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Periodontitis is a chronic inflammatory disease driven by dysbiosis in subgingival microbial communities leading to increased abundance of a limited number of pathobionts, including Porphyromonas gingivalis and Treponema denticola. Oral health, particularly periodontitis, is a modifiable risk factor for Alzheimer disease (AD) pathogenesis, with components of both these bacteria identified in postmortem brains of persons with AD. Repeated oral inoculation of mice with P. gingivalis results in brain infiltration of bacterial products, increased inflammation, and induction of AD-like biomarkers. P. gingivalis displays synergistic virulence with T. denticola during periodontitis. The aim of the current study was to determine the ability of P. gingivalis and T. denticola, grown in physiologically relevant conditions, individually and in combination, to induce AD-like pathology following chronic oral inoculation of female mice over 12 weeks. P. gingivalis alone significantly increased all 7 brain pathologies examined: neuronal damage, activation of astrocytes and microglia, expression of inflammatory cytokines interleukin 1β (IL-1β) and interleukin 6 and production of amyloid-β plaques and hyperphosphorylated tau, in the hippocampus, cortex and midbrain, compared to control mice. T. denticola alone significantly increased neuronal damage, activation of astrocytes and microglia, and expression of IL-1β, in the hippocampus, cortex and midbrain, compared to control mice. Coinoculation of P. gingivalis with T. denticola significantly increased activation of astrocytes and microglia in the hippocampus, cortex and midbrain, and increased production of hyperphosphorylated tau and IL-1β in the hippocampus only. The host brain response elicited by oral coinoculation was less than that elicited by each bacterium, suggesting coinoculation was less pathogenic., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Periodontitis is a common chronic inflammatory disease, affecting approximately 19% of the global adult population. A relationship between periodontal disease and Alzheimer disease has long been recognized, and recent evidence has been uncovered to link these 2 diseases mechanistically. Periodontitis is caused by dysbiosis in the subgingival plaque microbiome, with a pronounced shift in the oral microbiota from one consisting primarily of Gram-positive aerobic bacteria to one predominated by Gram-negative anaerobes, such as Porphyromonas gingivalis. A common phenomenon shared by all bacteria is the release of membrane vesicles to facilitate biomolecule delivery across long distances. In particular, the vesicles released by P gingivalis and other oral pathogens have been found to transport bacterial components across the blood-brain barrier, initiating the physiologic changes involved in Alzheimer disease. In this review, we summarize recent data that support the relationship between vesicles secreted by periodontal pathogens to Alzheimer disease pathology., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Variants in ABCA7, a member of the ABC transporter superfamily, have been associated with increased risk for developing late onset Alzheimer's disease (LOAD)., Methods: CRISPR-Cas9 was used to generate an Abca7 V1613M variant in mice, modeling the homologous human ABCA7 V1599M variant, and extensive characterization was performed., Results: Abca7 V1613M microglia show differential gene expression profiles upon lipopolysaccharide challenge and increased phagocytic capacity. Homozygous Abca7 V1613M mice display elevated circulating cholesterol and altered brain lipid composition. When crossed with 5xFAD mice, homozygous Abca7 V1613M mice display fewer Thioflavin S-positive plaques, decreased amyloid beta (Aβ) peptides, and altered amyloid precursor protein processing and trafficking. They also exhibit reduced Aβ-associated inflammation, gliosis, and neuronal damage., Discussion: Overall, homozygosity for the Abca7 V1613M variant influences phagocytosis, response to inflammation, lipid metabolism, Aβ pathology, and neuronal damage in mice. This variant may confer a gain of function and offer a protective effect against Alzheimer's disease-related pathology., Highlights: ABCA7 recognized as a top 10 risk gene for developing Alzheimer's disease. Loss of function mutations result in increased risk for LOAD. V1613M variant reduces amyloid beta plaque burden in 5xFAD mice. V1613M variant modulates APP processing and trafficking in 5xFAD mice. V1613M variant reduces amyloid beta-associated damage in 5xFAD mice., (© 2024 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

The cause of Alzheimer's disease (AD), and the pathophysiological mechanisms involved, remain major unanswered questions in medical science. Oral bacteria, especially those species associated with chronic periodontitis and particularly Porphyromonas gingivalis , are being linked causally to AD pathophysiology in a subpopulation of susceptible individuals. P. gingivalis produces large amounts of proteolytic enzymes, haem and iron capture proteins, adhesins and internalins that are secreted and attached to the cell surface and concentrated onto outer membrane vesicles (OMVs). These enzymes and adhesive proteins have been shown to cause host tissue damage and stimulate inflammatory responses. The ecological and pathophysiological roles of P. gingivalis OMVs, their ability to disperse widely throughout the host and deliver functional proteins lead to the proposal that they may be the link between a P. gingivalis focal infection in the subgingivae during periodontitis and neurodegeneration in AD. P. gingivalis OMVs can cross the blood brain barrier and may accelerate AD-specific neuropathology by increasing neuroinflammation, plaque/tangle formation and dysregulation of iron homeostasis, thereby inducing ferroptosis leading to neuronal death and neurodegeneration.

Background: Atherosclerosis and consequent risk of cardiovascular events or mortality can be accurately assessed by quantifying coronary artery calcium score (CACS) derived from computed tomography. HMG-CoA-reductase inhibitors (statins) are the primary pharmacotherapy used to reduce cardiovascular events, yet there is growing data that support statin use may increase coronary calcification. We set out to determine the likelihood of severe CACS in the context of chronic statin therapy., Methods: We established a retrospective, case-control study of 1,181 U.S. veterans without coronary artery disease (CAD) from a single site, the Providence VA Medical Center. Duration of statin therapy for primary prevention was divided into 5-year categorical increments. The primary outcome was CACS derived from low-dose lung cancer screening computed tomography (LCSCT), stratified by CACs severity (none = 0; mild = 1-99; moderate = 100-399; and severe ≥400 AU). Statin duration of zero served as the referent control. Ordinal logistic regression analysis determined the association between duration of statin use and CACS categories. Proportional odds assumption was tested using likelihood ratio test. Atherosclerotic cardiovascular disease (ASCVD) risk score, body mass index, and CKD (glomerular filtration rate of <60 ml/min/1.73 m2) were included in the adjustment models., Results: The mean age of the study population was 64.7±7.2 years, and 706 (60%) patients were prescribed a statin at baseline. Duration of statin therapy was associated with greater odds of having increased CACS (>0-5 years, OR: 1.71 [CI: 1.34-2.18], p<0.001; >5-10 years, OR: 2.80 [CI: 2.01-3.90], p<0.001; >10 years, OR: 5.30 [CI: 3.23-8.70], p<0.001), and the relationship between statin duration and CACS remained significant after multivariate adjustment (>0-5 years, OR: 1.49 [CI: 1.16-1.92], p = 0.002; >5-10 years, OR: 2.38 [CI: 1.7-3.35], p<0.001; >10 years, OR: 4.48 [CI: 2.7-7.43], p<0.001)., Conclusions: Long-term use of statins is associated with increased likelihood of severe CACS in patients with significant smoking history. The use of CACS to interpret cardiovascular event risk may require adjustment in the context of chronic statin therapy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The Arg-specific gingipains of Porphyromonas gingivalis RgpA and RgpB have 97% identical sequences in their catalytic domains yet their propeptides are only 76% identical. RgpA isolates as a proteinase-adhesin complex (HRgpA) which hinders direct kinetic comparison of RgpA cat as a monomer with monomeric RgpB. We tested modifications of rgpA identifying a variant that enabled us to isolate histidine-tagged monomeric RgpA (rRgpAH). Kinetic comparisons between rRgpAH and RgpB used benzoyl-L-Arg-4-nitroanilide with and without cysteine and glycylglycine acceptor molecules. With no glycylglycine, values of K m , V max , k cat and k cat /K m for each enzyme were similar, but with glycylglycine K m decreased, V max increased and k cat increased ~ twofold for RgpB but ~ sixfold for rRgpAH. The k cat /K m for rRgpAH was unchanged whereas that of RgpB more than halved. Recombinant RgpA propeptide inhibited rRgpAH and RgpB with K i 13 nM and 15 nM K i respectively slightly more effectively than RgpB propeptide which inhibited rRgpAH and RgpB with K i 22 nM and 29 nM respectively (p < 0.0001); a result that may be attributable to the divergent propeptide sequences. Overall, the data for rRgpAH reflected observations previously made by others using HRgpA, indicating rRgpAH fidelity and confirming the first production and isolation of functional affinity tagged RgpA., (© 2023. The Author(s).)

