FipB, an essential virulence factor of Francisella tularensis subsp. tularensis, has dual roles in disulfide bond formation.

FipB, an essential virulence factor of Francisella tularensis, is a lipoprotein with two conserved domains that have similarity to disulfide bond formation A (DsbA) proteins and the amino-terminal dimerization domain of macrophage infectivity potentiator (Mip) proteins, which are proteins with peptidyl-prolyl cis/trans isomerase activity. This combination of conserved domains is unusual, so we further characterized the enzymatic activity and the importance of the Mip domain and lipid modification in virulence. Unlike typical DsbA proteins, which are oxidases, FipB exhibited both oxidase and isomerase activities. FipA, which also shares similarity with Mip proteins, potentiated the isomerase activity of FipB in an in vitro assay and within the bacteria, as measured by increased copper sensitivity. To determine the importance of the Mip domain and lipid modification of FipB, mutants producing FipB proteins that lacked either the Mip domain or the critical cysteine necessary for lipid modification were constructed. Both strains replicated within host cells and retained virulence in mice, though there was some attenuation. FipB formed surface-exposed dimers that were sensitive to dithiothreitol (DTT), dependent on the Mip domain and on at least one cysteine in the active site of the DsbA-like domain. However, these dimers were not essential for virulence, because the Mip deletion mutant, which failed to form dimers, was still able to replicate intracellularly and retained virulence in mice. Thus, the Mip domains of FipB and FipA impart additional isomerase functionality to FipB, but only the DsbA-like domain and oxidase activity are essential for its critical virulence functions.

Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms: carbon and energy flow contribute to the distinct biofilm growth state.

BACKGROUND: Desulfovibrio vulgaris Hildenborough is a sulfate-reducing bacterium (SRB) that is intensively studied in the context of metal corrosion and heavy-metal bioremediation, and SRB populations are commonly observed in pipe and subsurface environments as surface-associated populations. In order to elucidate physiological changes associated with biofilm growth at both the transcript and protein level, transcriptomic and proteomic analyses were done on mature biofilm cells and compared to both batch and reactor planktonic populations. The biofilms were cultivated with lactate and sulfate in a continuously fed biofilm reactor, and compared to both batch and reactor planktonic populations. RESULTS: The functional genomic analysis demonstrated that biofilm cells were different compared to planktonic cells, and the majority of altered abundances for genes and proteins were annotated as hypothetical (unknown function), energy conservation, amino acid metabolism, and signal transduction. Genes and proteins that showed similar trends in detected levels were particularly involved in energy conservation such as increases in an annotated ech hydrogenase, formate dehydrogenase, pyruvate:ferredoxin oxidoreductase, and rnf oxidoreductase, and the biofilm cells had elevated formate dehydrogenase activity. Several other hydrogenases and formate dehydrogenases also showed an increased protein level, while decreased transcript and protein levels were observed for putative coo hydrogenase as well as a lactate permease and hyp hydrogenases for biofilm cells. Genes annotated for amino acid synthesis and nitrogen utilization were also predominant changers within the biofilm state. Ribosomal transcripts and proteins were notably decreased within the biofilm cells compared to exponential-phase cells but were not as low as levels observed in planktonic, stationary-phase cells. Several putative, extracellular proteins (DVU1012, 1545) were also detected in the extracellular fraction from biofilm cells. CONCLUSIONS: Even though both the planktonic and biofilm cells were oxidizing lactate and reducing sulfate, the biofilm cells were physiologically distinct compared to planktonic growth states due to altered abundances of genes/proteins involved in carbon/energy flow and extracellular structures. In addition, average expression values for multiple rRNA transcripts and respiratory activity measurements indicated that biofilm cells were metabolically more similar to exponential-phase cells although biofilm cells are structured differently. The characterization of physiological advantages and constraints of the biofilm growth state for sulfate-reducing bacteria will provide insight into bioremediation applications as well as microbially-induced metal corrosion.

Supervised Study: Required Independent Research at a Community College Supports Persistence in Science.

This study assesses the impacts of the Science program at Piedmont Virginia Community College and its flagship capstone research experience, Supervised Study, through psychosocial perceptions associated with persistence in science and through a comparative analysis of subsequent science bachelor's degree attainment. Supervised Study involves authentic, independent projects, a research methods course and learning community, and one-on-one faculty mentoring. The Persistence in the Sciences survey was used as a repeated-measures instrument in four semesters of Supervised Study. Positive trends were observed for self-efficacy, science identity, community values, and networking, while responses related to project ownership were mixed (n = 13). To contextualize these observations, transfer and bachelor's degree completion rates were analyzed. Students who earn an associate's degree in Science (n = 113 between 2012 and 2019) complete bachelor's degrees at high rates (66.4%). Moreover, they are two to four times more likely to major in physical and natural sciences than their science-oriented peers, who take many of the same courses, with the exception of Supervised Study. Notably, these comparison rates remain consistent between different demographic groups. These findings further describe a model for research at the community college level that supports persistence in undergraduate science for a broad group of students.

