Detailed landform mapping of key areas in the Vale of Pickering, supported by LiDAR interpretation, has produced sufficient evidence to establish a reinterpretation of the Mid to Late Pleistocene chronology of the Vale of Pickering by defining the margins of two temporally distinct proglacial lakes and reaching a new understanding of the origin of some well-documented geomorphological features. The main significance of the mapping has been to establish that the Hutton Buscel terrace probably originated by lateral erosion along the southern edge of the Corallian Group dip slope of the North York Moors prior to deposition of a broad alluvial plain below a 70 m strandline. Traces of a comparable feature were also located below the Chalk Group escarpment on the southern side of the Vale of Pickering. Perhaps of equal significance has been confirmation that the younger of the two lakes, which has a 45 m shoreline, was possibly connected to Lake Humber in the Vale of York through the Derwent Valley. Evidence for such a lake was provided by mapped shorelines at Malton and Pickering that appear compatible with shorelines in Lake Humber. To account for deep erosion of the Derwent and Mere valleys and the occurrence of laminated clays at c. 65 m, below a 70 m shoreline above Crambe, regional uplift has been evoked post the older 70 m lake. In-valley alluvial fans have been mapped for the first time in Newton Dale and Thornton Dale.

The invertebrate trace fossils Selenichnites rossendalensis and Crescentichnus tesiltus are recorded and described from the Middle Jurassic Gristhorpe Member of the Cloughton Formation of the Cleveland Basin. This is the first record of these ichnospecies from the basin and now completes the occurrence of these and other traces assumed to have been made by limulids from all three non-marine formations of the Ravenscar Group.

This article describes the development of medical competencies for oral medicine specialty training in the UK and Ireland by a collaborative working group using a modified Delphi technique. The current specialty training curriculum for oral medicine (OM) in the UK was developed by a working group including members of the British Society for Oral Medicine (BSOM) and members of the Specialty Advisory Committee for Additional Dental Specialties (SACADS) and adopted by the UK General Dental Council (GDC) in 2010. When the curriculum was developed, the entry requirements for specialty training in OM included undergraduate degrees in both dentistry and medicine. At the time of adoption, the requirement for a medical degree was removed. Medical competencies were assumed to have been delivered in medical undergraduate and postgraduate training. Accordingly, there was a need to define the medical competencies for OM specialty training to benefit trainees, trainers, and assessors. In 2018, a group comprising specialty trainers, recent former specialty trainees, and current specialty trainees in OM held face-to-face meetings in addition to email discussions and developed an updated curriculum document to better reflect the medical competencies required in specialty training. A collaborative modified Delphi approach was used to evaluate medical foundation competencies and to include only those that were considered relevant to OM specialty training. A list of relevant and achievable medical competencies was determined that has been approved by SACADS and will be incorporated into a revised OM curriculum from the UK GDC. The newly agreed-upon document for medical competencies in OM specialty training will serve as a reference for trainees, trainers, and assessors and reflects a successful use of a modified Delphi approach.

The aims of this study were to qualitatively assess dental public health (DPH) residents’ perspectives on teaching methods for DPH competencies and to develop and implement a case-based simulation to address those competencies, constructed on the basis of the qualitative assessment. Focus group discussions were conducted with 18 DPH residents enrolled in two university-based DPH programs. Topic areas discussed in the two focus groups were perceived value of DPH competencies, ways to acquire new DPH skills/abilities, and additional skills/abilities needed by DPH residents. The focus groups’ responses showed that the residents felt competent in the analytical thinking competencies such as research methodology and critiquing literature. They emphasized the importance of learning leadership skills and reported feeling somewhat uncertain about their mastery of the policy and advocacy and system evaluation competencies. Of the two distinct categories of DPH skills and competencies— analytical/critical thinking and practical competencies—these residents reported that a greater proportion of time needed to be devoted to integrating the practical competencies into their education. Based on the residents’ feedback, the authors developed a structured seminar series taking a case-based approach to simulate real-world DPH problems, using real and semi-hypothetical planning projects to meet the residents’ perceived needs and covering gaps between didactic learning and practice.

Dental students’ ability to critique team performance in dental school team clinics is a key component of dental education. The aim of this study was to determine if students’ perceptions of their team leaders’ openness of communication, cooperative decision making, and well-defined goals were positively related to the students’ improvement-oriented voice behavior and willingness to raise concerns in the clinical environment. This study used a voluntary 12-question survey, distributed via email to all 311 students at the University of Nevada, Las Vegas School of Dental Medicine after completion of the spring 2017 semester. Eighty-seven students responded, for a response rate of 28%. Responses were stratified by team, class year, and gender, and the quantitative distribution of answers to each question was correlated with each other. Team leader collaborative qualities, which included openness for communication, cooperative decision making, and well-defined goals, were found to have a significant positive relationship with students’ willingness to both raise concerns and make suggestions. Additionally, when measured by class year and gender, team differences in voice behavior assessment by students across the teams were found to be independent of class year, and no significant differences were found by gender. These results suggested that, to maintain high levels of communication, proper reporting of concerns, and a high standard of care, dental schools should encourage team leaders to enhance their capacity to present active collaborative behaviors in the school’s clinic. The study also highlighted potential opportunities for further study of faculty traits and development in the dental school team model.

Changes in U.S. health care delivery systems and Commission on Dental Accreditation standards provide impetus for interprofessional education (IPE) and collaborative practice, but roadmaps for engaging dental and dental hygiene faculty to incorporate IPE in a systematic manner are limited. The purpose of this report is to describe the process for creating a strategy and gathering a variety of baseline data to use for determining objectives and metrics and the subsequent development of an IPE strategic plan at the University of North Carolina (UNC) at Chapel Hill Adams School of Dentistry (SOD). SOD IPE committee members included representation from the UNC Schools of Dentistry, Medicine, Pharmacy, and Business. A three-phase framework was developed. Phase 1 (IPE assessment) was an internal environmental scan including a 2017 faculty survey, departmental mapping of IPE activities, comparison of UNC with national results on the IPE component of the American Dental Education Association (ADEA) survey of dental school seniors (2016 graduating class), identification of faculty joint/adjunct appointments at other UNC schools, and a strengths, weaknesses, opportunities, threats (SWOT) analysis. Phase 2 (visioning) consisted of development of IPE mission, vision, and priorities. In Phase 3 (implementation), priorities were developed. Data-gathering led to a strategic plan with three objectives: 1) increase faculty engagement and recognition, 2) develop predoctoral dentistry and dental hygiene IPE curricula, and 3) develop an infrastructure that supports IPE. Specific initiatives and activities, supporting metrics, and estimated costs were developed for each objective. The framework guided a systematic, transparent, and organized process for collecting and monitoring the evidence and directing activities. A three-year strategic plan for IPE was developed in 2017, and implementation is ongoing.

Clinical teaching is a cornerstone of health sciences education; it is also the most challenging aspect. The University of Pittsburgh Schools of Dental Medicine, Nursing, and Pharmacy developed a new evidence-based interprofessional course framed as a faculty learning community (FLC) around the principles of learning in a clinical environment. The aim of this study was to assess the overall effectiveness of this two-semester FLC at four health professions schools in academic year 2014-15. The assessment included anonymous participant surveys in each session and an anonymous end-of-course survey. Thirty-five faculty members from dental, health and rehabilitation sciences, nursing, and pharmacy enrolled in the FLC, with six to 32 enrollees attending each session. All attendees at each session completed the session evaluation surveys, but the attendance rate at each session ranged from 17.1% to 91.4%. Sixteen participants (46%) completed the end-of-course survey. The results showed overall positive responses to the FLC and changes in the participants’ self-reported knowledge. Session surveys showed that the participants found the FLC topics helpful and appreciated the opportunity to learn from each other and the interprofessional nature of the FLC. Responses to the end-of-course survey were in alignment with the individual session surveys and cited specific benefits as being the content, teaching materials, and structured discussions. In additional feedback, participants reported interest to continue as a cohort and to extend the peer-support system beyond the FLC. This outcomes assessment of the first round of the FLC confirmed that this cohort-based faculty development in an interprofessional setting was well received by its participants. Their feedback provided valuable insights for changes to future offerings.

