) and protein were measured by reverse‐transcription polymerase chain reaction (RT‐PCR) and western blot analysis. Effects of cholestasis on NTCP and ER stress were assessed in response to 3, 5‐diethoxycarbonyl‐1, 4‐dihydrocollidine (DDC) feeding or bile duct ligation in FXR–/– mice after 7 or 3 days, respectively. Novel NTCP‐interacting proteins were identified by mass spectrometry (MS), interaction verified, and assessed by co‐immunoprecipitation and TCA uptake for functional relevance in relation to ER stress. ER stress induction strongly reduced NTCP protein expression, plasma membrane abundance, and NTCP‐mediated bile acid uptake. This was not controlled by FXR or through a single unfolded protein response (UPR) pathway but mainly depended on the interaction of NTCP with calnexin, an ER chaperone. In mice, expression of both NTCP and calnexin was reduced by thapsigargin or cholestasis‐induced ER stress. Calnexin down‐regulation in vitro recapitulated the effect of ER stress on NTCP. Conclusion: ER stress‐induced down‐regulation of calnexin provides an additional mechanism to dampen NTCP‐mediated bile acid uptake and protect hepatocytes against bile acid overload during cholestasis.

The B subunit of the subtilase cytotoxin (SubB) recognises N-glycolylneuraminic acid (Neu5Gc) containing glycans, the most prominent form of aberrant glycosylation in human cancers. We have previously engineered SubB by construction of a SubBΔS106/ΔT107 mutant (SubB2M) for greater specificity and enhanced recognition of Neu5Gc containing glycans. In this study, we further explore the utility of SubB2M as a Neu5Gc lectin by showing its improved specificity and recognition for Neu5Gc containing glycans over the wild-type SubB protein and an anti-Neu5Gc IgY antibody in a N-acetylneuraminic acid (Neu5Ac)/Neu5Gc glycan array and by surface plasmon resonance. Far-western blot analysis showed that SubB2M preferentially binds to bovine serum glycoproteins over human serum glycoproteins. SubB2M was also able to detect Neu5Gc containing bovine glycoproteins spiked into normal human serum with greater sensitivity than the wild-type SubB and the anti-Neu5Gc IgY antibody. These results suggest that SubB2M will be a useful tool for the testing of serum and other bodily fluids for cancer diagnosis and prognosis.

International audience; Loss-of-function mutations of the protein kinase PERK (EIF2AK3) in humans and mice cause permanent neonatal diabetes and severe proinsulin aggregation in the endoplasmic reticulum (ER), highlighting the essential role of PERK in insulin production in pancreatic cells. As PERK is generally known as a translational regulator of the unfolded protein response (UPR), the underlying cause of these cell defects has often been attributed to derepression of proinsulin synthesis, resulting in proinsulin overload in the ER. Using high-resolution imaging and standard protein fractionation and immunological methods we have examined the PERK-dependent phenotype more closely. We found that whereas proinsulin aggregation requires new protein synthesis, global protein and proinsulin synthesis are down-regulated in PERK-inhibited cells, strongly arguing against proinsulin overproduction being the root cause of their aberrant ER phenotype. Furthermore, we show that PERK regulates proinsulin proteostasis by modulating ER chaperones, including BiP and ERp72. Transgenic overexpression of BiP and BiP knockdown (KD) both promoted proinsulin aggregation, whereas ERp72 overexpression and knockdown rescued it. These findings underscore the importance of ER chaperones working in concert to achieve control of insulin production and identify a role for PERK in maintaining a functional balance among these chaperones.

Streptococcus pneumoniae is the leading infectious cause of death in children in the world. However, the mechanisms that drive the progression from asymptomatic colonization to disease are poorly understood. Two virulence-associated genomic accessory regions (ARs) were deleted in a highly virulent serotype 1 clinical isolate (strain 4496) and examined for their contribution to pathogenesis. Deletion of a prophage encoding a platelet-binding protein (PblB) resulted in reduced adherence, biofilm formation, reduced initial infection within the lungs, and a reduction in the number of circulating platelets in infected mice. However, the region's overall contribution to the survival of mice was not significant. In contrast, deletion of the variable region of pneumococcal pathogenicity island 1 (vPPI1) was also responsible for a reduction in adherence and biofilm formation but also reduced survival and invasion of the pleural cavity, blood, and lungs. While the 4496ΔPPI1 strain induced higher expression of the genes encoding interleukin-10 (IL-10) and CD11b in the lungs of challenged mice than the wild-type strain, very few other genes exhibited altered expression. Moreover, while the level of IL-10 protein was increased in the lungs of 4496ΔPPI1 mutant-infected mice compared to strain 4496-infected mice, the levels of gamma interferon (IFN-γ), CXCL10, CCL2, and CCL4 were not different in the two groups. However, the 4496ΔPPI1 mutant was found to be more susceptible than the wild type to phagocytic killing by a macrophage-like cell line. Therefore, our data suggest that vPPI1 may be a major contributing factor to the heightened virulence of certain serotype 1 strains, possibly by influencing resistance to phagocytic killing.