Triggering receptor on myeloid cells 2 (TREM2) is an innate immune receptor, upregulated on the surface of microglia associated with amyloid plaques in Alzheimer's disease (AD). Individuals heterozygous for the R47H variant of TREM2 have greatly increased risk of developing AD. We examined the effects of wild-type (WT), R47H and knock-out (KO) of human TREM2 expression in three microglial cell systems. Addition of mouse BV-2 microglia expressing R47H TREM2 to primary mouse neuronal cultures caused neuronal loss, not observed with WT TREM2. Neuronal loss was prevented by using annexin V to block exposed phosphatidylserine, an eat-me signal and ligand of TREM2, suggesting loss was mediated by microglial phagocytosis of neurons exposing phosphatidylserine. Addition of human CHME-3 microglia expressing R47H TREM2 to LUHMES neuronal-like cells also caused loss compared to WT TREM2. Expression of R47H TREM2 in BV-2 and CHME-3 microglia increased their uptake of phosphatidylserine-beads and synaptosomes versus WT TREM2. Human iPSC-derived microglia with heterozygous R47H TREM2 had increased phagocytosis of synaptosomes vs common-variant TREM2. Additionally, phosphatidylserine liposomes increased activation of human iPSC-derived microglia expressing homozygous R47H TREM2 versus common-variant TREM2. Finally, overexpression of TREM2 in CHME-3 microglia caused increased expression of cystatin F, a cysteine protease inhibitor, and knock-down of cystatin F increased CHME-3 uptake of phosphatidylserine-beads. Together, these data suggest that R47H TREM2 may increase AD risk by increasing phagocytosis of synapses and neurons via greater activation by phosphatidylserine and that WT TREM2 may decrease microglial phagocytosis of synapses and neurons via cystatin F., (© 2022 The Authors. GLIA published by Wiley Periodicals LLC.)

The human oral microbiome is becoming recognized as playing roles in health and disease well beyond the oral cavity over the lifetime of the individual. The oral microbiome is hypothesized to result from specific colonization events followed by a reproducible and ordered development of complex bacterial communities. Colonization events, proliferation, succession and subsequent community development are dependent on a range of host and environmental factors, most notably the neonate diet. It is now becoming apparent that early childhood and prenatal influences can have long term effects on the development of human oral microbiomes. In this review, the temporal development of the infant human oral microbiome is examined, with the effects of prenatal and postnatal influences and the roles of specific bacteria. Dietary and environmental factors, especially breastfeeding, have a significant influence on the development of the infant oral microbiome. The evidence available regarding the roles and functions of early colonizing bacteria is still limited, and gaps in knowledge where further research is needed to elucidate these specific roles in relation to health and disease still exist.

Background: Human microbiomes assemble in an ordered, reproducible manner yet there is limited information about early colonisation and development of bacterial communities that constitute the oral microbiome., Aim: The aim of this study was to determine the effect of exposure to breastmilk on assembly of the infant oral microbiome during the first 20 months of life., Methods: The oral microbiomes of 39 infants, 13 who were never breastfed and 26 who were breastfed for more than 10 months, from the longitudinal VicGeneration birth cohort study, were determined at four ages. In total, 519 bacterial taxa were identified and quantified in saliva by sequencing the V4 region of the bacterial 16S rRNA genes., Results: There were significant differences in the development of the oral microbiomes of never breastfed and breastfed infants. Bacterial diversity was significantly higher in never breastfed infants at 2 months, due largely to an increased abundance of Veillonella and species from the Bacteroidetes phylum compared with breastfed infants., Conclusion: These differences likely reflect breastmilk playing a prebiotic role in selection of early-colonising, health-associated oral bacteria, such as the Streptococcus mitis group. The microbiomes of both groups became more heterogenous following the introduction of solid foods., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM β-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins.

Calreticulin is a chaperone, normally found in the endoplasmic reticulum, but can be released by macrophages into the extracellular medium. It is also found in cerebrospinal fluid bound to amyloid beta (Aβ). We investigated whether brain cells release calreticulin, and whether extracellular calreticulin had any effects on microglia and neurons relevant to neuroinflammation and neurodegeneration. We found that microglia release nanomolar levels of calreticulin when inflammatory-activated with lipopolysaccharide, when endoplasmic reticulum stress was induced by tunicamycin, or when cell death was induced by staurosporine, and that neurons release calreticulin when crushed. Addition of nanomolar levels of extracellular calreticulin was found to chemoattract microglia, and activate microglia to release cytokines TNF-α, IL-6 and IL-1β, as well as chemokine (C-C motif) ligand 2. Calreticulin blocked Aβ fibrillization and modified Aβ oligomerization, as measured by thioflavin T fluorescence and transmission electron microscopy. Extracellular calreticulin also altered microglial morphology and proliferation, and prevented Aβ-induced neuronal loss in primary neuron-glial cultures. Thus, calreticulin is released by microglia and neurons, and acts: as an alarmin to recruit and activate microglia, as an extracellular chaperone to prevent Aβ aggregation, and as a neuroprotectant against Aβ neurotoxicity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Reid, Kitchener, Butler, Cockram and Brown.)

Aim: Periodontitis is a site-specific, chronic disease treated by non-surgical debridement of subgingival plaque. We aimed to determine the microbiome of sites that did not respond to this treatment (NR) compared with paired good responding (GR) sites before and after treatment., Materials and Methods: In a longitudinal cohort study, clinical parameters of disease and biological samples were taken prior to and 3 months after treatment. Twelve NR sites from six participants were paired with GR sites within the same participant. Subgingival plaque samples were subjected to bacterial community analysis using 16S rRNA gene sequencing., Results: There were no significant differences in clinical parameters and microbial communities at baseline between GR and NR sites. Bacterial communities in deep pockets were dominated by a small number of species, notably Porphyromonas gingivalis and Treponema denticola . In NR sites three months after treatment there was no significant change in bacterial composition whilst there was a collapse in the abundance of pathobionts in GR sites., Conclusion: NR sites were not identifiable prior to treatment by clinical or microbiological parameters. Treatment failed to disrupt pathogenic bacterial community in NR sites. Targeted suppression of particular species should be considered to initiate community collapse and aid disease resolution., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

The type IX secretion system (T9SS) transports cargo proteins through the outer membrane of Bacteroidetes and attaches them to the cell surface for functions including pathogenesis, gliding motility, and degradation of carbon sources. The T9SS comprises at least 20 different proteins and includes several modules: the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, the outer membrane Sov translocon, and the cell attachment complex. However, the spatial organization of these modules is unknown. We have characterized the protein interactome of the Sov translocon in Porphyromonas gingivalis and identified Sov-PorV-PorA as well as Sov-PorW-PorN-PorK to be novel networks. PorW also interacted with PGN&#95;1783 (PorD), which was required for maximum secretion efficiency. The identification of PorW as the missing link completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore. IMPORTANCE The T9SS is a newly identified protein secretion system of the Fibrobacteres - Chlorobi - Bacteroidetes superphylum used by pathogens associated with diseases of humans, fish, and poultry for the secretion and cell surface attachment of virulence factors. The T9SS comprises three known modules: (i) the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, (ii) the outer membrane Sov translocon, and (iii) the cell surface attachment complex. The spatial organization and interaction of these modules have been a mystery. Here, we describe the protein interactome of the Sov translocon in the human pathogen Porphyromonas gingivalis and have identified PorW as the missing link which bridges PorN with Sov and so completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing, for the first time, a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore.