The aim of this cross-sectional study was to examine the faculty mentoring practices in seven dental schools in the U.S. A 34-item survey was administered electronically to dental faculty members of all ranks, tracks, and job categories in seven dental schools using faculty listservs. Survey questions addressed current mentoring practices in which the faculty members were involved; their perceptions of those mentoring practices; their perceived characteristics of an ideal mentoring program, mentor, and mentee; perceived best practices; and respondents’ demographics. The survey was conducted from October 2017 to February 2018. A total of 154 surveys were completed (response rate 22%). Over 58% (90/154) of the respondents reported receiving no mentoring; 31.9% (49/154) said they received informal mentoring; and 9.7% (15/154) received formal mentoring. Of the 64 respondents who received mentoring, both formal and informal, 92.2% (59/64) were full-time faculty, and 7.8% (5/64) were part-time faculty (p=0.001). Approximately 39% of the respondents indicated that their mentoring program was not overseen by anyone and that participation was voluntary. The top three perceived benefits of mentoring were increased overall professional development, development of a career plan, and increased professional networks. The three most important characteristics of an ideal mentoring program for the respondents were a program based on the needs of the mentee, a mentor who has the desire to help the mentee, and a mentee who is eager to learn. The results of this study showed a very low level of formal or informal faculty mentoring programs in the dental schools surveyed. Future studies are needed to determine best practices and strategies to expand and enhance mentoring of faculty members.

The number of citations an article receives is an important indicator to quantify its influence in its field. The aim of this study was to identify and analyze the characteristics of the 50 top-cited articles addressing dental education published in two journals dedicated to dental education (European Journal of Dental Education and Journal of Dental Education). The Web of Science database was searched to retrieve the 50 most-cited articles from the two journals in December 2018. The top-cited articles were analyzed for journal of publication, number of citations, institution and country of origin, year of publication, study type, keywords, theme and subtheme, and international collaborations. The results showed the 50 top-cited articles were cited between 24 and 146 times each. The majority of these top-cited articles (n=34) were published in the Journal of Dental Education. Half (n=25) of the articles were by authors in the U.S. The most common study types were surveys (n=26) and reviews (n=10). The main themes of these top-cited articles were curriculum and learner characteristics. This bibliometric analysis can serve as a reference for recognizing studies with the most impact in the scholarship of dental education.

Interprofessional education (IPE) is based on collaborative practices that increase the occasions for communication among those in various health professions. However, there is a paucity of literature about the effectiveness of IPE programs in health professions education. The aim of this systematic review and meta-analysis was to objectively assess the literature on the effectiveness of IPE in improving health professions students’ attitudes after training. The major scholarly databases were searched for relevant IPE studies involving predoctoral health professions students. Two independent researchers selected the studies, extracted the data, and assessed the quality of the studies. Meta-analyses of the outcomes were performed using random effects models. Sixteen articles were ultimately selected for detailed review and meta-analysis. The meta-analysis showed that IPE training had a significant influence on students’ understanding of collaboration and resulted in better attitudes about interprofessional teamwork. Subscale analysis showed that one subscale score (roles and responsibilities) did not statistically significantly improve after IPE training (p=0.06), whereas the other four subscale items showed statistically significant improvements (p<0.01). The test for overall effects showed that IPE training had a significantly positive influence on students’ attitudes about IPE (Z=6.85, p<0.01). Subgroup results showed that medical students had more positive attitudes about IPE than did dental students. Regardless of profession, women students responded with significantly more positive feedback than did men students (p=0.02). These results suggest that intervention through IPE training has had positive effects in health professions education. Gender was an important factor impacting the outcomes of IPE. However, further clinical practice interventions may be helpful to enhance the IPE competence of health professions students.

Purpose: The purpose of this study was to identify current requirements for initial licensure and entry into the dental hygiene profession across state dental and dental hygiene licensing boards in the United States.Methods: A non-experimental study design was used to study dental and dental hygiene board licensing requirements in the United States, Puerto Rico and the Virgin Islands. Each regulatory board website was searched for requirements for entry-level dental hygiene licensure. Requirements were recorded on an Excel spreadsheet. State dental practice acts were reviewed to gather further information and 20 regulatory bodies were contacted to verify accuracy. Descriptive statistics were used to analyze data.Results: Information from a total of 52 dental boards (n=52) was examined for this study. Nearly all boards (n=51, 98.1%), with the exception of Alabama, required completion of entry-level education from a CODA accredited dental hygiene program and successful completion of the National Board Dental Hygiene Examination. Most states (n=51, 98.1%), except Delaware, also required a live-patient, a clinical board examination. Application fees ranged from $47.70 to $600. States varied considerably in terms of requirements for background checks, age, military status, and infection control training.Conclusion: Although the majority of regulatory bodies require completion of entry-level dental hygiene education from a CODA accredited program and successful completion of national board and a live-patient, clinical examination, there is considerable variation in other additional requirements for initial dental hygiene licensure.

Purpose: The purpose of this study was to determine strength of muscles involved with instrumentation (scaling) by dental hygienists and the additive effects of cellular (mobile) phone usage, as indicated by measurements of muscular force generation.Methods: A convenience sample of licensed dental hygienists currently in clinical practice (n=16) and an equal number of individuals not currently using devices/tools repetitively for work (n=16), agreed to participate in this pilot study. All participants completed a modified cell phone usage questionnaire to determine their use pattern and frequency. Upon completion of the questionnaire, participants' force production in six muscle groups was measured using a hand-held dynamometer. Descriptive statistics were used to analyze the data.Results: A total of 16 licensed dental hygienists (n=16) and 16 participants with no history of using tools/devices repetitively for work (n=16), comprised the experimental and control groups, repectively. The control group generated greater muscle force than the experimental group for the abductor pollicis longus (p=0.045). Significant differences were identified when comparing the low mobile phone users in the experimental group to the control group for the flexor pollicis brevis (p=0.031), abductor pollicis longus (p=0.031), and flexor digitorum (p=0.006), with the control group demonstrating higher muscle force. Years in clinical practice and mobile phone use was shown to have a significant effect on muscular force generation for the flexor pollicis brevis (F=3.645, df=3, p=0.020) and flexor digitorum (F=3.560, df=3, p=0.022); subjects who practiced dental hygiene the longest produced the least amount of muscle force.Conclusion: Results from this pilot study indicate there are no significant additive effects of cell phone use and dental hygiene practice on finger muscles used for instrumentation. However, results indicate that dental hygiene practice demonstrated significant effects on muscular strength as compared to individuals who do not use tools/devices repetitively for work. The small sample size may have impacted results and the study should be repeated with a larger sample.