Role of alcohols in growth, lipid composition, and membrane fluidity of yeasts, bacteria, and archaea.

Increased membrane fluidity, which causes cofactor leakage and loss of membrane potential, has long been documented as a cause for decreased cell growth during exposure to ethanol, butanol, and other alcohols. Reinforcement of the membrane with more complex lipid components is thus thought to be beneficial for the generation of more tolerant organisms. In this study, organisms with more complex membranes, namely, archaea, did not maintain high growth rates upon exposure to alcohols, indicating that more complex lipids do not necessarily fortify the membrane against the fluidizing effects of alcohols. In the presence of alcohols, shifts in lipid composition to more saturated and unbranched lipids were observed in most of the organisms tested, including archaea, yeasts, and bacteria. However, these shifts did not always result in a decrease in membrane fluidity or in greater tolerance of the organism to alcohol exposure. In general, organisms tolerating the highest concentrations of alcohols maintained membrane fluidity after alcohol exposure, whereas organisms that increased membrane rigidity were less tolerant. Altered lipid composition was a common response to alcohol exposure, with the most tolerant organisms maintaining a modestly fluid membrane. Our results demonstrate that increased membrane fluidity is not the sole cause of growth inhibition and that alcohols may also denature proteins within the membrane and cytosol, adversely affecting metabolism and decreasing cell growth.

Mental contrasting with implementation intentions increases study time for university students.

BACKGROUND: Goal setting is a core aspect of human behaviour that drives action. The intention to achieve one's goals, however, does not necessarily translate into desired outcomes. Although the mental contrasting with implementation intentions (MCII) strategy has demonstrated strong efficacy, limited investigations have been conducted in a university academic goal-setting context. AIMS: The current study sought to investigate the efficacy of MCII in facilitating academic goal attainment in university students. METHOD: Fifty-eight students from Macquarie University, Australia, were randomly allocated to either MCII or stress management training, and were assessed on their progress towards the target goal of increased hours of study four weeks later. Goal attainment scaling (GAS) facilitated the generation of tailored specific goals and was the primary outcome measure. RESULTS: An analysis of covariance indicated that students trained in MCII achieved significantly better goal outcomes than those trained in stress management for both broad (p = .038) and course- or unit-specific (p = .005) study goals. CONCLUSIONS: Results highlight the efficacy of using MCII and GAS in combination to promote increased study time for university students.

A Group Intervention for Individuals With Obesity and Comorbid Binge Eating Disorder: Results From a Feasibility Study.

Purpose: A common challenge among a subgroup of individuals with obesity is binge eating, that exists on a continuum from mild binge eating episodes to severe binge eating disorder (BED). BED is common among bariatric patients and the prevalence of disordered eating and ED in bariatric surgery populations is well known. Conventional treatments and assessment of obesity seldom address the underlying psychological mechanisms of binge eating and subsequent obesity. This study, titled PnP (People need People) is a psychoeducational group pilot intervention for individuals with BED and obesity including patients with previous bariatric surgery. Design, feasibility, and a broad description of the study population is reported. Material and Methods: A total of 42 patients were from an obesity clinic referred to assessment and treatment with PnP in a psychoeducational group setting (3-hour weekly meetings for 10 weeks). Of these, 6 (14.3%) patients had a previous history of bariatric surgery. Feasibility was assessed by tracking attendance, potentially adverse effects and outcome measures including body mass index (BMI), eating disorder pathology, overvaluation of shape and weight, impairment, self-reported childhood difficulties, alexithymia, internalized shame as well as health related quality of life (HRQoL). Results: All 42 patients completed the intervention, with no adverse effects and a high attendance rate with a median attendance of 10 sessions, 95% CI (8.9,9.6) and 0% attrition. Extent of psychosocial impairment due to eating disorder pathology, body dissatisfaction and severity of ED symptoms were high among the patients at baseline. Additionally, self-reported childhood difficulties, alexithymia, and internalized shame were high among the patients and indicate a need to address underlying psychological mechanisms in individuals with BED and comorbid obesity. Improvement of HRQoL and reduction of binge eating between baseline and the end of the intervention was observed with a medium effect. Conclusion: This feasibility study supports PnP as a potential group psychoeducational intervention for patients living with BED and comorbid obesity. Assessments of BED and delivery of this intervention may optimize selection of candidates and bariatric outcomes. These preliminary results warrant further investigation via a randomized control trial (RCT) to examine the efficacy and effectiveness of PnP.