Background: Reduction in glucocorticoid exposure is the primary benefit of new biologic treatments in severe asthma, but there is currently no evidence that reduction in glucocorticoid exposure corresponds to a proportionate reduction in associated toxicity., Objectives: To use the validated Glucocorticoid Toxicity Index (GTI) to assess change in glucocorticoid toxicity after 12 months treatment with mepolizumab, and compare toxicity change to glucocorticoid reduction and change in patient-reported outcome measures (PROMs)., Methods: A longitudinal, real-world prospective cohort of 101 consecutive patients with severe asthma commenced on mepolizumab in a specialist UK regional severe asthma clinic. GTI toxicity assessment, cumulative glucocorticoid exposure and PROMs were recorded on commencing mepolizumab (V1), and after 12 months treatment (V2)., Results: There was significant reduction in oral glucocorticoid exposure (V1 median 4280 mg prednisolone per year (interquartile range 3083-5475 mg) versus V2 2450 mg prednisolone per year (1243-3360 mg), p<0.001). Substantial improvements in individual toxicities were observed, but did not correlate with oral glucocorticoid reduction. Mean±sd GTI aggregate improvement score (AIS) was -35.7±57.8 with a wide range in toxicity change at individual patient level (AIS range -165 to +130); 70% (71 out of 101) had a reduction in toxicity (AIS <0); 3% (three out of 101) had no change (AIS=0); and 27% (27 out of 101) an increase in overall toxicity. 62% (62 out of 101) of patients met the AIS minimally clinically important difference of ≤-10, but AIS did not correlate with glucocorticoid reduction or change in PROMs., Conclusion: Mepolizumab resulted in substantial oral glucocorticoid reduction, but this did not correlate with reduction in oral glucocorticoid toxicity, which varies widely at the individual patient level. Oral glucocorticoid reduction is not a comprehensive measure of response to mepolizumab., Competing Interests: Conflict of interest: P.J. McDowell reports personal fees for lectures from GlaxoSmithKline, outside the submitted work. Conflict of interest: J.H. Stone reports that the Massachusetts General Hospital owns the copyright to the Glucocorticoid Toxicity Index. Conflict of interest: Y. Zhang has nothing to disclose. Conflict of interest: K. Honeyford has nothing to disclose. Conflict of interest: L. Dunn has nothing to disclose. Conflict of interest: R.J. Logan has nothing to disclose. Conflict of interest: L.P.A. McGarvey reports personal fees for lectures and advisory board work from GlaxoSmithKline, personal fees for lectures and advisory board work, and non-financial support from Chiesi, outside the submitted work. Conflict of interest: C.A. Butler reports other (conference registrations) from Chiesi and Napp, personal fees for lectures from GlaxoSmithKline, personal fees for lectures and advisory board work from AstraZeneca, outside the submitted work. Conflict of interest: L.G. Heaney reports non-financial support from GlaxoSmithKline, during the conduct of the study; grants from Amgen, AstraZeneca, Medimmune, Janssen, Novartis, Roche/Genentech, GlaxoSmithKline plc., Boehringer Ingelheim, Aerocrine and Vitalograph, other (support for advisory board work/lectures) from Novartis, Hoffman la Roche/Genentech Inc, Evelo Biosciences, Sanofi, GlaxoSmithKline, AstraZeneca, Teva, Theravance and Circassia, other (travel funding support to attend meetings; institution renumerated) from AstraZeneca, Boehringer Ingelheim, Chiesi, GSK and Napp Pharmaceutical, other (project grant funding) from Medimmune, Novartis UK, Roche/Genentech and GlaxoSmithKline plc., other (clinical trials for which his institution was remunerated) from AstraZeneca, GlaxoSmithKline, Schering Plough, Synairgen, Novartis and Roche/Genentech, outside the submitted work., (Copyright ©The authors 2022.)

Coronary artery disease caused by atherosclerosis is a major cause of morbidity and mortality around the world. Data from preclinical and clinical studies support the belief that atherosclerosis is an inflammatory disease that is mediated by innate and adaptive immune signaling mechanisms. This review sought to highlight the role of Rac-mediated inflammatory signaling in the mechanisms driving atherosclerotic calcification. In addition, current clinical treatment strategies that are related to targeting hypercholesterolemia as a critical risk factor for atherosclerotic vascular disease are addressed in relation to the effects on Rac immune signaling and the implications for the future of targeting immune responses in the treatment of calcific atherosclerosis.

Rationale: The utility of fractional exhaled nitric oxide ( F ENO ) suppression (FeNOSuppT) to identify non-adherence to inhaled corticosteroid (ICS) treatment has previously been reported, but whether it can predict clinical outcome remains unclear., Objectives: We examined the utility of FeNOSuppT in prediction of progression to biologic agents or discharge from specialist care., Methods: FeNOSuppT was measured at home using remote monitoring technology of inhaler use alongside daily F ENO measurement over 7 days. Long-term clinical outcomes in terms of progression to biologic agent or discharge from specialist care were compared for non-suppressors and suppressors., Measurements and Main Results: Of the 162 subjects, 135 successfully completed the test with 81 (60%) positive F ENO suppression tests. Subjects with a negative FeNOSuppT were more likely to proceed to biologic therapy (39 of 54 patients, 72%) compared to those with a positive FeNOSuppT (35 of 81 patients, 43%, p=0.001). In subjects with a positive FeNOSuppT, predictors of progression to biologic therapy included higher dose of maintenance steroid at initial assessment and prior intensive care unit admission. These subjects had a significant rise in F ENO between post-suppression test and follow-up (median, 33 (IQR 25-55) versus 71 (IQR 24-114); p=0.009), which was not explained by altered corticosteroid dose., Conclusions: A negative FeNOSuppT correlates with progression to biologic therapy. A positive FeNOSuppT, with subsequent maintenance of "optimised" F ENO , predicts a subgroup of patients in whom asthma control is preserved with adherence to high-dose ICS/long-acting β2 agonist and who can be discharged from specialist care., Competing Interests: Conflict of interest: C.A. Butler reports Vitalograph provided support for the INCA device used to assess adherence during the conduct of the study; and fees for lectures from AstraZeneca and GlaxoSmithKline, and conference support from NAPP and Chiesi, outside the submitted work. Conflict of interest: A.J. McMichael has nothing to disclose. Conflict of interest: K. Honeyford has nothing to disclose. Conflict of interest: L. Wright has nothing to disclose. Conflict of interest: J. Logan has nothing to disclose. Conflict of interest: J. Holmes has nothing to disclose. Conflict of interest: J. Busby has nothing to disclose. Conflict of interest: C.E. Hanratty has nothing to disclose. Conflict of interest: F. Yang has nothing to disclose. Conflict of interest: S.J. Smith has nothing to disclose. Conflict of interest: K. Murray has nothing to disclose. Conflict of interest: R. Chaudhuri reports advisory board meeting fees, a research grant, speaker fees and conference travel support from AstraZeneca; advisory board meeting and speaker fees from GSK, Teva, Novartis and Chiesi; and conference travel support from Napp Pharmaceuticals, all outside the submitted work. Conflict of interest: L.G. Heaney reports sponsorship for attending international scientific meetings from AstraZeneca, Boehringer Ingelheim, Chiesi, GSK and Napp Pharmaceuticals; personal fees for lectures and advisory boards from Novartis, Hoffman la Roche/Genentech Inc., Sanofi, Evelo Biosciences, GlaxoSmithKline, AstraZeneca, Teva, Theravance and Circassia; and institutional grant funding from Medimmune, Novartis UK, Roche/Genentech Inc. and GlaxoSmithKline; and is Academic Lead for the Medical Research Council Stratified Medicine UK Consortium in Severe Asthma, which involves Industrial Partnerships with Amgen, Genentech/Hoffman la Roche, AstraZeneca, Medimmune, GlaxoSmithKline, Aerocrine and Vitalograph, all outside the submitted work., (Copyright ©The authors 2021.)