Purpose: Untreated and poorly controlled diabetes causes increased levels of blood glucose associated with poor periodontal disease outcomes. Dental hygienists can play a significant role in screening patients for diabetes mellitus, leading to referral and early diagnosis. The purpose of this study was to determine the knowledge, attitudes, practices, and barriers faced by clinical dental hygienists regarding diabetes risk assessment and screenings.Methods: A mixed method design was used with a convenience sample of dental hygienists in clinical practice (n=316). A 32 item, electronic survey was validated at item-level, and participants were recruited through multiple dental hygiene Facebook groups. Descriptive statistics were used to analyze the data. The survey also included two open-ended attitude questions that were interpreted using thematic analysis to pinpoint common patterns within the data.Results: Dental hygienists had high knowledge scores regarding diabetes and oral health, although many were unaware of their states' specific statutes and regulations for screening practices. Nearly all (95.9%), were likely to educate and refer patients (82%), although fewer than half (40.9%), were likely to perform chairside screening for diabetes. Emergent themes for barriers to screening were time, money, patient acceptance/willingness, lack of education, not having the proper tools, and states' rules and regulations.Conclusion: Despite high knowledge scores regarding diabetes and oral health, there is a gap in regards to dental hygienists' willingness to perform diabetes screenings in a clinical setting. Dental hygienists should be capable of integrating chairside diabetes screening practices into the process of care with proper training.

Purpose: The purpose of this study was to analyze associations between the oral health literacy of refugees and two oral health outcomes: dental care utilization and oral health self-efficacy.Methods: A convenience sample of refugees in the greater Los Angeles area attending English as a second language (ESL) classes sponsored by two refugee assistance organizations was used for this cross-sectional, correlational study. Participants responded to a questionnaire using items from the Health Literacy in Dentistry (HeLD) scale, in addition to items concerning dental care utilization and oral health self-efficacy. Descriptive statistics, chi-square and Fisher's Exact tests were used to analyze results.Results: Sixty-two refugees volunteered to participate (n=62). A majority of the respondents were female from Iraq or Syria, and selected the item “with little difficulty” for all oral health literacy tasks. In regards to dental care utilization, more than half of the respondents were considered high utilizers (63%, n=34) meaning they had visited a dental office within the last year; while a little more than one-third (37%, n=20), were low utilizers, indicating they had either never been to a dental office or it had been more than one year since they had dental treatment. Statistical analysis showed associations between oral health literacy and dental care utilization. However, few associations between oral health literacy and oral health self-efficacy were identified (p=0.0045).Conclusions: Results support the provision of easily obtainable and understandable oral health information to increase oral health literacy and dental care utilization among refugee populations. Future research is needed to examine the oral health literacy among refugees resettling in the United States.

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the "inhibitors" previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

Geranylgeranoic acid (GGA) originally was identified in some animals and has been developed as an agent for preventing second primary hepatoma. We previously have also identified GGA as an acyclic diterpenoid in some medicinal herbs. Recently, we reported that in human hepatoma-derived HuH-7 cells, GGA is metabolically labeled from ¹³C-mevalonate. Several cell-free experiments have demonstrated that GGA is synthesized through geranylgeranial by oxygen-dependent oxidation of geranylgeraniol (GGOH), but the exact biochemical events giving rise to GGA in hepatoma cells remain unclear. Monoamine oxidase B (MOAB) has been suggested to be involved in GGOH oxidation. Here, using two human hepatoma cell lines, we investigated whether MAOB contributes to GGA biosynthesis. Using either HuH-7 cell lysates or recombinant human MAOB, we found that: 1) the MAO inhibitor tranylcypromine dose-dependently downregulates endogenous GGA levels in HuH-7 cells; and 2) siRNA-mediated MAOB silencing reduces intracellular GGA levels in HuH-7 and Hep3B cells. Unexpectedly, however, CRISPR/Cas9-generated MAOB-KO human hepatoma Hep3B cells had GGA levels similar to those in MAOB-WT cells. A sensitivity of GGA levels to siRNA-mediated MAOB downregulation was recovered when the MAOB-KO cells were transfected with a MAOB-expression plasmid, suggesting that MAOB is the enzyme primarily responsible for GGOH oxidation and that some other latent metabolic pathways may maintain endogenous GGA levels in the MAOB-KO hepatoma cells. Along with the previous findings, these results provide critical insights into the biological roles of human MAOB and provide evidence that hepatic MAOB is involved in endogenous GGA biosynthesis via GGOH oxidation.

Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (^–/–) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.­

The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K₂ subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.

Adipocytes take up long chain FAs through diffusion and protein-mediated transport, whereas FA efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating FA flux in adipocytes, the expression levels of 55 membrane transporters without known function were screened in subcutaneous adipose samples from obese patients before and after bariatric surgery using branched DNA methodology. Among the 33 solute carrier (SLC) transporter family members screened, the expression of 14 members showed significant changes before and after bariatric surgery. One of them, Slc43a3, increased about 2.5-fold after bariatric surgery. Further investigation demonstrated that Slc43a3 is highly expressed in murine adipose tissue and induced during adipocyte differentiation in primary preadipocytes and in OP9 cells. Knockdown of Slc43a3 with siRNA in differentiated OP9 adipocytes reduced both basal and forskolin-stimulated FA efflux, while also increasing FA uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased FA uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake.

Acne is one of the most common dermatological conditions, but the details of its pathology are unclear, and current management regimens often have adverse effects. Cutibacterium acnes is known as a major acne-associated bacterium that derives energy from lipase-mediated sebum lipid degradation. C. acnes is commensal, but lipase activity has been observed to differ among C. acnes types. For example, higher populations of the type IA strains are present in acne lesions with higher lipase activity. In the present study, we examined a conserved lipase in types IB and II that was truncated in type IA C. acnes strains. Closed, blocked, and open structures of C. acnes ATCC11828 lipases were elucidated by X-ray crystallography at 1.6–2.4 Å. The closed crystal structure, which is the most common form in aqueous solution, revealed that a hydrophobic lid domain shields the active site. By comparing closed, blocked, and open structures, we found that the lid domain-opening mechanisms of C. acnes lipases (_CAlipases) involve the lid-opening residues, Phe-179 and Phe-211. To the best of our knowledge, this is the first structure-function study of _CAlipases, which may help to shed light on the mechanisms involved in acne development and may aid in future drug design.

Fatty liver involves ectopic lipid accumulation and dysregulated hepatic oxidative metabolism, which can progress to a state of elevated inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH). The factors that control progression from simple steatosis to NASH are not fully known. Here, we tested the hypothesis that dietary vitamin E (VitE) supplementation would prevent NASH progression and associated metabolic alterations induced by a Western diet (WD). Hyperphagic melanocortin-4 receptor-deficient (MC4R^–/–) mice were fed chow, chow+VitE, WD, or WD+VitE starting at 8 or 20 weeks of age. All groups exhibited extensive hepatic steatosis by the end of the study (28 weeks of age). WD feeding exacerbated liver disease severity without inducing proportional changes in liver triglycerides. Eight weeks of WD accelerated liver pyruvate cycling, and 20 weeks of WD extensively upregulated liver glucose and oxidative metabolism assessed by ²H/¹³C flux analysis. VitE supplementation failed to reduce the histological features of NASH. Rather, WD+VitE increased the abundance and saturation of liver ceramides and accelerated metabolic flux dysregulation compared with 8 weeks of WD alone. In summary, VitE did not limit NASH pathogenesis in genetically obese mice, but instead increased some indicators of metabolic dysfunction.

The pregnane X receptor (PXR) is a nuclear receptor that can be activated by numerous drugs and xenobiotic chemicals. PXR thereby functions as a xenobiotic sensor to coordinately regulate host responses to xenobiotics by transcriptionally regulating many genes involved in xenobiotic metabolism. We have previously reported that PXR has pro-atherogenic effects in animal models, but how PXR contributes to atherosclerosis development in different tissues or cell types remains elusive. In this study, we generated an LDL receptor-deficient mouse model with myeloid-specific PXR deficiency (PXR^MyeLDLR^–/–) to elucidate the role of macrophage PXR signaling in atherogenesis. The myeloid PXR deficiency did not affect metabolic phenotypes and plasma lipid profiles, but PXR^MyeLDLR^–/– mice had significantly decreased atherosclerosis at both aortic root and brachiocephalic arteries compared with control littermates. Interestingly, the PXR deletion did not affect macrophage adhesion and migration properties, but reduced lipid accumulation and foam cell formation in the macrophages. PXR deficiency also led to decreased expression of the scavenger receptor CD36 and impaired lipid uptake in macrophages of the PXR^MyeLDLR^–/– mice. Further, RNA-Seq analysis indicated that treatment with a prototypical PXR ligand affects the expression of many atherosclerosis-related genes in macrophages in vitro. These findings reveal a pivotal role of myeloid PXR signaling in atherosclerosis development and suggest that PXR may be a potential therapeutic target in atherosclerosis management.