Enterohemorrhagic Escherichia coli (EHEC) is an important cause of diarrhea and hemolytic-uremic syndrome (HUS) worldwide. Australia's worst outbreak of HUS occurred in Adelaide in 1995 and was one of the first major HUS outbreaks attributed to a non-O157 Shiga-toxigenic E. coli (STEC) strain. Molecular analyses conducted at the time suggested that the outbreak was caused by an O111:H − clone, with strains from later in the outbreak harboring an extra copy of the genes encoding the potent Shiga toxin 2 (Stx2). Two decades later, we have used next-generation sequencing to compare two isolates from early and late in this important outbreak. We analyzed genetic content, single-nucleotide polymorphisms (SNPs), and prophage insertion sites; for the latter, we demonstrate how paired-end sequence data can be leveraged to identify such insertion sites. The two strains are genetically identical except for six SNP differences and the presence of not one but two additional Stx2-converting prophages in the later isolate. Isolates from later in the outbreak were associated with higher levels of morbidity, suggesting that the presence of the additional Stx2-converting prophages is significant in terms of the virulence of this clone.

•River flows to an inverted estuary were diminished during prolonged drought.•Extreme hypersalinity led to >50% reduction in the range of smallmouth hardyhead.•Salinity exceeded the osmoregulatory limit of the fish in much of its range.•Salinity returned to pre-drought levels following the return of river flows.•The smallmouth hardyhead population recovered to its former range after 2-years.

Streptococcus pneumoniae is responsible for significant mortality and morbidity worldwide and causes invasive pneumococcal diseases including pneumococcal meningitis. Pyroptosis is caspase-1-dependent inflammatory cell death and is known to be induced by various microbial infections. In the present study, we investigated the molecular mechanisms that regulate pyroptosis induced by S. pneumoniae in microglia. Our results revealed that S. pneumoniae induced pyroptosis through caspase-1 activation and IL-1β production. We also found that the activation of caspase-1 and the maturation of IL-1β and IL-18 in the S. pneumoniae-triggered pyroptotic cell death process were mediated by NLRP3 inflammasome. In addition, pneumococcal infection increased the expression of autophagy-related genes and induced autophagosome formation. We also showed that the inhibition of autophagy promoted pneumococcus-induced pyroptosis. Furthermore, ROS was generated by pneumococcal infection and inhibited caspase-1 activation within 4 h of infection. However, in the late phase of infection, IL-1β secretion and caspase-1-dependent cell death were induced by ROS. These results suggest that autophagy induction transiently delay pyroptosis induced by S. pneumoniae in microglia. Our study also revealed that the activation of caspase-1 and the production of IL-1β were induced by pneumolysin and that pneumolysin triggered pyroptosis in microglial cells. Similar to the in vitro results, S. pneumoniae induced caspase-1 activation and caspase-1-dependent cytokine maturation in the mouse meningitis model. Thus, the present data demonstrate that S. pneumoniae induces pyroptosis in murine microglia and that NLRP3 inflammasome is critical for caspase-1 activation during the process. Furthermore, the induction of autophagy could transiently protect microglia from pyroptosis.

Haemophilus influenzae and Streptococcus pneumoniae exist together as common commensals of the healthy human nasopharynx, but both are important aetiological agents of different diseases, including the paediatric disease otitis media. It was recently shown that the formation of a multispecies biofilm of H. influenzae and S. pneumoniae is the cause of chronic forms of otitis media. However, the interactions between the two species are not clearly defined. Using a defined and kinetic analysis, our study has shown that while co-existence of the two species occurs, S. pneumoniae is also able to convert H. influenzae to a non-culturable state. We determined that this process was dependent on growth phase and pH. To analyse the H. influenzae/S. pneumoniae interactions in more depth, we investigated the growth and transcriptional profile in a pH-defined batch culture model, as well as in a growth phase independent flow cell system. Transcriptomics has shown that there are changes in gene expression in each of the species when grown in co-culture, intriguingly inducing the S. pneumoniae bacteriocin transport genes, and phage-associated genes in both species. Importantly, we have shown vast changes in gene expression in a group of S. pneumoniae metabolic genes, including those encoding lactose utilisation, glycerol utilisation and sugar transport proteins; we have shown that the expression of these genes depends not only on the presence of H. influenzae, but also on the growth system utilised.