There is growing evidence that excessive microglial phagocytosis of neurons and synapses contributes to multiple brain pathologies. RNA-seq and genome-wide association (GWAS) studies have linked multiple phagocytic genes to neurodegenerative diseases, and knock-out of phagocytic genes has been found to protect against neurodegeneration in animal models, suggesting that excessive microglial phagocytosis contributes to neurodegeneration. Here, we review recent evidence that microglial phagocytosis of live neurons and synapses causes neurodegeneration in animal models of Alzheimer's disease and other tauopathies, Parkinson's disease, frontotemporal dementias, multiple sclerosis, retinal degeneration and neurodegeneration induced by ischaemia, infection or ageing. We also review factors regulating microglial phagocytosis of neurons, including: nucleotides, frackalkine, phosphatidylserine, calreticulin, UDP, CD47, sialylation, complement, galectin-3, Apolipoprotein E, phagocytic receptors, Siglec receptors, cytokines, microglial epigenetics and expression profile. Some of these factors may be potential treatment targets to prevent neurodegeneration mediated by excessive microglial phagocytosis of live neurons and synapses., (© 2021 International Society for Neurochemistry.)

CD33 is a Siglec (sialic acid-binding immunoglobulin-type lectin) receptor on microglia. Human CD33 can be alternatively spliced into two isoforms: the long isoform (CD33M) and a shorter isoform (CD33m) that lacks the sialic acid-binding site. CD33m appears to protect against Alzheimer's disease; however, it remains unclear how. To investigate potential mechanisms by which CD33m may confer protection, we expressed the CD33m and CD33M isoforms of human CD33 in mouse BV-2 and human CHME3 microglial cells and assessed microglia functions. In the BV-2 cells, CD33M inhibited microglial phagocytosis of beads, synapses, debris and dead cells, while CD33m increased phagocytosis of beads, debris and cells. RNAi knockdown of the endogenous mouse CD33 increased phagocytosis and prevented CD33m's (but not CD33M's) effect on phagocytosis. CD33M increased cell attachment but inhibited cell proliferation, while CD33m did the opposite. We also found that CD33M inhibited cell migration. In human CHME3 cells, CD33M increased cell attachment, but inhibited phagocytosis, proliferation and migration, whereas CD33m did the opposite. We conclude that CD33M inhibits microglial phagocytosis, inhibits migration and increases adhesion, while CD33m increases phagocytosis, proliferation and inhibits adhesion. Thus, CD33m might protect against Alzheimer's disease by increasing microglial proliferation, movement and phagocytosis of debris and dead cells., (© 2021 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Porphyromonas gingivalis , a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface, enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to anionic lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled in situ for correct secretion and A-LPS modification of cargo proteins, combinations of nonpigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Reacquisition of pigmentation occurred only between an A-LPS mutant and a T9SS mutant, which coincided with A-LPS modification of cargo proteins detected by Western blotting and coimmunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis , leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two-strain crosses involving only the five T9SS OM component mutants produced black pigmentation, implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation, and hence, a more ordered temporal in situ assembly of T9SS components may be required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host. IMPORTANCE Porphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions., (Copyright © 2021 American Society for Microbiology.)

Introduction: Research has highlighted relationships between the micro-organisms that inhabit our gastrointestinal tract (oral and gut microbiota) with host mood and gastrointestinal functioning. Mental health disorders and functional gastrointestinal disorders co-occur at high rates, although the mechanisms underlying these associations remain unclear. The Bugs and Brains Study aims to investigate complex relationships between anxiety/depression and irritable bowel syndrome (IBS) in two ways. First, its primary component will compare the gut and oral microbiota in females with anxiety/depression and/or IBS relative to controls, and investigate underlying physiological, endocrine and immune factors, as well as associations with diet and psychosocial factors. In an ancillary component, the study will also investigate gastrointestinal and mental health symptoms in a larger sample, and explore relationships with diet, exercise, oral health, substance use, medical history, early life adversity and psychosocial factors., Methods and Analysis: The Bugs and Brains Study aims to recruit 160 females to the primary component: (1) 40 controls; (2) 40 participants with a depressive/anxiety disorder, but no IBS; (3) 40 participants with IBS, but no depressive/anxiety disorder and (4) 40 participants with both depressive/anxiety disorder and IBS. Participation is completed within 1 month, and involves comprehensive questionnaires, anthropometrics, a diagnostic clinical interview, collection of two saliva samples, and stool, urine and hair samples. This study aims to use a systems biology approach to characterise oral and gut microbial composition and function using 16S rRNA gene sequencing and nuclear MR spectroscopy. As part of the ancillary component, it will collect questionnaire data from 1000 participants aged 18-40 years, capturing mental health, gastrointestinal health, oral health, diet and psychosocial factors., Ethics and Dissemination: Approval was granted by the University of Melbourne Human Research Ethics Committee (#1749221). All participants voluntarily provided informed consent. Results will be published in peer-reviewed journals and presented at scientific conferences., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose of Review: Despite increased clinician awareness, nonadherence to inhaled corticosteroid treatment presents a major challenge to successful asthma management and risks inappropriate treatment escalation, particularly in severe disease. In patients with Type-2 mediated biology, fractional exhaled nitric oxide (FeNO) has a role in assessment and monitoring of adherence to inhaled corticosteroids., Recent Findings: Asthmatic patients with elevated FeNO are at an increased risk of exacerbation. High FeNO is often secondary to suboptimal adherence to inhaled corticosteroid treatment, whether intentional or nonintentional. FENO-suppression can 'unmask' underlying adherence issues and is a useful test in the presence of Type-2 biology in the 'difficult-to-control' asthma population. Identification of nonadherence can improve asthma control and prevent inappropriate commencement of costly biologic therapies., Summary: Assessment of adherence and FeNO response to monitored inhaled corticosteroid in Type-2 biomarker high asthmatic individuals may prevent unnecessary escalation to biologic therapy. Establishing an 'optimised' FeNO may alert clinicians to the possibility of underlying nonadherence at future clinical assessments., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Glucocorticoid (GC)-associated morbidity in severe asthma (SA) is well recognized but varies in individual patients; systematic measurement of GC toxicity is important to measure improvement with steroid-sparing monoclonal antibodies., Objective: To describe for the first time individual patient GC toxicity in steroid-dependent SA using the Glucocorticoid Toxicity Index (GTI)., Methods: An observational consecutive patient cohort study was performed at a UK Regional SA Specialist clinic for systematic assessment of GC-associated morbidity using the GTI in routine clinical care. GTI was correlated with commonly used patient-reported outcome measures. An approach to GTI scoring, calculation of minimal clinically important difference (MCID), and development of digital GTI application in routine clinical care are described., Results: All patients had significant oral GC exposure (cumulative prednisolone/prior year, 4280 [3083, 5475] mg) with wide distribution of toxicity in individual patients (mean GTI score, 177.5 [73.7]). GTI score had only modest correlation with recent prednisolone exposure: maintenance prednisolone dose (rho = 0.26, P = .01), cumulative exposure/prior year (rho = 0.38, P < .001), and GC boosts/prior year (rho = 0.25, P = .01). GTI toxicity demonstrated stronger associations with asthma-related quality of life (mini-Asthma Quality of Life Questionnaire [mini-AQLQ] r = -0.50, P < .001 and St. George's Respiratory Questionnaire r = 0.42, P < .001). GTI MCID was calculated as 10 points. Multiple linear regression demonstrated that age and mini-AQLQ were strongest predictors of GC toxicity., Conclusions: The GTI is a useful tool to systematically capture and quantify GC toxicity at the individual patient level. GC toxicity varies widely between individual patients with SA and correlated only modestly with GC exposure over the preceding year. Age and mini-AQLQ are better predictors of GC toxicity. The GTI and MCID will facilitate assessment of individual SA response to steroid-sparing agents in clinical trials and routine care., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background:   Porphyromonas gingivalis  and  Treponema denticola  are proteolytic periodontopathogens that co-localize in polymicrobial subgingival plaque biofilms, display  in vitro  growth symbiosis and synergistic virulence in animal models of disease. These symbioses are underpinned by a range of metabolic interactions including cooperative hydrolysis of glycine-containing peptides to produce free glycine, which  T. denticola  uses as a major energy and carbon source., Objective:  To characterize the  P. gingivalis  gene products essential for these interactions. Methods: The  P. gingivalis  transcriptome exposed to cell-free  T. denticola  conditioned medium was determined using RNA-seq.  P. gingivalis  proteases potentially involved in hydrolysis of glycine-containing peptides were identified using a bioinformatics approach., Results:  One hundred and thirty-twogenes displayed differential expression, with the pattern of gene expression consistent with succinate cross-feeding from  T. denticola  to  P. gingivalis  and metabolic shifts in the  P. gingivalis  folate-mediated one carbon superpathway. Interestingly, no  P. gingivalis  proteases were significantly up-regulated. Three  P. gingivalis  proteases were identified as candidates and inactivated to determine their role in the release of free glycine.  P. gingivalis  PG0753 and PG1788 but not PG1605 are involved in the hydrolysis of glycine-containing peptides, making free glycine available for  T. denticola  utilization., Conclusion:  Collectively these metabolic interactions help to partition resources and engage synergistic interactions between these two species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Chronic periodontitis has a polymicrobial biofilm etiology and interactions between key oral bacterial species, such as Porphyromonas gingivalis and Treponema denticola contribute to disease progression. P. gingivalis and T. denticola are co-localized in subgingival plaque and have been previously shown to exhibit strong synergy in growth, biofilm formation and virulence in an animal model of disease. The motility of T. denticola , although not considered as a classic virulence factor, may be involved in synergistic biofilm development between P. gingivalis and T. denticola . We determined the role of T. denticola motility in polymicrobial biofilm development using an optimized transformation protocol to produce two T. denticola mutants targeting the motility machinery. These deletion mutants were non-motile and lacked the gene encoding the flagellar hook protein of the periplasmic flagella (Δ flgE ) or a component of the stator motor that drives the flagella (Δ motB ). The specificity of these gene deletions was determined by whole genome sequencing. Quantitative proteomic analyses of mutant strains revealed that the specific inactivation of the motility-associated gene, motB , had effects beyond motility. There were 64 and 326 proteins that changed in abundance in the Δ flgE and Δ motB mutants, respectively. In the Δ flgE mutant, motility-associated proteins showed the most significant change in abundance confirming the phenotype change for the mutant was related to motility. However, the inactivation of motB as well as stopping motility also upregulated cellular stress responses in the mutant indicating pleiotropic effects of the mutation. T. denticola wild-type and P. gingivalis displayed synergistic biofilm development with a 2-fold higher biomass of the dual-species biofilms than the sum of the monospecies biofilms. Inactivation of T. denticola flgE and motB reduced this synergy. A 5-fold reduction in dual-species biofilm biomass was found with the motility-specific Δ flgE mutant suggesting that T. denticola periplasmic flagella are essential in synergistic biofilm formation with P. gingivalis ., (Copyright © 2019 Ng, Slakeski, Butler, Veith, Chen, Liu, Hoffmann, Dashper and Reynolds.)