Lipid rafts regulate the initiation of cellular metabolic and signaling pathways by organizing the pathway components in ordered microdomains on the cell surface. Cellular responses regulated by lipid rafts range from physiological to pathological, and the success of a therapeutic approach targeting "pathological" lipid rafts depends on the ability of a remedial agent to recognize them and disrupt pathological lipid rafts without affecting normal raft-dependent cellular functions. In this article, concluding the Thematic Review Series on Biology of Lipid Rafts, we review current experimental therapies targeting pathological lipid rafts, including examples of inflammarafts and clusters of apoptotic signaling molecule-enriched rafts. The corrective approaches include regulation of cholesterol and sphingolipid metabolism and membrane trafficking by using HDL and its mimetics, LXR agonists, ABCA1 overexpression, and cyclodextrins, as well as a more targeted intervention with apoA-I binding protein. Among others, we highlight the design of antagonists that target inflammatory receptors only in their activated form of homo- or heterodimers, when receptor dimerization occurs in pathological lipid rafts. Other therapies aim to promote raft-dependent physiological functions, such as augmenting caveolae-dependent tissue repair. The overview of this highly dynamic field will provide readers with a view on the emerging concept of targeting lipid rafts as a therapeutic strategy.

Cellular membranes are not homogenous mixtures of proteins; rather, they are segregated into microdomains on the basis of preferential association between specific lipids and proteins. These microdomains, called lipid rafts, are well known for their role in receptor signaling on the plasma membrane (PM) and are essential to such cellular functions as signal transduction and spatial organization of the PM. A number of disease states, including atherosclerosis and other cardiovascular disorders, may be caused by dysfunctional maintenance of lipid rafts. Lipid rafts do not occur only in the PM but also have been found in intracellular membranes and extracellular vesicles (EVs). Here, we focus on discussing newly discovered functions of lipid rafts and microdomains in intracellular membranes, including lipid and protein trafficking from the ER, Golgi bodies, and endosomes to the PM, and we examine lipid raft involvement in the production and composition of EVs. Because lipid rafts are small and transient, visualization remains challenging. Future work with advanced techniques will continue to expand our knowledge about the roles of lipid rafts in cellular functioning.

Lipid rafts are highly ordered regions of the plasma membrane that are enriched in cholesterol and sphingolipids and play important roles in many cells. In hematopoietic stem and progenitor cells (HSPCs), lipid rafts house receptors critical for normal hematopoiesis. Lipid rafts also can bind and sequester kinases that induce negative feedback pathways to limit proliferative cytokine receptor cycling back to the cell membrane. Modulation of lipid rafts occurs through an array of mechanisms, with optimal cholesterol efflux one of the major regulators. As such, cholesterol homeostasis also regulates hematopoiesis. Increased lipid raft content, which occurs in response to changes in cholesterol efflux in the membrane, can result in prolonged receptor occupancy in the cell membrane and enhanced signaling. In addition, certain diseases, like diabetes, may contribute to lipid raft formation and affect cholesterol retention in rafts. In this review, we explore the role of lipid raft-related mechanisms in hematopoiesis and CVD (specifically, atherosclerosis) and discuss how defective cholesterol efflux pathways in HSPCs contribute to expansion of lipid rafts, thereby promoting myelopoiesis and thrombopoiesis. We also discuss the utility of cholesterol acceptors in contributing to lipid raft regulation and disruption, and highlight the potential to manipulate these pathways for therapeutic gain in CVD as well as other disorders with aberrant hematopoiesis.

Activation of microglia and astrocytes secondary to inflammatory processes contributes to the development and perpetuation of pain with a neuropathic phenotype. This pain state presents as a chronic debilitating condition and affects a large population of patients with conditions like rheumatoid arthritis and diabetes, or after surgery, trauma, or chemotherapy. Here, we review the regulation of lipid rafts in glial cells and the role they play as a key component of neuroinflammatory sensitization of central pain signaling pathways. In this context, we introduce the concept of an inflammaraft (i-raft), enlarged lipid rafts harboring activated receptors and adaptor molecules and serving as an organizing platform to initiate inflammatory signaling and the cellular response. Characteristics of the inflammaraft include increased relative abundance of lipid rafts in inflammatory cells, increased content of cholesterol per raft, and increased levels of inflammatory receptors, such as toll-like receptor (TLR)4, adaptor molecules, ion channels, and enzymes in lipid rafts. This inflammaraft motif serves an important role in the membrane assembly of protein complexes, for example, TLR4 dimerization. Operating within this framework, we demonstrate the involvement of inflammatory receptors, redox molecules, and ion channels in the inflammaraft formation and the regulation of cholesterol and sphingolipid metabolism in the inflammaraft maintenance and disruption. Strategies for targeting inflammarafts, without affecting the integrity of lipid rafts in noninflammatory cells, may lead to developing novel therapies for neuropathic pain states and other neuroinflammatory conditions.

Lipid rafts are small, dynamic membrane areas characterized by the clustering of selected membrane lipids as the result of the spontaneous separation of glycolipids, sphingolipids, and cholesterol in a liquid-ordered phase. The exact dynamics underlying phase separation of membrane lipids in the complex biological membranes are still not fully understood. Nevertheless, alterations in the membrane lipid composition affect the lateral organization of molecules belonging to lipid rafts. Neural lipid rafts are found in brain cells, including neurons, astrocytes, and microglia, and are characterized by a high enrichment of specific lipids depending on the cell type. These lipid rafts seem to organize and determine the function of multiprotein complexes involved in several aspects of signal transduction, thus regulating the homeostasis of the brain. The progressive decline of brain performance along with physiological aging is at least in part associated with alterations in the composition and structure of neural lipid rafts. In addition, neurodegenerative conditions, such as lysosomal storage disorders, multiple sclerosis, and Parkinson’s, Huntington’s, and Alzheimer’s diseases, are frequently characterized by dysregulated lipid metabolism, which in turn affects the structure of lipid rafts. Several events underlying the pathogenesis of these diseases appear to depend on the altered composition of lipid rafts. Thus, the structure and function of lipid rafts play a central role in the pathogenesis of many common neurodegenerative diseases.

Cholesterol/sphingolipid-rich membrane domains, known as lipid rafts or membrane rafts, play a critical role in the compartmentalization of signaling pathways. Physical segregation of proteins in lipid rafts may modulate the accessibility of proteins to regulatory or effector molecules. Thus, lipid rafts serve as sorting platforms and hubs for signal transduction proteins. Cancer cells contain higher levels of intracellular cholesterol and lipid rafts than their normal non-tumorigenic counterparts. Many signal transduction processes involved in cancer development (insulin-like growth factor system and phosphatidylinositol 3-kinase-AKT) and metastasis [cluster of differentiation (CD)44] are dependent on or modulated by lipid rafts. Additional proteins playing an important role in several malignant cancers (e.g., transmembrane glycoprotein mucin 1) are also being detected in association with lipid rafts, suggesting a major role of lipid rafts in tumor progression. Conversely, lipid rafts also serve as scaffolds for the recruitment and clustering of Fas/CD95 death receptors and downstream signaling molecules leading to cell death-promoting raft platforms. The partition of death receptors and downstream signaling molecules in aggregated lipid rafts has led to the formation of the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER, which leads to apoptosis amplification and can be pharmacologically modulated. These death-promoting rafts can be viewed as a linchpin from which apoptotic signals are launched. In this review, we discuss the involvement of lipid rafts in major signaling processes in cancer cells, including cell survival, cell death, and metastasis, and we consider the potential of lipid raft modulation as a promising target in cancer therapy.