Streptococcus pneumoniae is a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. We have previously reported that S. pneumoniae serotype 3 isolates belonging to the same multilocus sequence type (MLST) differed markedly in in vitro and in vivo phenotypes, in accordance with the clinical site of isolation, suggesting stable niche adaptation within a clonal lineage. In the present study, we have extended our analysis to serotype 14 clinical isolates from cases of sepsis or otitis media that belong to the same MLST (ST15). In a murine intranasal challenge model, five ST15 isolates (three from blood and two from ears) colonized the nasopharynx to similar extents. However, blood and ear isolates exhibited significant differences in bacterial loads in other host niches (lungs, ear, and brain) at both 24 and 72 h postchallenge. In spite of these differences, blood and ear isolates were present in the lungs at similar levels at 6 h postchallenge, suggesting that early immune responses may underpin the distinct virulence phenotypes. Transcriptional analysis of lung tissue from mice infected for 6 h with blood isolates versus ear isolates revealed 8 differentially expressed genes. Two of these were exclusively expressed in response to infection with the ear isolate. These results suggest a link between the differential capacities to elicit early innate immune responses and the distinct virulence phenotypes of clonally related S. pneumoniae strains.

Group A Streptococcus (Streptococcus pyogenes), group B Streptococcus (Streptococcus agalactiae) and Streptococcus pneumoniae (pneumococcus) are host-adapted bacterial pathogens among the leading infectious causes of human morbidity and mortality. These microbes and related members of the genus Streptococcus produce an array of toxins that act against human cells or tissues, resulting in impaired immune responses and subversion of host physiological processes to benefit the invading microorganism. This toxin repertoire includes haemolysins, proteases, superantigens and other agents that ultimately enhance colonization and survival within the host and promote dissemination of the pathogen.

Shiga-toxigenic Escherichia coli (STEC) causes severe gastrointestinal infections in humans that may lead to life-threatening systemic sequelae, such as the hemolytic uremic syndrome (HUS). Rapid diagnosis of STEC infection early in the course of disease opens a window of opportunity for therapeutic intervention, for example, by administration of agents that neutralize Shiga toxin (Stx) in the gut lumen. We previously developed a recombinant bacterium that expresses a mimic of the Stx receptor globotriaosyl ceramide (Gb3) on its surface through modification of the lipopolysaccharide (A. W. Paton, R. Morona, and J. C. Paton, Nat Med 6: 265–270, 2000, http://dx.doi.org/10.1038/73111 ). This construct was highly efficacious in vivo , protecting mice from otherwise fatal STEC disease, but the fact that it is a genetically modified organism (GMO) has been a barrier to clinical development. In the present study, we have overcome this issue by development of Gb3 receptor mimic bacterial ghosts (BGs) that are not classified as GMOs. Gb3-BGs neutralized Stx1 and Stx2 in vitro with high efficiency, whereas alternative Gb3-expressing non-GMO subbacterial particles (minicells and outer membrane blebs) were ineffective. Gb3-BGs were highly efficacious in a murine model of STEC disease. All mice (10/10) treated with Gb3-BGs survived challenge with a highly virulent O113:H21 STEC strain and showed no pathological signs of renal injury. In contrast, 6/10 mice treated with control BGs succumbed to STEC challenge, and survivors exhibited significant weight loss, neutrophilia, and histopathological evidence of renal damage. Thus, Gb3-BGs offer a non-GMO approach to treatment of STEC infection in humans, particularly in an outbreak setting.

Nearshore waters provide very important habitat for sea ducks (Tribe Mergini) during migration and winter, but gathering information on sea duck use of shallow nearshore waters is challenging because traditional aerial and boat-based surveys are expensive, are usually conducted infrequently, and are often not feasible near the coast. The objective of this study was to use land-based surveys to characterize spatiotemporal variation in the abundance and behavior (e.g., foraging, flying) of Common Eider (Somateria mollissima) and scoters (Melanitta spp.) in nearshore waters of southern New England. Surveys (60–120 min per survey, n = 1,044 surveys) were conducted throughout the day from February 2009 to July 2010 to assess diurnal and seasonal variation in sea duck behavior and spatial distribution at nine sites in southern Rhode Island. The density of sea ducks resting or foraging on the water exhibited little diurnal variation, whereas flight activity dramatically increased nearer to sunrise. Sea...

Human activity is one of the most important factors affecting disturbance to birds that use coastal barrier beaches in southern New England. The barrier beach at Napatree Point, RI, provides important breeding habitat for several bird species, key stop- over habitat for thousands of migrating shorebirds, and is also a popular destination for people. Anecdotal evidence suggested that walkers, joggers, dogs, and watercraft were disrupting birds that foraged and roosted at this site. Our objectives were to character- ize the frequency and sources of disturbance to birds and use this information to develop management recommendations to minimize the frequency of human disturbances to birds at Napatree Point. We conducted 106 hours of observation from May through August in 2013. Of 211 flight responses, the most common sources of disturbance to birds were beach walkers (57.8%), motorboats (8.5%), kayaks (8.5%), bird watchers (7.6%), and an- glers (6.2%). Birds typically flushed when pedestrians (e.g., walkers, bird watchers, and anglers) were within 39 ± 24 m (median ± inter-quartile range) and watercraft (e.g., mo- torboats and kayaks) were within 38 ± 33 m. Flight responses were positively correlated with the number of people on the beach and the number of boats in the area. Disturbances to birds peaked in July when human visitation was highest. Using a spatially explicit den- sity map of flight-initiation distance vectors, we identified the most important area to set as a buffer zone for human access if managers seek to reduce the frequency of human dis- turbances to birds at Napatree Point.