Elucidating mechanisms of solvent toxicity in ethanologenic Escherichia coli.

Ethanol toxicity and its effect on ethanol production by the recombinant ethanologenic Escherichia coli strain KO11 were investigated in batch and continuous fermentation. During batch growth, ethanol produced by KO11 reduced both the specific cell growth rate (micro) and the cell yield (Y(X/S)). The extent of inhibition increased with the production of both acetate and lactate. Subsequent accumulation of these metabolites and ethanol resulted in cessation of cell growth, redirection of metabolism to reduce ethanol production, and increased requirements for cell maintenance. These effects were found to depend on both the glycolytic flux and the flux from pyruvate to ethanol. Pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) activities measured during the batch fermentation suggested that decreased ethanol production resulted from enzyme inhibition rather than down-regulation of genes in the ethanol-producing pathway. Ethanol was added in continuous fermentation to provide an ethanol concentration of either 17 or 27 g/L, triggering sustained oscillations in the cell growth rate. Cell concentrations oscillated in-phase with ethanol and acetate concentrations. The amplitude of oscillations depended on the concentration of ethanol in the fermentor. Through multiple oscillatory cycles, the yield (Y(P/S)) and concentration of ethanol decreased, while production of acetate increased. These results suggest that KO11 favorably adapted to improve growth by synthesizing more ATP though acetate production, and recycling NADH by producing more lactate and less ethanol. Implications of these results for strategies to improve ethanol production are described.

Fluoride Use in Caries Prevention in the Primary Care Setting.

Dental caries remains the most common chronic disease of childhood in the United States. Caries is a largely preventable condition, and fluoride has proven effectiveness in caries prevention. This clinical report aims to clarify the use of available fluoride modalities for caries prevention in the primary care setting and to assist pediatricians in using fluoride to achieve maximum protection against dental caries, while minimizing the likelihood of enamel fluorosis. Fluoride varnish application is now considered the standard of care in pediatric primary care. This report highlights administration, billing, and payment information regarding the fluoride varnish procedure.

Oral Development and Pathology.

BACKGROUND: The mouth is integral in the development of feeding, the initiation of digestion, and for speech and socialization. Signs of systemic disease and nutritional deficiencies often manifest in the mouth, and poor oral health can exacerbate many systemic conditions. METHODS: This review addresses the fetal development of the mouth, major anomalies, common minor physical findings, and pathologic conditions and their management. RESULTS: Pediatric practitioners have historically been poorly trained in diagnosis and management of oral conditions, so this article provides an overview of oral embryology and pathology, with a focus on hard and soft tissue disease identification, triage, and management. For primary prevention to be effective, pediatric providers must be knowledgeable about the process of dental caries, prevention of the disease, and available interventions, including fluoride. CONCLUSION: The embryology and anatomy of the oral cavity are complex, and the mouth is crucial to many physiologic processes. Pediatric primary care providers are uniquely positioned to prevent, identify, and triage dental caries, the most common chronic disease of childhood.

Curriculum influence on interdisciplinary oral health education and practice.

OBJECTIVES: Oral diseases are very prevalent across the lifespan and impact overall health, yet are largely preventable. The Smiles for Life (SFL) curriculum was created to educate healthcare providers about oral disease and support integration of oral health and primary care. This study examines SFL's influence on clinical practice and education. METHODS: Surveys were sent to registered users of SFL. Users who self-identified as direct care providers (DCPs), or educators, were included in the analysis. RESULTS: Survey response rate was 18 percent, with 87 percent identifying as DCPs and 13 percent as educators. Across professions, 85 percent of DCPs reported SFL influencing their practice to some degree, with variance among profession type and experience. DCPs most commonly reported that SFL led them to improve how they conduct oral health activities, with 60 percent performing the activity more skillfully following completion of SFL. Fluoride varnish application was the most common practice behavior initiated, and caries risk assessments was the oral health activity affected to the greatest degree. A majority of educators (94 percent) reported that SFL led them to incorporate or enhance oral health in their teaching. SFL helped educators emphasize the importance of oral health, improved their ability to teach content, raised motivation, and reduced barriers to teaching oral health. CONCLUSIONS: Data supports that SFL is positively influencing oral health practice across professions, especially in areas of caries risk assessment and fluoride varnish application. SFL improves the frequency and quality with which DCPs and educators participate in oral health activities, and facilitates oral health inclusion in primary care.