Lipid rafts, solid regions of the plasma membrane enriched in cholesterol and glycosphingolipids, are essential parts of a cell. Functionally, lipid rafts present a platform that facilitates interaction of cells with the outside world. However, the unique properties of lipid rafts required to fulfill this function at the same time make them susceptible to exploitation by pathogens. Many steps of pathogen interaction with host cells, and sometimes all steps within the entire lifecycle of various pathogens, rely on host lipid rafts. Such steps as binding of pathogens to the host cells, invasion of intracellular parasites into the cell, the intracellular dwelling of parasites, microbial assembly and exit from the host cell, and microbe transfer from one cell to another all involve lipid rafts. Interaction also includes modification of lipid rafts in host cells, inflicted by pathogens from both inside and outside the cell, through contact or remotely, to advance pathogen replication, to utilize cellular resources, and/or to mitigate immune response. Here, we provide a systematic overview of how and why pathogens interact with and exploit host lipid rafts, as well as the consequences of this interaction for the host, locally and systemically, and for the microbe. We also raise the possibility of modulation of lipid rafts as a therapeutic approach against a variety of infectious agents.

Lipid rafts are organized plasma membrane microdomains, which provide a distinct level of regulation of cellular metabolism and response to extracellular stimuli, affecting a diverse range of physiologic and pathologic processes. This Thematic Review Series focuses on Biology of Lipid Rafts rather than on their composition or structure. The aim is to provide an overview of ideas on how lipid rafts are involved in regulation of different pathways and how they interact with other layers of metabolic regulation. Articles in the series will review the involvement of lipid rafts in regulation of hematopoiesis, production of extracellular vesicles, host interaction with infection, and the development and progression of cancer, neuroinflammation, and neurodegeneration, as well as the current outlook on therapeutic targeting of lipid rafts.

ABSTRACT

The luminous marine Gram-negative bacterium Vibrio (Aliivibrio) fischeri is the natural light organ symbiont of several squid species, including the Hawaiian bobtail squid, Euprymna scolopes, and the Japanese bobtail squid, Euprymna morsei. Work with E. scolopes has shown how the bacteria establish their niche in the light organ of the newly hatched host. Two types of V. fischeri strains have been distinguished based upon their behavior in cocolonization competition assays in juvenile E. scolopes, i.e., (i) niche-sharing or (ii) niche-dominant behavior. This study aimed to determine whether these behaviors are observed with other V. fischeri strains or whether they are specific to those isolated from E. scolopes light organs. Cocolonization competition assays between V. fischeri strains isolated from the congeneric squid E. morsei or from other marine animals revealed the same sharing or dominant behaviors. In addition, whole-genome sequencing of these strains showed that the dominant behavior is polyphyletic and not associated with the presence or absence of a single gene or genes. Comparative genomics of 44 squid light organ isolates from around the globe led to the identification of symbiosis-specific candidates in the genomes of these strains. Colonization assays using genetic derivatives with deletions of these candidates established the importance of two such genes in colonization. This study has allowed us to expand the concept of distinct colonization behaviors to strains isolated from a number of squid and fish hosts.

IMPORTANCE There is an increasing recognition of the importance of strain differences in the ecology of a symbiotic bacterial species and, in particular, how these differences underlie crucial interactions with their host. Nevertheless, little is known about the genetic bases for these differences, how they manifest themselves in specific behaviors, and their distribution among symbionts of different host species. In this study, we sequenced the genomes of Vibrio fischeri isolated from the tissues of squids and fishes and applied comparative genomics approaches to look for patterns between symbiont lineages and host colonization behavior. In addition, we identified the only two genes that were exclusively present in all V. fischeri strains isolated from the light organs of sepiolid squid species. Mutational studies of these genes indicated that they both played a role in colonization of the squid light organ, emphasizing the value of applying a comparative genomics approach in the study of symbioses.

ABSTRACT

Obesity is associated with increased disease severity, elevated viral titers in exhaled breath, and significantly prolonged viral shed during influenza A virus infection. Due to the mutable nature of RNA viruses, we questioned whether obesity could also influence influenza virus population diversity. Here, we show that minor variants rapidly emerge in obese mice. The variants exhibit increased viral replication, resulting in enhanced virulence in wild-type mice. The increased diversity of the viral population correlated with decreased type I interferon responses, and treatment of obese mice with recombinant interferon reduced viral diversity, suggesting that the delayed antiviral response exhibited in obesity permits the emergence of a more virulent influenza virus population. This is not unique to obese mice. Obesity-derived normal human bronchial epithelial (NHBE) cells also showed decreased interferon responses and increased viral replication, suggesting that viral diversity also was impacted in this increasing population.

IMPORTANCE Currently, 50% of the adult population worldwide is overweight or obese. In these studies, we demonstrate that obesity not only enhances the severity of influenza infection but also impacts viral diversity. The altered microenvironment associated with obesity supports a more diverse viral quasispecies and affords the emergence of potentially pathogenic variants capable of inducing greater disease severity in lean hosts. This is likely due to the impaired interferon response, which is seen in both obese mice and obesity-derived human bronchial epithelial cells, suggesting that obesity, aside from its impact on influenza virus pathogenesis, permits the stochastic accumulation of potentially pathogenic viral variants, raising concerns about its public health impact as the prevalence of obesity continues to rise.

ABSTRACT

Synthesis and cleavage of the cell wall polymer peptidoglycan (PG) are carefully orchestrated processes and are essential for the growth and survival of bacteria. Yet, the function and importance of many enzymes that act on PG in Mycobacterium tuberculosis remain to be elucidated. We demonstrate that the activity of the N-acetylmuramyl-l-alanine amidase Ami1 is dispensable for cell division in M. tuberculosis in vitro yet contributes to the bacterium’s ability to persist during chronic infection in mice. Furthermore, the d,l-endopeptidase RipA, a predicted essential enzyme, is dispensable for the viability of M. tuberculosis but required for efficient cell division in vitro and in vivo. Depletion of RipA sensitizes M. tuberculosis to rifampin and to cell envelope-targeting antibiotics. Ami1 helps sustain residual cell division in cells lacking RipA, but the partial redundancy provided by Ami1 is not sufficient during infection, as depletion of RipA prevents M. tuberculosis from replicating in macrophages and leads to dramatic killing of the bacteria in mice. Notably, RipA is essential for persistence of M. tuberculosis in mice, suggesting that cell division is required during chronic mouse infection. Despite the multiplicity of enzymes acting on PG with redundant functions, we have identified two PG hydrolases that are important for M. tuberculosis to replicate and persist in the host.

IMPORTANCE Tuberculosis (TB) is a major global heath burden, with 1.6 million people succumbing to the disease every year. The search for new drugs to improve the current chemotherapeutic regimen is crucial to reducing this global health burden. The cell wall polymer peptidoglycan (PG) has emerged as a very successful drug target in bacterial pathogens, as many currently used antibiotics target the synthesis of this macromolecule. However, the multitude of genes encoding PG-synthesizing and PG-modifying enzymes with apparent redundant functions has hindered the identification of novel drug targets in PG synthesis in Mycobacterium tuberculosis. Here, we demonstrate that two PG-cleaving enzymes are important for virulence of M. tuberculosis. In particular, the d,l-endopeptidase RipA represents a potentially attractive drug target, as its depletion results in the clearance of M. tuberculosis from the host and renders the bacteria hypersusceptible to rifampin, a frontline TB drug, and to several cell wall-targeting antibiotics.