Anthropogenic modification of catchments and river flow can significantly alter estuarine habitats, hydrology and nutrient delivery with implications for fisheries productivity. The Coorong estuary at the terminus of Australia’s River Murray supports an economically important fishery as well as being recognised internationally as a critical site for migratory birds. Salinity near the Murray Mouth varies between fresh and marine depending upon river flow, but the Coorong becomes increasingly saline along its 120 km length. Freshwater flow to the Coorong is naturally variable but has significantly reduced by extraction for irrigated agriculture and domestic use upstream. Extreme drought from 2000 to 2010 and over-allocation of water resources resulted in the cessation of freshwater flow to the Coorong, significantly increasing salinity. During this period the diversity and abundance of organisms in the Coorong declined which reduced food web complexity. During lower flows the system generally becomes less p...

Free fatty acids hold dual roles during infection, serving to modulate the host immune response while also functioning directly as antimicrobials. Of particular importance are the long chain polyunsaturated fatty acids, which are not commonly found in bacterial organisms, that have been proposed to have antibacterial roles. Arachidonic acid (AA) is a highly abundant long chain polyunsaturated fatty acid and we examined its effect upon Streptococcus pneumoniae. Here, we observed that in a murine model of S. pneumoniae infection the concentration of AA significantly increases in the blood. The impact of AA stress upon the pathogen was then assessed by a combination of biochemical, biophysical and microbiological assays. In vitro bacterial growth and intra-macrophage survival assays revealed that AA has detrimental effects on pneumococcal fitness. Subsequent analyses demonstrated that AA exerts antimicrobial activity via insertion into the pneumococcal membrane, although this did not increase the susceptibility of the bacterium to antibiotic, oxidative or metal ion stress. Transcriptomic profiling showed that AA treatment also resulted in a dramatic down-regulation of the genes involved in fatty acid biosynthesis, in addition to impacts on other metabolic processes, such as carbon-source utilization. Hence, these data reveal that AA has two distinct mechanisms of perturbing the pneumococcal membrane composition. Collectively, this work provides a molecular basis for the antimicrobial contribution of AA to combat pneumococcal infections. Refereed/Peer-reviewed

Pleural infection/empyema is common and its incidence continues to rise. Streptococcus pneumoniae is the commonest bacterial cause of empyema in children and among the commonest in adults. The mesothelium represents the first line of defense against invading microorganisms, but mesothelial cell responses to common empyema pathogens, including S. pneumoniae, have seldom been studied. We assessed mesothelial cell viability in vitro following exposure to common empyema pathogens. Clinical isolates of S. pneumoniae from 25 patients with invasive pneumococcal disease and three reference strains were tested. All potently induced death of cultured mesothelial cells (MeT-5A) in a dose- and time-dependent manner (>90% at 107 CFU/mL after 24 hours). No significant mesothelial cell killing was observed when cells were co-cultured with Staphylococcus aureus, Streptococcus sanguinis and Streptococcus milleri group bacteria. S. pneumoniae induced mesothelial cell death via secretory product(s) as cytotoxicity could be: i) reproduced using conditioned media derived from S. pneumoniae and ii) in transwell studies when the bacteria and mesothelial cells were separated. No excess cell death was seen when heat-killed S. pneumoniae were used. Pneumolysin, a cytolytic S. pneumoniae toxin, induced cell death in a time- and dose-dependent manner. S. pneumoniae lacking the pneumolysin gene (D39 ΔPLY strain) failed to kill mesothelial cells compared to wild type (D39) controls, confirming the necessity of pneumolysin in D39-induced mesothelial cell death. However, pneumolysin gene mutation in other S. pneumoniae strains (TIGR4, ST3 and ST23F) only partly abolished their cytotoxic effects, suggesting different strains may induce cell death via different mechanisms.

Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however, its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of the unfolded protein response (UPR), is a bona fide substrate of the Sel1L-Hrd1 ERAD complex. ERAD-mediated IRE1α degradation occurs under basal conditions in a BiP-dependent manner, requires both the intramembrane hydrophilic residues of IRE1α and the lectin protein OS9, and is attenuated by ER stress. ERAD deficiency causes IRE1α protein stabilization, accumulation and mild activation both in vitro and in vivo. Although enterocyte-specific Sel1L-knockout mice (Sel1L(ΔIEC)) are viable and seem normal, they are highly susceptible to experimental colitis and inflammation-associated dysbiosis, in an IRE1α-dependent but CHOP-independent manner. Hence, Sel1L-Hrd1 ERAD serves a distinct, essential function in restraint of IRE1α signalling in vivo by managing its protein turnover.