Components of the type six secretion system are substrates of Francisella tularensis Schu S4 DsbA-like FipB protein.

FipB, an essential virulence factor in the highly virulent Schu S4 strain of F. tularensis subsp. tularensis, shares sequence similarity with Disulfide Bond formation (Dsb) proteins, which can have oxidoreductase, isomerase, or chaperone activity. To further explore FipB's role in virulence potential substrates were identified by co-purification and 2D gel electrophoresis, followed by protein sequencing using mass spectrometry. A total of 119 potential substrates were identified. Proteins with predicted enzymatic activity were prevalent, and there were 19 proteins that had been previously identified as impacting virulence. Among the potential substrates were IglC, IglB, and PdpB, three components of the Francisella Type Six Secretion System (T6SS), which is also essential for virulence. T6SS are widespread in Gram-negative pathogens, but have not been reported to be dependent on Dsb-like proteins for assembly or function. The presented results suggest that FipB affects IglB and IglC substrates differently. In a fipB mutant there were differences in free sulfhydryl accessibility of IglC, but not IglB, when compared to wild-type bacteria. However, for both proteins FipB appears to act as a chaperone that facilitates proper folding and conformation. Understanding the role FipB plays the assembly and structure in this T6SS may reveal critical aspects of assembly that are common and novel among this widely distributed class of secretion systems.

Integrating Oral Health Into Overall Health Care to Prevent Early Childhood Caries: Need, Evidence, and Solutions.

Medicaid data shows that few one- to two-year-olds receive a preventive dental visit, indicating our limited success implementing the existing policy paradigm of dental home establishment by 12 months of age. Few pediatricians refer children for early dental care, few dentists are comfortable seeing children younger than two-years-old, fewer still provide restorative care, and many dentists do not accept Medicaid insurance. These realities mandate new strategies to meet the needs of children and families and effectively tackle early childhood caries (ECC). Primary care medical providers have frequent contact with families, providing opportunities to incorporate oral health promotion and prevention in non-dental settings. Components of such an approach include: screening; risk assessment; oral health counseling; fluoride varnish application; successful referral for children needing intense intervention; policy support; and financial incentives to sustain change. Current research indicates that oral health counseling, particularly motivational interviewing, and fluoride varnish applied in the non-dental setting positively affect patient outcomes. Cost savings may only be realized if ECC prevention programs use: support professionals; integrative disease management; and innovative insurance structures. The purpose of this paper was to examine the evidence for the effectiveness of the provision of oral health preventive services in the primary care setting.

Sangre de grado Croton palanostigma induces apoptosis in human gastrointestinal cancer cells.

Sangre de grado is an ethnomedicinal red tree sap obtained from Croton spp. that is used to treat gastrointestinal ulcers, cancer and to promote wound healing. To evaluate the potential role of sangre de grado (SdG) in cancer we examined its effects on human cancer cells, AGS (stomach), HT29 and T84 (colon). Viability of cells treated with SdG (10-200 microg/ml) decreased (P<0.01) in a dose dependent manner measured over a 24-h period. Cell proliferation at 48 h decreased (P<0.01) in all cells treated with SdG (>100 microg/ml). When cells in suspension were treated with SdG (100 microg/ml) cell adherence was severely compromised (>85%). Cells treated with SdG (100 microg/ml) underwent apoptosis as detected by nucleus condensation and DNA fragmentation determined by ELISA, and flow cytometry. Morphological changes as assessed by acridine orange. These effects were similar to that observed with Taxol (30 microM). A significant alteration of microtubular architecture was equally observed in both stomach and colon cancer cells exposed to SdG (100 microg/ml). The induction of apoptosis and microtubule damage in AGS, HT29 and T84 cells suggest that sangre de grado should be evaluated further as a potential source of anti-cancer agents.

Fluoride use in caries prevention in the primary care setting.

Dental caries remains the most common chronic disease of childhood in the United States. Caries is a largely preventable condition, and fluoride has proven effectiveness in the prevention of caries. The goals of this clinical report are to clarify the use of available fluoride modalities for caries prevention in the primary care setting and to assist pediatricians in using fluoride to achieve maximum protection against dental caries while minimizing the likelihood of enamel fluorosis.

A pediatric case of severe pandysautonomia responsive to plasmapheresis.