ABSTRACT

The synergy between Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV-1) interferes with therapy and facilitates the pathogenesis of both human pathogens. Fundamental mechanisms by which M. tuberculosis exacerbates HIV-1 infection are not clear. Here, we show that exosomes secreted by macrophages infected with M. tuberculosis, including drug-resistant clinical strains, reactivated HIV-1 by inducing oxidative stress. Mechanistically, M. tuberculosis-specific exosomes realigned mitochondrial and nonmitochondrial oxygen consumption rates (OCR) and modulated the expression of host genes mediating oxidative stress response, inflammation, and HIV-1 transactivation. Proteomics analyses revealed the enrichment of several host factors (e.g., HIF-1α, galectins, and Hsp90) known to promote HIV-1 reactivation in M. tuberculosis-specific exosomes. Treatment with a known antioxidant—N-acetyl cysteine (NAC)—or with inhibitors of host factors—galectins and Hsp90—attenuated HIV-1 reactivation by M. tuberculosis-specific exosomes. Our findings uncover new paradigms for understanding the redox and bioenergetics bases of HIV-M. tuberculosis coinfection, which will enable the design of effective therapeutic strategies.

IMPORTANCE Globally, individuals coinfected with the AIDS virus (HIV-1) and with M. tuberculosis (causative agent of tuberculosis [TB]) pose major obstacles in the clinical management of both diseases. At the heart of this issue is the apparent synergy between the two human pathogens. On the one hand, mechanisms induced by HIV-1 for reactivation of TB in AIDS patients are well characterized. On the other hand, while clinical findings clearly identified TB as a risk factor for HIV-1 reactivation and associated mortality, basic mechanisms by which M. tuberculosis exacerbates HIV-1 replication and infection remain poorly characterized. The significance of our research is in identifying the role of fundamental mechanisms such as redox and energy metabolism in catalyzing HIV-M. tuberculosis synergy. The quantification of redox and respiratory parameters affected by M. tuberculosis in stimulating HIV-1 will greatly enhance our understanding of HIV-M. tuberculosis coinfection, leading to a wider impact on the biomedical research community and creating new translational opportunities.

ABSTRACT

Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug-resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we used two distinct genomic approaches, namely, transposon library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage-infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra- and interbacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together, our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities, such as the host microbiota, during phage therapy.

IMPORTANCE We lack fundamental understanding of how phage infection influences bacterial gene expression and, consequently, how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole-genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside their intended host targets.

ABSTRACT

In the absence of a vaccine, multidrug-resistant Neisseria gonorrhoeae has emerged as a major human health threat, and new approaches to treat gonorrhea are urgently needed. N. gonorrhoeae pili are posttranslationally modified by a glycan that terminates in a galactose. The terminal galactose is critical for initial contact with the human cervical mucosa via an interaction with the I-domain of complement receptor 3 (CR3). We have now identified the I-domain galactose-binding epitope and characterized its galactose-specific lectin activity. Using surface plasmon resonance and cellular infection assays, we found that a peptide mimic of this galactose-binding region competitively inhibited the N. gonorrhoeae-CR3 interaction. A compound library was screened for potential drugs that could similarly prohibit the N. gonorrhoeae-CR3 interaction and be repurposed as novel host-targeted therapeutics for multidrug-resistant gonococcal infections in women. Two drugs, methyldopa and carbamazepine, prevented and cured cervical cell infection by multidrug-resistant gonococci by blocking the gonococcal-CR3 I-domain interaction.

IMPORTANCE Novel therapies that avert the problem of Neisseria gonorrhoeae with acquired antibiotic resistance are urgently needed. Gonococcal infection of the human cervix is initiated by an interaction between a galactose modification made to its surface appendages, pili, and the I-domain region of (host) complement receptor 3 (CR3). By targeting this crucial gonococcal–I-domain interaction, it may be possible to prevent cervical infection in females. To this end, we identified the I-domain galactose-binding epitope of CR3 and characterized its galactose lectin activity. Moreover, we identified two drugs, carbamazepine and methyldopa, as effective host-targeted therapies for gonorrhea treatment. At doses below those currently used for their respective existing indications, both carbamazepine and methyldopa were more effective than ceftriaxone in curing cervical infection ex vivo. This host-targeted approach would not be subject to N. gonorrhoeae drug resistance mechanisms. Thus, our data suggest a long-term solution to the growing problem of multidrug-resistant N. gonorrhoeae infections.

ABSTRACT

Host-associated microbial communities are shaped by extrinsic and intrinsic factors to the holobiont organism. Environmental factors and microbe-microbe interactions act simultaneously on the microbial community structure, making the microbiome dynamics challenging to predict. The coral microbiome is essential to the health of coral reefs and sensitive to environmental changes. Here, we develop a dynamic model to determine the microbial community structure associated with the surface mucus layer (SML) of corals using temperature as an extrinsic factor and microbial network as an intrinsic factor. The model was validated by comparing the predicted relative abundances of microbial taxa to the relative abundances of microbial taxa from the sample data. The SML microbiome from Pseudodiploria strigosa was collected across reef zones in Bermuda, where inner and outer reefs are exposed to distinct thermal profiles. A shotgun metagenomics approach was used to describe the taxonomic composition and the microbial network of the coral SML microbiome. By simulating the annual temperature fluctuations at each reef zone, the model output is statistically identical to the observed data. The model was further applied to six scenarios that combined different profiles of temperature and microbial network to investigate the influence of each of these two factors on the model accuracy. The SML microbiome was best predicted by model scenarios with the temperature profile that was closest to the local thermal environment, regardless of the microbial network profile. Our model shows that the SML microbiome of P. strigosa in Bermuda is primarily structured by seasonal fluctuations in temperature at a reef scale, while the microbial network is a secondary driver.

IMPORTANCE Coral microbiome dysbiosis (i.e., shifts in the microbial community structure or complete loss of microbial symbionts) caused by environmental changes is a key player in the decline of coral health worldwide. Multiple factors in the water column and the surrounding biological community influence the dynamics of the coral microbiome. However, by including only temperature as an external factor, our model proved to be successful in describing the microbial community associated with the surface mucus layer (SML) of the coral P. strigosa. The dynamic model developed and validated in this study is a potential tool to predict the coral microbiome under different temperature conditions.

ABSTRACT

Horizontal gene transfer (HGT) promotes the spread of genes within bacterial communities. Among the HGT mechanisms, natural transformation stands out as being encoded by the bacterial core genome. Natural transformation is often viewed as a way to acquire new genes and to generate genetic mixing within bacterial populations. Another recently proposed function is the curing of bacterial genomes of their infectious parasitic mobile genetic elements (MGEs). Here, we propose that these seemingly opposing theoretical points of view can be unified. Although costly for bacterial cells, MGEs can carry functions that are at points in time beneficial to bacteria under stressful conditions (e.g., antibiotic resistance genes). Using computational modeling, we show that, in stochastic environments, an intermediate transformation rate maximizes bacterial fitness by allowing the reversible integration of MGEs carrying resistance genes, although these MGEs are costly for host cell replication. Based on this dual function (MGE acquisition and removal), transformation would be a key mechanism for stabilizing the bacterial genome in the long term, and this would explain its striking conservation.