Objectives: To determine if chewing gum containing casein phosphopeptide stabilised amorphous calcium phosphate (CPP-ACP) promoted an increase in the abundance of Streptococcus sanguinis and other species associated with dental health in supragingival plaque in a clinical study., Materials and Methods: Nineteen participants were recruited for a three-leg cross-over, randomised, controlled clinical trial. Participants chewed a sugar-free gum with or without CPP-ACP six times daily for 20 min over two weeks. The study also involved no gum chewing (no gum) for the same two week period. Participants were randomly assigned to one of the test gums or no gum for each intervention period. Participants abstained from oral hygiene and had washout periods of two weeks between intervention periods. After each intervention period, supragingival plaque was collected and analysed for bacterial composition by sequencing the V4 variable region of the 16S rRNA gene. Data were analysed using a linear mixed model., Results: The CPP-ACP gum intervention produced a significant (p < 0.01) increase in the proportions of S. sanguinis (112%), as well as the commensal species Rothia dentocariosa (127%), Corynebacterium durum (80%) and Streptococcus mitis (55%) when compared with the no gum intervention. All the species that were promoted by the CPP-ACP gum are known to possess one or both of the alkali-producing enzymes arginine deiminase and nitrate reductase., Conclusion: This clinical study demonstrated that chewing a sugar-free gum containing CPP-ACP promoted prebiosis by significantly increasing the proportion of S. sanguinis and other health-associated bacterial species in supragingival plaque., Clinical Significance: Regular chewing of CPP-ACP sugar-free gum increases the proportions of health-associated commensal species in supragingival plaque to promote prebiosis and oral homeostasis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Dental caries is associated with plaque dysbiosis, leading to an increase in the proportions of acidogenic and aciduric bacteria at the expense of alkali-generating commensal species. Stannous fluoride (SnF 2 ) slows the progression of caries by remineralization of early lesions but has also been suggested to inhibit glycolysis of aciduric bacteria. Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) promotes fluoride remineralization by acting as a salivary biomimetic that releases bioavailable calcium and phosphate ions, and the peptide complex has also been suggested to modify plaque composition. We developed a polymicrobial biofilm model of caries using 6 bacterial species representative of supragingival plaque that were cultured on sound human enamel and pulsed with sucrose 4 times a day to produce a high cariogenic challenge. We used this model to explore the mechanisms of action of SnF 2 and CPP-ACP. Bacterial species in the biofilms were enumerated with 16S rRNA gene sequence analyses, and mineral loss and lesion formation were determined in the enamel directly under the polymicrobial biofilms via transverse microradiography. The model tested the twice-daily addition of SnF 2 , CPP-ACP, or both. SnF 2 treatment reduced demineralization by 50% and had a slight effect on the composition of the polymicrobial biofilm. CPP-ACP treatment caused a similar inhibition of enamel demineralization (50%), a decrease in Actinomyces naeslundii and Lactobacillus casei abundance, and an increase in Streptococcus sanguinis and Fusobacterium nucleatum abundance in the polymicrobial biofilm. A combination of SnF 2 and CPP-ACP resulted in a greater suppression of the acidogenic and aciduric bacteria and a significant 72% inhibition of enamel demineralization.

The aim of this study was to explore how people's sexual history affects their attractiveness. Using an Internet survey, 188 participants rated their willingness to engage in a relationship with a hypothetical individual with a specified number of past sexual partners, ranging from 0 to 60+. The effect of past partner number was very large. Average willingness ratings initially rose as past partner number rose, but then fell dramatically. For short-term relationships, men were more willing than women to get involved (although the difference was not large). For long-term relationships, in contrast, there was virtually no sex difference. Thus, contrary to the idea that male promiscuity is tolerated but female promiscuity is not, both sexes expressed equal reluctance to get involved with someone with an overly extensive sexual history. Finally, participants with an unrestricted sociosexual orientation (high SO participants) were more tolerant than low SO participants of prospective mates with higher numbers of past sexual partners but were also less tolerant of prospective mates with low numbers of past sexual partners.

Severe asthma represents a major unmet clinical need; understanding the pathophysiology is essential for the development of new therapies. Using microarray analysis, we previously found three immunological clusters in asthma: Th2-high, Th17-high, and Th2/17-low. Although new therapies are emerging for Th2-high disease, identifying molecular pathways in Th2-low disease remains an important goal. Further interrogation of our previously described microarray dataset revealed upregulation of gene expression for carcinoembryonic Ag cell adhesion molecule (CEACAM) family members in the bronchi of patients with severe asthma. Our aim was therefore to explore the distribution and cellular localization of CEACAM6 using immunohistochemistry on bronchial biopsy tissue obtained from patients with mild-to-severe asthma and healthy control subjects. Human bronchial epithelial cells were used to investigate cytokine and corticosteroid in vitro regulation of CEACAM6 gene expression. CEACAM6 protein expression in bronchial biopsies was increased in airway epithelial cells and lamina propria inflammatory cells in severe asthma compared with healthy control subjects. CEACAM6 in the lamina propria was localized to neutrophils predominantly. Neutrophil density in the bronchial mucosa was similar across health and the spectrum of asthma severity, but the percentage of neutrophils expressing CEACAM6 was significantly increased in severe asthma, suggesting the presence of an altered neutrophil phenotype. CEACAM6 gene expression in cultured epithelial cells was upregulated by wounding and neutrophil elastase. In summary, CEACAM6 expression is increased in severe asthma and primarily associated with airway epithelial cells and tissue neutrophils. CEACAM6 may contribute to the pathology of treatment-resistant asthma via neutrophil and airway epithelial cell-dependent pathways., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA&#95;C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS., Competing Interests: The authors have declared that no competing interests exist.