Background/aims There are documented barriers to work experience in medicine for children from low socio-economic backgrounds. This can adversely affect their chances of successfully obtaining a medical school place Academic ability is not the only factor medical school applicants should demonstrate, but they should also understand what a career in medicine involves NHS Lanarkshire currently provides a three-day in-hospital work experience programme to local schoolchildren. In order to provide further opportunities to experience medical careers first hand, a widening access simulation initiative has been developed. The emphasis is on exposure to simulated surgical activities in hands-on theatre environment, as this is challenging to access during traditional work experience programmes due to safety concerns for both schoolchildren and patients resulting in a passive observational experience. This work describes the evolution of 9Fix the Fracture9, a simulated widening access orthopaedic theatre session. Project description Creating a simulated orthopaedic theatre that is both safe for schoolchildren to gain hand-on experience yet provides an authentic surgical experience is challenging. Due to health and safety concerns, real orthopaedic equipment could not be used due to the likelihood of injury. The same issues arose when considering what material to best use to simulate broken bones, as training sessions with postgraduates had previously used broken animal bones, but again this poses a health and safety issue to schoolchildren. Results We created an authentic theatre environment using basic surgical equipment and a simulation mannequin whose plastic legs had been replaced by legs filled with polyester and an open fracture created by inserting pre-broken plastic bones with pre-drilled holes. Moulage was used to create an open fracture skin effect. Surgical drapes were used to cover the leg for authenticity. The bones were repaired by the schoolchildren using a toy model construction system that consisted of reusable metal strips, plates and bolts. This minimised any risk of injury and providing the opportunity to 9repair9 a fracture in an authentic orthopaedic theatre environment. Conclusions and recommendations Using simulation in widening access is a novel approach to work experience supplementation, but is one where the only boundary is the imagination. This descriptive project has shown that there are low cost, reusable options to simulating theatre environments. This enables schoolchildren to get hands on theatre experience, augmenting their work experience and providing them with an inspirational and memorable experience to take forward to medical school applications and interviews. Reference . Nicholls, G.

Empyema is defined by the presence of bacteria and/or pus in pleural effusions. However, the biology of bacteria within human pleural fluid has not been studied. Streptococcus pneumoniae is the most common cause of pediatric and frequent cause of adult empyema. We investigated whether S. pneumoniae can proliferate within human pleural fluid and if growth is affected by the cellular content of the fluid and/or characteristics of pneumococcal surface proteins. Invasive S. pneumoniae isolates (n = 24) and reference strain recovered from human blood or empyema were inoculated (1.5×106CFU/mL) into sterile human malignant pleural fluid samples (n = 11). All S. pneumoniae (n = 25) strains proliferated rapidly, increasing by a median of 3009 (IQR 1063-9846) from baseline at 24hrs in all pleural effusions tested. Proliferation was greater than in commercial pneumococcal culture media and concentrations were maintained for 48hrs without autolysis. A similar magnitude of proliferation was observed in pleural fluid before and after removal of its cellular content, p = 0.728. S. pneumoniae (D39 strain) wild-type, and derivatives (n = 12), each with mutation(s) in a different gene required for full virulence were inoculated into human pleural fluid (n = 8). S. pneumoniae with pneumococcal surface antigen A (ΔpsaA) mutation failed to grow (2207-fold lower than wild-type), p

Pertussis-like toxins are secreted by several bacterial pathogens during infection. They belong to the AB5 virulence factors, which bind to glycans on host cell membranes for internalization. Host cell recognition and internalization are mediated by toxin B subunits sharing a unique pentameric ring-like assembly. Although the role of pertussis toxin in whooping cough is well-established, pertussis-like toxins produced by other bacteria are less studied, and their mechanisms of action are unclear. Here, we report that some extra-intestinal Escherichia coli pathogens (i.e. those that reside in the gut but can spread to other bodily locations) encode a pertussis-like toxin that inhibits mammalian cell growth in vitro. We found that this protein, EcPlt, is related to toxins produced by both nontyphoidal and typhoidal Salmonella serovars. Pertussis-like toxins are secreted as disulfide-bonded heterohexamers in which the catalytic ADP-ribosyltransferase subunit is activated when exposed to the reducing environment in mammalian cells. We found here that the reduced EcPlt exhibits large structural rearrangements associated with its activation. We noted that inhibitory residues tethered within the NAD+-binding site by an intramolecular disulfide in the oxidized state dissociate upon the reduction and enable loop restructuring to form the nucleotide-binding site. Surprisingly, although pertussis toxin targets a cysteine residue within the α subunit of inhibitory trimeric G-proteins, we observed that activated EcPlt toxin modifies a proximal lysine/asparagine residue instead. In conclusion, our results reveal the molecular mechanism underpinning activation of pertussis-like toxins, and we also identified differences in host target specificity.