IMPORTANCE Natural transformation is the acquisition, controlled by bacteria, of extracellular DNA and is one of the most common mechanisms of horizontal gene transfer, promoting the spread of resistance genes. However, its evolutionary function remains elusive, and two main roles have been proposed: (i) the new gene acquisition and genetic mixing within bacterial populations and (ii) the removal of infectious parasitic mobile genetic elements (MGEs). While the first one promotes genetic diversification, the other one promotes the removal of foreign DNA and thus genome stability, making these two functions apparently antagonistic. Using a computational model, we show that intermediate transformation rates, commonly observed in bacteria, allow the acquisition then removal of MGEs. The transient acquisition of costly MGEs with resistance genes maximizes bacterial fitness in environments with stochastic stress exposure. Thus, transformation would ensure both a strong dynamic of the bacterial genome in the short term and its long-term stabilization.

ABSTRACT

Population-level analyses are rapidly becoming inadequate to answer many of biomedical science and microbial ecology’s most pressing questions. The role of microbial populations within ecosystems and the evolutionary selective pressure on individuals depend fundamentally on the metabolic activity of single cells. Yet, many existing single-cell technologies provide only indirect evidence of metabolic specialization because they rely on correlations between transcription and phenotype established at the level of the population to infer activity. In this study, we take a top-down approach using isotope labels and secondary ion mass spectrometry to track the uptake of carbon and nitrogen atoms from different sources into biomass and directly observe dynamic changes in anabolic specialization at the level of single cells. We investigate the classic microbiological phenomenon of diauxic growth at the single-cell level in the model methylotroph Methylobacterium extorquens. In nature, this organism inhabits the phyllosphere, where it experiences diurnal changes in the available carbon substrates, necessitating an overhaul of central carbon metabolism. We show that the population exhibits a unimodal response to the changing availability of viable substrates, a conclusion that supports the canonical model but has thus far been supported by only indirect evidence. We anticipate that the ability to monitor the dynamics of anabolism in individual cells directly will have important applications across the fields of ecology, medicine, and biogeochemistry, especially where regulation downstream of transcription has the potential to manifest as heterogeneity that would be undetectable with other existing single-cell approaches.

IMPORTANCE Understanding how genetic information is realized as the behavior of individual cells is a long-term goal of biology but represents a significant technological challenge. In clonal microbial populations, variation in gene regulation is often interpreted as metabolic heterogeneity. This follows the central dogma of biology, in which information flows from DNA to RNA to protein and ultimately manifests as activity. At present, DNA and RNA can be characterized in single cells, but the abundance and activity of proteins cannot. Inferences about metabolic activity usually therefore rely on the assumption that transcription reflects activity. By tracking the atoms from which they build their biomass, we make direct observations of growth rate and substrate specialization in individual cells throughout a period of growth in a changing environment. This approach allows the flow of information from DNA to be constrained from the distal end of the regulatory cascade and will become an essential tool in the rapidly advancing field of single-cell metabolism.

ABSTRACT

Human genetics influence a range of pathological and clinical phenotypes in respiratory infections; however, the contributions of disease modifiers remain underappreciated. We exploited the Collaborative Cross (CC) mouse genetic-reference population to map genetic modifiers that affect the severity of Pseudomonas aeruginosa lung infection. Screening for P. aeruginosa respiratory infection in a cohort of 39 CC lines exhibits distinct disease phenotypes ranging from complete resistance to lethal disease. Based on major changes in the survival times, a quantitative-trait locus (QTL) was mapped on murine chromosome 3 to the genomic interval of Mb 110.4 to 120.5. Within this locus, composed of 31 protein-coding genes, two candidate genes, namely, dihydropyrimidine dehydrogenase (Dpyd) and sphingosine-1-phosphate receptor 1 (S1pr1), were identified according to the level of genome-wide significance and disease gene prioritization. Functional validation of the S1pr1 gene by pharmacological targeting in C57BL/6NCrl mice confirmed its relevance in P. aeruginosa pathophysiology. However, in a cohort of Canadian patients with cystic fibrosis (CF) disease, regional genetic-association analysis of the syntenic human locus on chromosome 1 (Mb 97.0 to 105.0) identified two single-nucleotide polymorphisms (rs10875080 and rs11582736) annotated to the Dpyd gene that were significantly associated with age at first P. aeruginosa infection. Thus, there is evidence that both genes might be implicated in this disease. Our results demonstrate that the discovery of murine modifier loci may generate information that is relevant to human disease progression.

IMPORTANCE Respiratory infection caused by P. aeruginosa is one of the most critical health burdens worldwide. People affected by P. aeruginosa infection include patients with a weakened immune system, such as those with cystic fibrosis (CF) genetic disease or non-CF bronchiectasis. Disease outcomes range from fatal pneumonia to chronic life-threatening infection and inflammation leading to the progressive deterioration of pulmonary function. The development of these respiratory infections is mediated by multiple causes. However, the genetic factors underlying infection susceptibility are poorly known and difficult to predict. Our study employed novel approaches and improved mouse disease models to identify genetic modifiers that affect the severity of P. aeruginosa lung infection. We identified candidate genes to enhance our understanding of P. aeruginosa infection in humans and provide a proof of concept that could be exploited for other human pathologies mediated by bacterial infection.

ABSTRACT

Cryptosporidium parvum and Cryptosporidium hominis have emerged as major enteric pathogens of infants in the developing world, in addition to their known importance in immunocompromised adults. Although there has been recent progress in identifying new small molecules that inhibit Cryptosporidium sp. growth in vitro or in animal models, we lack information about their mechanism of action, potency across the life cycle, and cidal versus static activities. Here, we explored four potent classes of compounds that include inhibitors that likely target phosphatidylinositol 4 kinase (PI4K), phenylalanine-tRNA synthetase (PheRS), and several potent inhibitors with unknown mechanisms of action. We utilized monoclonal antibodies and gene expression probes for staging life cycle development to define the timing of when inhibitors were active during the life cycle of Cryptosporidium parvum grown in vitro. These different classes of inhibitors targeted different stages of the life cycle, including compounds that blocked replication (PheRS inhibitors), prevented the segmentation of daughter cells and thus blocked egress (PI4K inhibitors), or affected sexual-stage development (a piperazine compound of unknown mechanism). Long-term cultivation of C. parvum in epithelial cell monolayers derived from intestinal stem cells was used to distinguish between cidal and static activities based on the ability of parasites to recover from treatment. Collectively, these approaches should aid in identifying mechanisms of action and for designing in vivo efficacy studies based on time-dependent concentrations needed to achieve cidal activity.

IMPORTANCE Currently, nitazoxanide is the only FDA-approved treatment for cryptosporidiosis; unfortunately, it is ineffective in immunocompromised patients, has varied efficacy in immunocompetent individuals, and is not approved in infants under 1 year of age. Identifying new inhibitors for the treatment of cryptosporidiosis requires standardized and quantifiable in vitro assays for assessing potency, selectivity, timing of activity, and reversibility. Here, we provide new protocols for defining which stages of the life cycle are susceptible to four highly active compound classes that likely inhibit different targets in the parasite. We also utilize a newly developed long-term culture system to define assays for monitoring reversibility as a means of defining cidal activity as a function of concentration and time of treatment. These assays should provide valuable in vitro parameters to establish conditions for efficacious in vivo treatment.