Severe asthma represents a major unmet clinical need. Eosinophilic inflammation persists in the airways of many patients with uncontrolled asthma, despite high-dose inhaled corticosteroid therapy. Suppressors of cytokine signalling (SOCS) are a family of molecules involved in the regulation of cytokine signalling via inhibition of the Janus kinase-signal transducers and activators of transcription pathway. We examined SOCS expression in the airways of asthma patients and investigated whether this is associated with persistent eosinophilia.Healthy controls, mild/moderate asthmatics and severe asthmatics were studied. Whole genome expression profiling, quantitative PCR and immunohistochemical analysis were used to examine expression of SOCS1, SOCS2 and SOCS3 in bronchial biopsies. Bronchial epithelial cells were utilised to examine the role of SOCS1 in regulating interleukin (IL)-13 signalling in vitroSOCS1 gene expression was significantly lower in the airways of severe asthmatics compared with mild/moderate asthmatics, and was inversely associated with airway eosinophilia and other measures of T-helper type 2 (Th2) inflammation. Immunohistochemistry demonstrated SOCS1 was predominantly localised to the bronchial epithelium. SOCS1 overexpression inhibited IL-13-mediated chemokine ligand (CCL) 26 (eotaxin-3) mRNA expression in bronchial epithelial cells.Severe asthma patients with persistent airway eosinophilia and Th2 inflammation have reduced airway epithelial SOCS1 expression. SOCS1 inhibits epithelial IL-13 signalling, supporting its key role in regulating Th2-driven eosinophilia in severe asthma., (Copyright ©ERS 2016.)

As health care increasingly emphasizes high value, the terms "population health" and "patient-centered care" have become common, but their application is less clear. Patient-centered care encourages using data to optimize care for an individual. Population health offers a framework to consider how to efficiently and effectively manage a condition for a population, how prevention affects large groups, and the specific distribution of a given disorder. Integrating both concepts into practice can facilitate required outcome-measure reporting and potentially improve patient outcomes. Clinical practice guidelines and appropriate use criteria are examples of reconciliation of these topics. By embracing attempts to decrease variation in treating musculoskeletal disorders while personalizing delivery to individual patients, surgeons may benefit from the improvement of both efficiency and patient experience., (Copyright © 2016 by The Journal of Bone and Joint Surgery, Incorporated.)

Chronic periodontitis has a polymicrobial biofilm aetiology. Polymicrobial biofilms are complex, dynamic microbial communities formed by two or more bacterial species that are important for the persistence and proliferation of participating microbes in the environment. Interspecies adherence, which often involves bacterial surface-associated molecules, and communications are essential in the spatial and temporal development of a polymicrobial biofilm, which in turn is necessary for the overall fitness of a well-organized multispecies biofilm community. In the oral cavity, interactions between key oral bacterial species, including Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, are essential for the progression of chronic periodontitis. In vivo, P. gingivalis and T. denticola are frequently found to co-exist in deep periodontal pockets and have been co-localized to the superficial layers of subgingival plaque as microcolony blooms adjacent to the pocket epithelium, suggesting possible interbacterial interactions that contribute towards disease. The motility and chemotactic ability of T. denticola, although not considered as classic virulence factors, are likely to be important in the synergistic biofilm formation with P. gingivalis. In vitro, P. gingivalis and T. denticola display a symbiotic relationship in nutrient utilization and growth promotion. Together these data suggest there is an intimate relationship between these two species that has evolved to enhance their survival and virulence., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

PgMntR is a predicted member of the DtxR family of transcriptional repressors responsive to manganese in the anaerobic periodontal pathogen Porphyromonas gingivalis. Our bioinformatic analyses predicted that PgMntR had divalent metal binding site(s) with elements of both manganous and ferrous ion specificity and that PgMntR has unusual twin C-terminal FeoA domains. We produced recombinant PgMntR and four variants to probe the specificity of metal binding and its impact on protein structure and DNA binding. PgMntR dimerised in the absence of a divalent transition metal cation. PgMntR bound three Mn(II) per monomer with an overall dissociation constant Kd 2.0 x 10(-11) M at pH 7.5. PgMntR also bound two Fe(II) with distinct binding affinities, Kd1 2.5 x 10(-10) M and Kd2 ≤ 6.0 x 10(-8) M at pH 6.8. Two of the metal binding sites may form a binuclear centre with two bound Mn2+ being bridged by Cys108 but this centre provided only one site for Fe2+. Binding of Fe2+ or Mn2+ did not have a marked effect on the PgMntR secondary structure. Apo-PgMntR had a distinct affinity for the promoter region of the gene encoding the only known P. gingivalis manganese transporter, FB2. Mn2+ increased the DNA binding affinity of PgMntR whilst Fe2+ destabilised the protein-DNA complex in vitro. PgMntR did not bind the promoter DNA of the gene encoding the characterised iron transporter FB1. The C-terminal FeoA domain was shown to be essential for PgMntR structure/function, as its removal caused the introduction of an intramolecular disulfide bond and abolished the binding of Mn2+ and DNA. These data indicate that PgMntR is a novel member of the DtxR family that may function as a transcriptional repressor switch to specifically regulate manganese transport and homeostasis in an iron-dependent manner.

Porphyromonas gingivalis is a Gram-negative anaerobe considered to be a keystone pathogen in the development of the bacterial-associated inflammatory oral disease chronic periodontitis. Although post-translational modifications (PTMs) of proteins are commonly found to modify protein function in eukaryotes and prokaryotes, PTMs such as lysine acetylation have not been examined in P. gingivalis. Lysine acetylation is the addition of an acetyl group to a lysine which removes this amino acid's positive charge and can induce changes in a protein's secondary structure and reactivity. A proteomics based approach combining immune-affinity enrichment with high sensitivity Orbitrap mass spectrometry identified 130 lysine acetylated peptides from 92 P. gingivalis proteins. The majority of these peptides (71) were attributed to 45 proteins with predicted metabolic activity; these proteins could be mapped to several P. gingivalis metabolic pathways where enzymes catalysing sequential reactions within the same pathway were often found acetylated. In particular, the catabolic pathways of complex anaerobic fermentation of amino acids to produce energy had 12 enzymes lysine acetylated. The results suggest that lysine acetylation may be an important mechanism in metabolic regulation in P. gingivalis, which is vital for P. gingivalis survival and adaptation of its metabolism throughout infection. Statement of significance. Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The ability of the pathogen to induce dysbiosis and disease is related to an array of specific virulence factors and metabolic regulation that enables the bacterium to proliferate in an inflamed periodontal pocket. The mechanisms P. gingivalis uses to adapt to a changing and hostile environment are poorly understood and here we show, for the first time, that enzymes of critical metabolic pathways for energy production in this bacterium were acetylated on certain lysine residues. These enzymes were often found catalysing sequential reactions within the same catabolic pathway. The results suggest that lysine acetylation is an important mechanism of metabolic regulation in P. gingivalis vital for its adaptation and proliferation to produce disease., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Increasing evidence suggests that asthma is a heterogeneous disorder regulated by distinct molecular mechanisms. In a cross-sectional study of asthmatics of varying severity (n = 51), endobronchial tissue gene expression analysis revealed three major patient clusters: TH2-high, TH17-high, and TH2/17-low. TH2-high and TH17-high patterns were mutually exclusive in individual patient samples, and their gene signatures were inversely correlated and differentially regulated by interleukin-13 (IL-13) and IL-17A. To understand this dichotomous pattern of T helper 2 (TH2) and TH17 signatures, we investigated the potential of type 2 cytokine suppression in promoting TH17 responses in a preclinical model of allergen-induced asthma. Neutralization of IL-4 and/or IL-13 resulted in increased TH17 cells and neutrophilic inflammation in the lung. However, neutralization of IL-13 and IL-17 protected mice from eosinophilia, mucus hyperplasia, and airway hyperreactivity and abolished the neutrophilic inflammation, suggesting that combination therapies targeting both pathways may maximize therapeutic efficacy across a patient population comprising both TH2 and TH17 endotypes., (Copyright © 2015, American Association for the Advancement of Science.)