Shiga toxigenicEscherichia coli(STEC) are important food-borne pathogens and a major cause of haemorrhagic colitis and haemolytic-uremic syndrome (HUS) worldwide. In 1995 a severe HUS outbreak in Adelaide occurred. A recent genomic analysis of STEC O111:H-strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes although the structure of the Stx2-converting prophages could not be fully resolved due to the fragmented assembly. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) long read sequencing to characterise the complete epigenome (genome and methylome) of 95JB1 and 95NR1. Using long reads we completely resolved the structure of two, tandemly inserted, stx2-converting phage in 95NR1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5’-CTGCm6AG-3’) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethalyted in the 95JB1, including at least 180 potential promoter regions that could explain regulatory differences between the strains. We identified a Type IIG methyltransferase encoded in both genomes in association with three additional genes in an operon-like arrangement. IS1203mediated disruption of this operon in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the enormous potential of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

To determine whether children with autism spectrum disorder (ASD) would tolerate an acupressure/acupuncture intervention and whether parents would adhere to a twice-weekly, 8-week intervention protocol. Second, to further understand best measures to use to capture impact of intervention on behavioral and regulatory functions.This is an observational pilot study with pre-, mid-, and postintervention measures.The intervention was carried out in a private practice office in a large metropolitan area.A total of 10 children of ages 3-10 years with ASD and one of their parents participated.A total of 16 biweekly treatment sessions of acupressure and/or acupuncture were carried out by a licensed acupuncturist, and a daily home-based acupressure intervention was carried out by a parent.Attendance, tolerance of intervention, parent compliance with home program, and parent compliance in completing daily diary and five standardized measures of behavioral and regulatory functions pre-, mid-, and postintervention were recorded.The 10 children in this observational study, collectively, tolerated the intervention and parents adhered to the 16 sessions, biweekly protocol, and home protocol, as well as completing daily diary and five standardized measures at three different time intervals. The five measurements appeared to be sensitive to behavioral and regulatory functions that may improve with this type of intervention.The results of this observational pilot study suggest that acupressure/acupuncture is a feasible intervention for children with ASD that merits rigorous evaluation through a randomized controlled trial.

Subtilase cytotoxin (SubAB) of Escherichia coli is an AB5 class bacterial toxin. The pentameric B subunit (SubB) binds the cellular carbohydrate receptor, α2–3-linked N-glycolylneuraminic acid (Neu5Gc). Neu5Gc is not expressed on normal human cells, but is expressed by cancer cells. Elevated Neu5Gc has been observed in breast, ovarian, prostate, colon and lung cancer. The presence of Neu5Gc is prognostically important, and correlates with invasiveness, metastasis and tumour grade. Neu5Gc binding by SubB suggests that it may have utility as a diagnostic tool for the detection Neu5Gc tumor antigens. Native SubB has 20-fold less binding to N-acetlylneuraminic acid (Neu5Ac); over 30-fold less if the Neu5Gc linkage was changed from α2–3 to α2–6. Using molecular modeling approaches, site directed mutations were made to reduce the α2–3 $${\boldsymbol{\gg }}$$ ≫ α2–6-linkage preference, while maintaining or enhancing the selectivity of SubB for Neu5Gc over Neu5Ac. Surface plasmon resonance and glycan array analysis showed that the SubBΔS106/ΔT107 mutant displayed improved specificity towards Neu5Gc and bound to α2–6-linked Neu5Gc. SubBΔS106/ΔT107 could discriminate NeuGc- over Neu5Ac-glycoconjugates in ELISA. These data suggest that improved SubB mutants offer a new tool for the testing of biological samples, particularly serum and other fluids from individuals with cancer or suspected of having cancer.

Endoplasmic reticulum (ER) stress that leads to the accumulation of misfolded proteins in the ER initiates the unfolded protein response (UPR). This homeostatic response activates signaling pathways that seek to reinstate a proper ER protein folding balance or induce apoptosis if ER stress persists. Recently, we and others identified human FICD (Filamentation induced by cyclic AMP domain-containing protein), an enzyme with adenylyltransferase (aka AMPylation) activity, as a new UPR target. Here, we demonstrate that FICD is functionally linked to the UPR, as evidenced by the finding that the adenylyltransferase activity of the protein induces ER stress, while FICD silencing increases sensitivity to ER stress. We identify BiP, an abundant ER chaperone and key regulator of the UPR, as the main substrate of FICD AMPylation in ER-derived microsomes, further emphasizing close functional connection of FICD to the UPR and in line with recent reports that AMPylation inactivates BiP. Notably, BiP overexpression increased the levels of BiP AMPylation as well as FICD auto-AMPylation, suggesting a homeostatic response that balances the pool of active BiP to modulate its functions in protein folding as well as UPR signaling. Finally, we show that overexpressed FICD forms a disulfide-bonded homo-dimer through Cys51 and Cys75 and demonstrate that mutation of these two cysteines in the context of a hyperactive FICD mutant leads to increased BiP AMPylation. This latter finding opens up the possibility that FICD activity is redox regulated and closely connected with ER redox homeostasis.