ABSTRACT

Anti-galactose-α-1,3-galactose (anti-α-Gal) antibody is naturally expressed at a high level in humans. It constitutes about 1% of immunoglobulins found in human blood. Here, we designed a live attenuated influenza virus vaccine that can generate α-Gal epitopes in infected cells in order to facilitate opsonization of infected cells, thereby enhancing vaccine-induced immune responses. In the presence of normal human sera, cells infected with this mutant can enhance phagocytosis of human macrophages and cytotoxicity of NK cells in vitro. Using a knockout mouse strain that allows expression of anti-α-Gal antibody in vivo, we showed that this strategy can increase vaccine immunogenicity and the breadth of protection. This vaccine can induce 100% protection against a lethal heterosubtypic group 1 (H5) or group 2 (mouse-adapted H3) influenza virus challenge in the mouse model. In contrast, its heterosubtypic protective effect in wild-type or knockout mice that do not have anti-α-Gal antibody expression is only partial, demonstrating that the enhanced vaccine-induced protection requires anti-α-Gal antibody upon vaccination. Anti-α-Gal-expressing knockout mice immunized with this vaccine produce robust humoral and cell-mediated responses upon a lethal virus challenge. This vaccine can stimulate CD11b^lo/– pulmonary dendritic cells, which are known to be crucial for clearance of influenza virus. Our approach provides a novel strategy for developing next-generation influenza virus vaccines.

IMPORTANCE Influenza A viruses have multiple HA subtypes that are antigenically diverse. Classical influenza virus vaccines are subtype specific, and they cannot induce satisfactory heterosubtypic immunity against multiple influenza virus subtypes. Here, we developed a live attenuated H1N1 influenza virus vaccine that allows the expression of α-Gal epitopes by infected cells. Anti-α-Gal antibody is naturally produced by humans. In the presence of this antibody, human cells infected with this experimental vaccine virus can enhance several antibody-mediated immune responses in vitro. Importantly, mice expressing anti-α-Gal antibody in vivo can be fully protected by this H1N1 vaccine against a lethal H5 or H3 virus challenge. Our work demonstrates a new strategy for using a single influenza virus strain to induce broadly cross-reactive immune responses against different influenza virus subtypes.

ABSTRACT

Over the last decade, innate immune priming has been evidenced in many invertebrate phyla. If mechanistic models have been proposed, molecular studies aiming to substantiate these models have remained scarce. We reveal here the transcriptional signature associated with immune priming in the oyster Crassostrea gigas. Oysters were fully protected against Ostreid herpesvirus 1 (OsHV-1), a major oyster pathogen, after priming with poly(I·C), which mimics viral double-stranded RNA. Global analysis through RNA sequencing of oyster and viral genes after immune priming and viral infection revealed that poly(I·C) induces a strong antiviral response that impairs OsHV-1 replication. Protection is based on a sustained upregulation of immune genes, notably genes involved in the interferon pathway and apoptosis, which control subsequent viral infection. This persistent antiviral alert state remains active over 4 months and supports antiviral protection in the long term. This acquired resistance mechanism reinforces the molecular foundations of the sustained response model of immune priming. It further opens the way to applications (pseudovaccination) to cope with a recurrent disease that causes dramatic economic losses in the shellfish farming industry worldwide.

IMPORTANCE In the last decade, important discoveries have shown that resistance to reinfection can be achieved without a functional adaptive immune system, introducing the concept of innate immune memory in invertebrates. However, this field has been constrained by the limited number of molecular mechanisms evidenced to support these phenomena. Taking advantage of an invertebrate species, the Pacific oyster (Crassostrea gigas), in which we evidenced one of the longest and most effective periods of protection against viral infection observed in an invertebrate, we provide the first comprehensive transcriptomic analysis of antiviral innate immune priming. We show that priming with poly(I·C) induced a massive upregulation of immune-related genes, which control subsequent viral infection, and it was maintained for over 4 months after priming. This acquired resistant mechanism reinforces the molecular foundations of the sustained response model of immune priming. It opens the way to pseudovaccination to prevent the recurrent diseases that currently afflict economically or ecologically important invertebrates.

ABSTRACT

The cell cycle is a critical component of cellular proliferation, differentiation, and response to stress, yet its role in the regulation of intracellular symbioses is not well understood. To explore host-symbiont cell cycle coordination in a marine symbiosis, we employed a model for coral-dinoflagellate associations: the tropical sea anemone Aiptasia (Exaiptasia pallida) and its native microalgal photosymbionts (Breviolum minutum and Breviolum psygmophilum). Using fluorescent labeling and spatial point-pattern image analyses to characterize cell population distributions in both partners, we developed protocols that are tailored to the three-dimensional cellular landscape of a symbiotic sea anemone tentacle. Introducing cultured symbiont cells to symbiont-free adult hosts increased overall host cell proliferation rates. The acceleration occurred predominantly in the symbiont-containing gastrodermis near clusters of symbionts but was also observed in symbiont-free epidermal tissue layers, indicating that the presence of symbionts contributes to elevated proliferation rates in the entire host during colonization. Symbiont cell cycle progression differed between cultured algae and those residing within hosts; the endosymbiotic state resulted in increased S-phase but decreased G₂/M-phase symbiont populations. These phenotypes and the deceleration of cell cycle progression varied with symbiont identity and host nutritional status. These results demonstrate that host and symbiont cells have substantial and species-specific effects on the proliferation rates of their mutualistic partners. This is the first empirical evidence to support species-specific regulation of the symbiont cell cycle within a single cnidarian-dinoflagellate association; similar regulatory mechanisms likely govern interpartner coordination in other coral-algal symbioses and shape their ecophysiological responses to a changing climate.

IMPORTANCE Biomass regulation is critical to the overall health of cnidarian-dinoflagellate symbioses. Despite the central role of the cell cycle in the growth and proliferation of cnidarian host cells and dinoflagellate symbionts, there are few studies that have examined the potential for host-symbiont coregulation. This study provides evidence for the acceleration of host cell proliferation when in local proximity to clusters of symbionts within cnidarian tentacles. The findings suggest that symbionts augment the cell cycle of not only their enveloping host cells but also neighboring cells in the epidermis and gastrodermis. This provides a possible mechanism for rapid colonization of cnidarian tissues. In addition, the cell cycles of symbionts differed depending on nutritional regime, symbiotic state, and species identity. The responses of cell cycle profiles to these different factors implicate a role for species-specific regulation of symbiont cell cycles within host cnidarian tissues.

ABSTRACT

The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3' untranslated regions (UTRs). The umbravirus Pea enation mosaic virus 2 (PEMV2) is a nonsegmented, positive-sense RNA virus with an unusually long 3' UTR that is susceptible to NMD. To establish a systemic infection, the PEMV2 long-distance movement protein p26 was previously shown to both stabilize viral RNAs and bind them for transport through the plant’s vascular system. The current study demonstrated that p26 protects both viral and nonviral messenger RNAs from NMD. Although p26 localizes to both the cytoplasm and nucleolus, p26 exerts its anti-NMD effects exclusively in the cytoplasm independently of long-distance movement. Using a transcriptome-wide approach in the model plant Nicotiana benthamiana, p26 protected a subset of cellular NMD target transcripts, particularly those containing long, structured, GC-rich 3' UTRs. Furthermore, transcriptome sequencing (RNA-seq) revealed that the NMD pathway is highly dysfunctional during PEMV2 infection, with 1,820 (48%) of NMD targets increasing in abundance. Widespread changes in the host transcriptome are common during plant RNA virus infections, and these results suggest that, in at least some instances, virus-mediated NMD inhibition may be a major contributing factor.

IMPORTANCE Nonsense-mediated decay (NMD) represents an RNA regulatory pathway that degrades both natural and faulty messenger RNAs with long 3' untranslated regions. NMD targets diverse families of RNA viruses, requiring that viruses counteract the NMD pathway for successful amplification in host cells. A protein required for long-distance movement of Pea enation mosaic virus 2 (PEMV2) is shown to also protect both viral and host mRNAs from NMD. RNA-seq analyses of the Nicotiana benthamiana transcriptome revealed that PEMV2 infection significantly impairs the host NMD pathway. RNA viruses routinely induce large-scale changes in host gene expression, and, like PEMV2, may use NMD inhibition to alter the host transcriptome in an effort to increase virus amplification.