Porphyromonas gingivalis is a Gram-negative pathogen associated with the biofilm-mediated disease chronic periodontitis. P. gingivalis biofilm formation is dependent on environmental heme for which P. gingivalis has an obligate requirement as it is unable to synthesize protoporphyrin IX de novo, hence P. gingivalis transports iron and heme liberated from the human host. Homeostasis of a variety of transition metal ions is often mediated in Gram-negative bacteria at the transcriptional level by members of the Ferric Uptake Regulator (Fur) superfamily. P. gingivalis has a single predicted Fur superfamily orthologue which we have designated Har (heme associated regulator). Recombinant Har formed dimers in the presence of Zn2+ and bound one hemin molecule per monomer with high affinity (Kd of 0.23 µM). The binding of hemin resulted in conformational changes of Zn(II)Har and residue 97Cys was involved in hemin binding as part of a predicted -97C-98P-99L- hemin binding motif. The expression of 35 genes was down-regulated and 9 up-regulated in a Har mutant (ECR455) relative to wild-type. Twenty six of the down-regulated genes were previously found to be up-regulated in P. gingivalis grown as a biofilm and 11 were up-regulated under hemin limitation. A truncated Zn(II)Har bound the promoter region of dnaA (PGN&#95;0001), one of the up-regulated genes in the ECR455 mutant. This binding decreased as hemin concentration increased which was consistent with gene expression being regulated by hemin availability. ECR455 formed significantly less biofilm than the wild-type and unlike wild-type biofilm formation was independent of hemin availability. P. gingivalis possesses a hemin-binding Fur orthologue that regulates hemin-dependent biofilm formation.

Background: The airway epithelium is exposed to a range of physical and chemical irritants in the environment that are known to trigger asthma. Transient receptor potential (TRP) cation channels play a central role in sensory responses to noxious physical and chemical stimuli. Recent genetic evidence suggests an involvement of transient receptor potential vanilloid 1 (TRPV1), one member of the vanilloid subfamily of TRP channels, in the pathophysiology of asthma. The functional expression of TRPV1 on airway epithelium has yet to be elucidated., Objective: In this study we examined the molecular, functional, and immunohistochemical expression of TRPV1 in asthmatic and healthy airways., Methods: Bronchial biopsy specimens and bronchial brushings were obtained from healthy volunteers (n = 18), patients with mild-to-moderate asthma (n = 24), and patients with refractory asthma (n = 22). Cultured primary bronchial epithelial cells from patients with mild asthma (n = 4), nonasthmatic coughers (n = 4), and healthy subjects (n = 4) were studied to investigate the functional role of TRPV1., Results: Quantitative immunohistochemistry revealed significantly more TRPV1 expression in asthmatic patients compared with healthy subjects, with the greatest expression in patients with refractory asthma (P = .001). PCR and Western blotting analysis confirmed gene and protein expression of TRPV1 in cultured primary bronchial epithelial cells. Patch-clamp electrophysiology directly confirmed functional TRPV1 expression in all 3 groups. In functional assays the TRPV1 agonist capsaicin induced dose-dependent IL-8 release, which could be blocked by the antagonist capsazepine. Reduction of external pH from 7.4 to 6.4 activated a capsazepine-sensitive outwardly rectifying membrane current., Conclusions: Functional TRPV1 channels are present in the human airway epithelium and overexpressed in the airways of patients with refractory asthma. These channels might represent a novel therapeutic target for the treatment of uncontrolled asthma., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Burkholderia cepacia complex organisms are important transmissible pathogens found in cystic fibrosis (CF) patients. In recent years, the rates of cross-infection of epidemic strains have declined due to effective infection control efforts. However, cases of sporadic B. cepacia complex infection continue to occur in some centers. The acquisition pathways and clinical outcomes of sporadic B. cepacia complex infection are unclear. We sought to determine the patient clinical characteristics, outcomes, incidence, and genotypic relatedness for all cases of B. cepacia complex infection at two CF centers. We also sought to study the external conditions that influence the acquisition of infection. From 2001 to 2011, 67 individual organisms were cultured from the respiratory samples of 64 patients. Sixty-five percent of the patients were adults, in whom chronic infections were more common (68%) (P = 0.006). The incidence of B. cepacia complex infection increased by a mean of 12% (95% confidence interval [CI], 3 to 23%) per year. The rates of transplantation and death were similar in the incident cases who developed chronic infection compared to those in patients with chronic Pseudomonas aeruginosa infection. Multilocus sequence typing revealed 50 individual strains from 65 isolates. Overall, 85% of the patients were infected with unique strains, suggesting sporadic acquisition of infection. The yearly incidence of nonepidemic B. cepacia complex infection was positively correlated with the amount of rainfall in the two sites examined: subtropical Brisbane (r = 0.65, P = 0.031) and tropical Townsville (r = 0.82, P = 0.002). This study demonstrates that despite strict cohort segregation, new cases of unrelated B. cepacia complex infection continue to occur. These data also support an environmental origin of infection and suggest that climate conditions may be associated with the acquisition of B. cepacia complex infections.

Background: The relationships between physicians and hospitals are viewed as central to the proposition of delivering high-quality health care at a sustainable cost. Over the last two decades, major changes in the scope, breadth, and complexities of these relationships have emerged. Despite understanding the need for physician-hospital alignment, identification and understanding the incentives and drivers of alignment prove challenging., Questions/purposes: Our review identifies the primary drivers of physician alignment with hospitals from both the physician and hospital perspectives. Further, we assess the drivers more specific to motivating orthopaedic surgeons to align with hospitals., Methods: We performed a comprehensive literature review from 1992 to March 2012 to evaluate published studies and opinions on the issues surrounding physician-hospital alignment. Literature searches were performed in both MEDLINE(®) and Health Business™ Elite., Results: Available literature identifies economic and regulatory shifts in health care and cultural factors as primary drivers of physician-hospital alignment. Specific to orthopaedics, factors driving alignment include the profitability of orthopaedic service lines, the expense of implants, and issues surrounding ambulatory surgery centers and other ancillary services., Conclusions: Evolving healthcare delivery and payment reforms promote increased collaboration between physicians and hospitals. While economic incentives and increasing regulatory demands provide the strongest drivers, cultural changes including physician leadership and changing expectations of work-life balance must be considered when pursuing successful alignment models. Physicians and hospitals view each other as critical to achieving lower-cost, higher-quality health care.

Rationale: Upregulation of glucocorticoid receptor β (GRβ) has been implicated in steroid resistance in severe asthma, although previous studies are conflicting. GRβ has been proposed as a dominant negative isoform of glucocorticoid receptor α (GRα) but it has also been suggested that GRβ can cause steroid resistance via reduced expression of histone deacetylase 2 (HDAC2), a key regulator of steroid responsiveness in the airway., Objectives: To examine GRβ, GRα, HDAC1 and HDAC2 expression at transcript and protein levels in bronchial biopsies from a large series of patients with severe asthma, and to compare the findings with those of patients with mild to moderate asthma and healthy volunteers., Methods: Bronchoscopic study in two UK centres with real-time PCR and immunohistochemistry performed on biopsies, western blotting of bronchial epithelial cells and immunoprecipitation with anti-GRβ antibody., Measurements and Main Results: Protein and mRNA expression for GRα and HDAC2 did not differ between groups. GRβ mRNA was detected in only 13 of 73 samples (seven patients with severe asthma), however immunohistochemistry showed widespread epithelial staining in all groups. Western blotting of bronchial epithelial cells with GRβ antibody detected an additional 'cross-reacting' protein, identified as clathrin. HDAC1 expression was increased in patients with severe asthma compared with healthy volunteers., Conclusions: GRβ mRNA is expressed at low levels in a minority of patients with severe asthma. HDAC1 and HDAC2 expression was not downregulated in severe asthma. These data do not support upregulated GRβ and resultant reduced HDAC expression as the principal mechanism of steroid resistance in severe asthma. Conflicting GRβ literature may be explained in part by clathrin cross-reactivity with commercial antibodies.