We describe a 13-year-old female with abrupt onset urinary retention progressing rapidly to pandysautonomia with symptoms of postural orthostatic tachycardia syndrome, gastroparesis, anhidrosis, pupillary dysfunction, and abdominal pain. Pandysautonomia has been reported frequently in adults, but is less commonly described in children. Autonomic nervous system dysfunction usually has a self-limiting course with gradual near-complete or complete recovery. Most patients with pure pandysautonomia produce an antibody targeted against the ganglionic nicotinic acetylcholine receptor and titers have been shown to correlate with symptom severity. The clinical presentation described in this report is consistent with a progressive form of acute autoimmune autonomic neuropathy, but she was initially seronegative for known autoantibodies. She responded promptly to plasmapheresis. This case report emphasizes the importance of recognizing features of autonomic nervous system dysfunction and discusses the medical evaluation and treatment options for pediatric patients based on symptom severity.

Nature versus nurture: developing enzymes that function under extreme conditions.

Many industrial processes used to produce chemicals and pharmaceuticals would benefit from enzymes that function under extreme conditions. Enzymes from extremophilic microorganisms have evolved to function in a variety of extreme environments, and bioprospecting for these microorganisms has led to the discovery of new enzymes with high tolerance to nonnatural conditions. However, bioprospecting is inherently limited by the diversity of enzymes evolved by nature. Protein engineering has also been successful in generating extremophilic enzymes by both rational mutagenesis and directed evolution, but screening for activity under extreme conditions can be difficult. This review examines the emerging synergy between bioprospecting and protein engineering in developing extremophilic enzymes. Specific topics include unnatural industrial conditions relevant to biocatalysis, biophysical properties of extremophilic enzymes, and industrially relevant extremophilic enzymes found either in nature or through protein engineering.

Identification and characterization of a multidomain hyperthermophilic cellulase from an archaeal enrichment.

Despite extensive studies on microbial and enzymatic lignocellulose degradation, relatively few Archaea are known to deconstruct crystalline cellulose. Here we describe a consortium of three hyperthermophilic archaea enriched from a continental geothermal source by growth at 90 °C on crystalline cellulose, representing the first instance of Archaea able to deconstruct lignocellulose optimally above 90 °C. Following metagenomic studies on the consortium, a 90 kDa, multidomain cellulase, annotated as a member of the TIM barrel glycosyl hydrolase superfamily, was characterized. The multidomain architecture of this protein is uncommon for hyperthermophilic endoglucanases, and two of the four domains of the enzyme have no characterized homologues. The recombinant enzyme has optimal activity at 109 °C, a half-life of 5 h at 100 °C, and resists denaturation in strong detergents, high-salt concentrations, and ionic liquids. Cellulases active above 100 °C may assist in biofuel production from lignocellulosic feedstocks by hydrolysing cellulose under conditions typically employed in biomass pretreatment.

Biofilm formation in Desulfovibrio vulgaris Hildenborough is dependent upon protein filaments.

Desulfovibrio vulgaris Hildenborough is a Gram-negative sulfate-reducing bacterium (SRB), and the physiology of SRBs can impact many anaerobic environments including radionuclide waste sites, oil reservoirs and metal pipelines. In an attempt to understand D. vulgaris as a population that can adhere to surfaces, D. vulgaris cultures were grown in a defined medium and analysed for carbohydrate production, motility and biofilm formation. Desulfovibrio vulgaris wild-type cells had increasing amounts of carbohydrate into stationary phase and approximately half of the carbohydrate remained internal. In comparison, a mutant that lacked the 200 kb megaplasmid, strain DeltaMP, produced less carbohydrate and the majority of carbohydrate remained internal of the cell proper. To assess the possibility of carbohydrate re-allocation, biofilm formation was investigated. Wild-type cells produced approximately threefold more biofilm on glass slides compared with DeltaMP; however, wild-type biofilm did not contain significant levels of exopolysaccharide. In addition, stains specific for extracellular carbohydrate did not reveal polysaccharide material within the biofilm. Desulfovibrio vulgaris wild-type biofilms contained long filaments as observed with scanning electron microscopy (SEM), and the biofilm-deficient DeltaMP strain was also deficient in motility. Biofilms grown directly on silica oxide transmission electron microscopy (TEM) grids did not contain significant levels of an exopolysaccharide matrix when viewed with TEM and SEM, and samples stained with ammonium molybdate also showed long filaments that resembled flagella. Biofilms subjected to protease treatments were degraded, and different proteases that were added at the time of inoculation inhibited biofilm formation. The data indicated that D. vulgaris did not produce an extensive exopolysaccharide matrix, used protein filaments to form biofilm between cells and silica oxide surfaces, and the filaments appeared to be flagella. It is likely that D. vulgaris used flagella for more than a means of locomotion to a surface, but also used flagella, or modified flagella, to establish and/or maintain biofilm structure.