The pneumococcus is the world's foremost respiratory pathogen, but the mechanisms allowing this pathogen to proceed from initial asymptomatic colonization to invasive disease are poorly understood. We have examined the early stages of invasive pneumococcal disease (IPD) by comparing host transcriptional responses to an invasive strain and a noninvasive strain of serotype 1 Streptococcus pneumoniae in the mouse lung. While the two strains were present in equal numbers in the lung 6 h after intranasal challenge, only the invasive strain (strain 1861) had invaded the pleural cavity at that time point; this correlated with subsequent development of bacteremia in mice challenged with strain 1861 but not the noninvasive strain (strain 1). Progression beyond the lung was associated with stronger induction of the type I interferon (IFN-I) response in the lung at 6 h. Suppression of the IFN-I response through administration of neutralizing antibody to IFNAR1 (the receptor for type I interferons) led to significantly reduced invasion of the pleural cavity by strain 1861 at 6 h postchallenge. Our data suggest that strong induction of the IFN-I response is a key factor in early progression of invasive serotype 1 strain 1861 beyond the lung during development of IPD.

Subtilase cytotoxin (SubAB) is the prototype of a recently emerged family of AB5 cytotoxins produced by Shiga-toxigenic Escherichia coli (STEC). Its mechanism of action involves highly specific A-subunit-mediated proteolytic cleavage of the essential endoplasmic reticulum (ER) chaperone BiP. Our previous in vivo studies showed that intraperitoneal injection of purified SubAB causes a major redistribution of leukocytes and elevated leukocyte apoptosis in mice, as well as profound splenic atrophy. In the current study, we investigated selected chemokine and proinflammatory cytokine responses to treatment with SubAB, a nontoxic derivative (SubA A272 B), or Shiga toxin 2 (Stx2) in human macrophage (U937), brain microvascular endothelial (HBMEC), and colonic epithelial (HCT-8) cell lines, at the levels of secreted protein, cell-associated protein, and gene expression. Stx2 treatment upregulated expression of chemokines and cytokines at both the protein and mRNA levels. In contrast, SubAB induced significant decreases in secreted interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in all three tested cell lines and a significant decrease in secreted IL-6 in HBMECs. The downregulation of secreted chemokines or cytokines was not observed in SubA A272 B-treated cells, indicating a requirement for BiP cleavage. The downregulation of secreted chemokines and cytokines by SubAB was not reflected at the mRNA and cell-associated protein levels, suggesting a SubAB-induced export defect.

Early-onset torsion dystonia (EOTD) is a neurological disorder characterized by involuntary and sustained muscle contractions that can lead to paralysis and abnormal posture. EOTD is associated with the deletion of a glutamate (ΔE) in torsinA, an endoplasmic reticulum (ER) resident AAA(+) ATPase. To date, the effect of ΔE on torsinA and the reason that this mutation results in EOTD are unclear. Moreover, there are no specific therapeutic options to treat EOTD. To define the underlying biochemical defects associated with torsinAΔE and to uncover factors that might be targeted to offset defects associated with torsinAΔE, we developed a yeast torsinA expression system and tested the roles of ER chaperones in mediating the folding and stability of torsinA and torsinAΔE. We discovered that the ER lumenal Hsp70, BiP, an associated Hsp40, Scj1, and a nucleotide exchange factor, Lhs1, stabilize torsinA and torsinAΔE. BiP also maintained torsinA and torsinAΔE solubility. Mutations predicted to compromise specific torsinA functional motifs showed a synthetic interaction with the ΔE mutation and destabilized torsinAΔE, suggesting that the ΔE mutation predisposes torsinA to defects in the presence of secondary insults. In this case, BiP was required for torsinAΔE degradation, consistent with data that specific chaperones exhibit either pro-degradative or pro-folding activities. Finally, using two independent approaches, we established that BiP stabilizes torsinA and torsinAΔE in mammalian cells. Together, these data define BiP as the first identified torsinA chaperone, and treatments that modulate BiP might improve symptoms associated with EOTD.

Biologists now use a variety of survey platforms to assess the spatial distribution and abundance of marine birds, yet few attempts have been made to integrate data from multiple survey platforms to improve model accuracy or precision. We used density surface models (DSMs) to incorporate data from two survey platforms to predict the distribution and abundance of a diving marine bird, the Common Loon (Gavia immer). We conducted strip transect surveys from a multiengine, fixed-wing aircraft and line surveys from a 28 m ship during winter 2009–2010 in a 3,800 km2 study area off the coast of Rhode Island, USA. We accounted for imperfect detection and availability bias due to Common Loon diving behavior. We incorporated spatially explicit environmental covariates (water depth and latitude) to provide predictions of the spatial distribution and abundance of wintering Common Loons. The combined-platform DSM estimated the highest Common Loon densities (>20 individuals km−2) in nearshore waters