Background: Public reporting of patient health outcomes offers the potential to incentivize quality improvement by fostering increased accountability among providers. Voluntary reporting of risk-adjusted outcomes in cardiac surgery, for example, is viewed as a "watershed event" in healthcare accountability. However, public reporting of outcomes, cost, and quality information in orthopaedic surgery remains limited by comparison, attributable in part to the lack of standard assessment methods and metrics, provider fear of inadequate adjustment of health outcomes for patient characteristics (risk adjustment), and historically weak market demand for this type of information., Questions/purposes: We review the origins of public reporting of outcomes in surgical care, identify existing initiatives specific to orthopaedics, outline the challenges and opportunities, and propose recommendations for public reporting of orthopaedic outcomes., Methods: We performed a comprehensive review of the literature through a bibliographic search of MEDLINE and Google Scholar databases from January 1990 to December 2010 to identify articles related to public reporting of surgical outcomes., Results: Orthopaedic-specific quality reporting efforts include the early FDA adverse event reporting MedWatch program and the involvement of surgeons in the Physician Quality Reporting Initiative. Issues that require more work include balancing different stakeholder perspectives on quality reporting measures and methods, defining accountability and attribution for outcomes, and appropriately risk-adjusting outcomes., Conclusions: Given the current limitations associated with public reporting of quality and cost in orthopaedic surgery, valuable contributions can be made in developing specialty-specific evidence-based performance measures. We believe through leadership and involvement in policy formulation and development, orthopaedic surgeons are best equipped to accurately and comprehensively inform the quality reporting process and its application to improve the delivery and outcomes of orthopaedic care.

Synthesis of the largest cytochrome c oxidase (CcO) subunit, Cox1, on yeast mitochondrial ribosomes is coupled to assembly of CcO. The translational activator Mss51 is sequestered in early assembly intermediate complexes by an interaction with Cox14 that depends on the presence of newly synthesized Cox1. If CcO assembly is prevented, the level of Mss51 available for translational activation is reduced. We deleted the C-terminal 11 or 15 residues of Cox1 by site-directed mutagenesis of mtDNA. Although these deletions did not prevent respiratory growth of yeast, they eliminated the assembly-feedback control of Cox1 synthesis. Furthermore, these deletions reduced the strength of the Mss51-Cox14 interaction as detected by co-immunoprecipitation, confirming the importance of the Cox1 C-terminal residues for Mss51 sequestration. We surveyed a panel of mutations that block CcO assembly for the strength of their effect on Cox1 synthesis, both by pulse labeling and expression of the ARG8(m) reporter fused to COX1. Deletion of the nuclear gene encoding Cox6, one of the first subunits to be added to assembling CcO, caused the most severe reduction in Cox1 synthesis. Deletion of the C-terminal 15 amino acids of Cox1 increased Cox1 synthesis in the presence of each of these mutations, except pet54. Our data suggest a novel activity of Pet54 required for normal synthesis of Cox1 that is independent of the Cox1 C-terminal end.

This paper presents a simple analytical circuit-like model to study the transmission of electromagnetic waves through stacked two-dimensional (2-D) conducting meshes. When possible the application of this methodology is very convenient since it provides a straightforward rationale to understand the physical mechanisms behind measured and computed transmission spectra of complex geometries. Also, the disposal of closed-form expressions for the circuit parameters makes the computation effort required by this approach almost negligible. The model is tested by proper comparison with previously obtained numerical and experimental results. The experimental results are explained in terms of the behavior of a finite number of strongly coupled Fabry-Pérot resonators. The number of transmission peaks within a transmission band is equal to the number of resonators. The approximate resonance frequencies of the first and last transmission peaks are obtained from the analysis of an infinite structure of periodically stacked resonators, along with the analytical expressions for the lower and upper limits of the pass-band based on the circuit model.

Functional interactions of the translational activator Mss51 with both the mitochondrially encoded COX1 mRNA 5'-untranslated region and with newly synthesized unassembled Cox1 protein suggest that it has a key role in coupling Cox1 synthesis with assembly of cytochrome c oxidase. Mss51 is present at levels that are near rate limiting for expression of a reporter gene inserted at COX1 in mitochondrial DNA, and a substantial fraction of Mss51 is associated with Cox1 protein in assembly intermediates. Thus, sequestration of Mss51 in assembly intermediates could limit Cox1 synthesis in wild type, and account for the reduced Cox1 synthesis caused by most yeast mutations that block assembly. Mss51 does not stably interact with newly synthesized Cox1 in a mutant lacking Cox14, suggesting that the failure of nuclear cox14 mutants to decrease Cox1 synthesis, despite their inability to assemble cytochrome c oxidase, is due to a failure to sequester Mss51. The physical interaction between Mss51 and Cox14 is dependent upon Cox1 synthesis, indicating dynamic assembly of early cytochrome c oxidase intermediates nucleated by Cox1. Regulation of COX1 mRNA translation by Mss51 seems to be an example of a homeostatic mechanism in which a positive effector of gene expression interacts with the product it regulates in a posttranslational assembly process.

Members of the Oxa1/YidC/Alb3 family of protein translocases are essential for assembly of energy-transducing membrane complexes. In Saccharomyces cerevisiae, Oxa1 and its paralog, Cox18, are required for assembly of Cox2, a mitochondrially encoded subunit of cytochrome c oxidase. Oxa1 is known to be required for cotranslational export of the Cox2 N-terminal domain across the inner mitochondrial membrane, while Cox18 is known to be required for post-translational export of the Cox2 C-tail domain. We find that overexpression of Oxa1 does not compensate for the absence of Cox18 at the level of respiratory growth. However, it does promote some translocation of the Cox2 C-tail domain across the inner membrane and causes increased accumulation of Cox2, which remains unassembled. This result suggests that Cox18 not only translocates the C-tail, but also must deliver it in a distinct state competent for cytochrome oxidase assembly. We identified respiring mutants from a cox18Delta strain overexpressing OXA1, whose respiratory growth requires overexpression of OXA1. The recessive nuclear mutations allow some assembly of Cox2 into cytochrome c oxidase. After failing to identify these mutations by methods based on transformation, we successfully located them to MGR1 and MGR3 by comparative hybridization to whole-genome tiling arrays and microarray-assisted bulk segregant analysis followed by linkage mapping. While Mgr1 and Mgr3 are known to associate with the Yme1 mitochondrial inner membrane i-AAA protease and to participate in membrane protein degradation, their absence does not appear to stabilize Cox2 under these conditions. Instead, Yme1 probably chaperones the folding and/or assembly of Oxa1-exported Cox2 in the absence of Mrg1 or Mgr3, since respiratory growth and cytochrome c oxidase assembly in a cox18 mgr3 double-mutant strain overexpressing OXA1 is YME1 dependent.

Porphyromonas gingivalis is an anaerobic, asaccharolytic, gram-negative bacterium that has essential requirements for both iron and protoporphyrin IX, which it preferentially obtains as heme. A combination of large-scale quantitative proteomic analysis using stable isotope labeling strategies and mass spectrometry, together with transcriptomic analysis using custom-made DNA microarrays, was used to identify changes in P. gingivalis W50 protein and transcript abundances on changing from heme-excess to heme-limited continuous culture. This approach identified 160 genes and 70 proteins that were differentially regulated by heme availability, with broad agreement between the transcriptomic and proteomic data. A change in abundance of the enzymes of the aspartate and glutamate catabolic pathways was observed with heme limitation, which was reflected in organic acid end product levels of the culture fluid. These results demonstrate a shift from an energy-efficient anaerobic respiration to a less efficient process upon heme limitation. Heme limitation also resulted in an increase in abundance of a protein, PG1374, which we have demonstrated, by insertional inactivation, to have a role in epithelial cell invasion. The greater abundance of a number of transcripts/proteins linked to invasion of host cells, the oxidative stress response, iron/heme transport, and virulence of the bacterium indicates that there is a broad response of P. gingivalis to heme availability.