Assessments of possible adverse effects of transmitters on marked individuals is an important component of individual-based tracking studies, particularly for species that are listed under the U.S. Endangered Species Act. The breeding and post-breeding movements of adult Piping Plovers (Charadrius melodus) from the federally-threatened Atlantic Coast Population were studied by gluing miniature, 1.0-g, digital VHF radio-transmitters on their interscapular region. Mark-resighting data from 2015-2018 was used to estimate apparent survival rates for 289 adult Piping Plovers in Massachusetts, Rhode Island, and New Jersey in order to compare survival estimates between individuals with a transmitter attached and control individuals without a transmitter. Cormack–Jolly–Seber models were used for live-encounter data in a Bayesian framework to estimate apparent survival rates based on resightings of uniquely marked individuals. There was no evidence that mean apparent survival rates differed between adults with transmitters (0.756; 95% CI = 0.611 – 0.877) and without transmitters (0.673; 95% CI = 0.607 – 0.740). In addition, there was no evidence of differences in apparent survival rates between breeding location (state) or years. This study provides further evidence that radio transmitters glued temporarily to the inter-scapular region can be an effective tool to monitor local and regional movements of sensitive shorebirds, such as Piping Plovers.

The southern New England continental shelf is an important region for black scoters (Melanitta americana) during winter and migratory staging periods and a priority area for developing offshore wind energy facilities. However, little is known about the migration phenology and habitat use of black scoters in this portion of their range and this information is necessary to assess potential risks to black scoters during the marine spatial planning process. In this regional black scoter study over 2 winters, we used satellite telemetry and spatial modeling techniques to estimate migratory timing and length of stay, quantify winter homerangesize andsitefidelitybetweenwinters,examinekeyhabitatcharacteristics associated with core-use areas, and map relative probabilities of use across a 3,800-km 2 marine spatial planning area for 2 proposed offshorerenewableenergyfacilities.Black scoters spentnearly 5monthsinsouthernNew England,with wide variation among individuals in the size of winter utilization distributions (range 16-12,367km 2 ). Approximately 50% of the tagged birds returned to southern New England during the subsequent winter and had variable fidelity to core-use areas occupied the previous winter. During both winters, black scoter core- use areas were located closer to shore, at shallower water depths, with coarser sediment grain size and higher probability of hard-bottom occurrence relative to available areas. Resource selection functions classified the majority of a nearshore 5-turbine, 34-km 2 renewable energy zone off Block Island as high probability of use by black scoters, whereas an offshore 200-turbine, 667-km 2 federal lease block zone was classified as low to medium-low probability of selection. Wind energy facilities, such as the Block Island site, constructed in relatively shallow (

Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zinc-binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxy-terminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.

There are currently no offshore wind energy developments (OWEDs) in North America, although numerous OWEDs have been proposed along the Atlantic Coast. Development pressure has been a catalyst for marine spatial planning (MSP) to identify suitable areas for OWED. However, integrating complex ecological information to guide OWED siting remains a substantial challenge. We developed spatial distribution models of marine birds from aerial surveys that we conducted from 2010 to 2012 throughout a 3800 km 2 area off the coast of Rhode Island. For seven groups of marine birds, we constructed either a density surface model or a presence–absence model that incorporated relevant environmental covariates. We integrated our spatial models, along with uncertainty, using spatial conservation prioritization (SCP) software. This identified sites with high marine bird conservation priority that aided evaluation of proposed OWED sites. We found that shallow nearshore waters had the highest conservation priority overall, but we also detected key offshore areas of high priority. Hypothetical OWEDs placed in conservation priority areas significantly reduced the overall distribution of focal species. Currently proposed OWED sites are located in areas of relatively low conservation priority and so would not substantially reduce the overall distribution of marine birds. This SCP approach when combined with quantitative models of bird distribution given relevant environmental covariates provides a robust framework that satisfies the principles of ecosystem-based MSP. Thus, this combined SCP-distribution modeling framework should be extremely helpful to decision makers as they evaluate proposed siting locations of OWEDs in the context of a dynamic marine system.

The relative stability of divalent first-row transition metal ion complexes, as defined by the Irving-Williams series, poses a fundamental chemical challenge for selectivity in bacterial metal ion acquisition. Here we show that although the substrate-binding protein of Streptococcus pneumoniae, PsaA, is finely attuned to bind its physiological substrate manganese, it can also bind a broad range of other divalent transition metal cations. By combining high-resolution structural data, metal-binding assays and mutational analyses, we show that the inability of open-state PsaA to satisfy the preferred coordination chemistry of manganese enables the protein to undergo the conformational changes required for cargo release to the Psa permease. This is specific for manganese ions, whereas zinc ions remain bound to PsaA. Collectively, these findings suggest a new ligand binding and release mechanism for PsaA and related substrate-binding proteins that facilitate specificity for divalent cations during competition from zinc ions, which are more abundant in biological systems.