Discovery and structural characterization of the D-box, a conserved TonB motif that couples an inner-membrane motor to outer-membrane transport.

Gram-negative bacteria use TonB-dependent transport to take up nutrients from the external environment, employing the Ton complex to import a variety of nutrients that are either scarce or too large to cross the outer membrane unaided. The Ton complex contains an inner-membrane motor (ExbBD) that generates force, as well as nutrient-specific transport proteins on the outer membrane. These two components are coupled by TonB, which transmits the force from the inner to the outer membrane. TonB contains an N-terminus anchored in the inner membrane, a C-terminal domain that binds the outer-membrane transporter, and a proline-rich linker connecting the two. While much is known about the interaction between TonB and outer-membrane transporters, the critical interface between TonB and ExbBD is less well understood. Here, we identify a conserved motif within TonB that we term the D-box, which serves as an attachment point for ExbD. We characterize the interaction between ExbD and the D-box both functionally and structurally, showing that a homodimer of ExbD captures one copy of the D-box peptide via beta-strand recruitment. We additionally show that both the D-box motif and ExbD are conserved in a range of Gram-negative bacteria, including members of the ESKAPE group of pathogens. The ExbD:D-box interaction is likely to represent an important aspect of force transduction between the inner and outer membranes. Given that TonB-dependent transport is an important contributor to virulence, this interaction is an intriguing potential target for novel antibacterial therapies.

Dental Care Needs of Male versus Female Children Visiting a School-based Mobile Dental Facility in West Virginia.

Purpose: Many school-aged children have not received dental care in West Virginia, despite mandated statewide requirements of a dental evaluation and dental treatment before entering school, and the provision of Medicaid/CHIP insurance coverage for children from families below the federal poverty level. An innovative mobile oral health program to educate children, provide preventive care, and bring technology to public schools was developed for West Virginia children in a need shortage area. It was unknown if the unmet dental needs challenge was greater for male or female children residing in that area. The purpose of this study was to determine whether there was a difference by sex in the number of attendees and the incidence of dental caries for children who visited a school-based mobile dental facility.Methods: School-aged children who had not had a dental examination within the previous year were offered school-based examinations/assessments, preventive care, and oral health education via a mobile oral health program following parental/guardian consent. Data were collected concerning the number of current carious teeth in need of restoration. Descriptive statistics and chi square analyses were conducted to analyze the data.Results: There were 429 students evaluated at the school-based mobile dental facility. Half (50.3%) were male. Referrals for additional necessary oral/medical care were made for 214 (50.1%) children; 45.9% of males and 53.3% of females (p= 0.287) had dental caries.Conclusion: Results from this study indicate that sex was not a statistically significant factor in school-based mobile dental facility attendance nor in current dental caries incidence among school-aged children in an underserved area of West Virginia.

Ste24: An Integral Membrane Protein Zinc Metalloprotease with Provocative Structure and Emergent Biology.

Ste24, an integral membrane protein zinc metalloprotease, is found in every kingdom of eukaryotes. It was discovered approximately 20 years ago by yeast genetic screens identifying it as a factor responsible for processing the yeast mating a-factor pheromone. In animals, Ste24 processes prelamin A, a component of the nuclear lamina; mutations in the human ortholog of Ste24 diminish its activity, giving rise to genetic diseases of accelerated aging (progerias). Additionally, lipodystrophy, acquired from the standard highly active antiretroviral therapy used to treat AIDS patients, likely results from off-target interactions of HIV (aspartyl) protease inhibitor drugs with Ste24. Ste24 possesses a novel "α-barrel" structure, consisting of a ring of seven transmembrane α-helices enclosing a large (>12,000 Å3) interior volume that contains the active-site and substrate-binding region; this "membrane-interior reaction chamber" is unprecedented in integral membrane protein structures. Additionally, the surface of the membrane-interior reaction chamber possesses a strikingly large negative electrostatic surface potential, adding additional "functional mystery." Recent publications implicate Ste24 as a key factor in several endoplasmic reticulum processes, including the unfolded protein response, a cellular stress response of the endoplasmic reticulum, and removal of misfolded proteins from the translocon. Ste24, with its provocative structure, enigmatic mechanism, and recently emergent new biological roles including "translocon unclogger" and (non-enyzmatic) broad-spectrum viral restriction factor, presents far differently than before 2016, when it was viewed as a "CAAX protease" responsible for cleavage of prenylated (farnesylated or geranylgeranylated) substrates. The emphasis of this review is on Ste24 of the "Post-CAAX-Protease Era."

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24.

Ste24 enzymes, a family of eukaryotic integral membrane proteins, are zinc metalloproteases (ZMPs) originally characterized as "CAAX proteases" targeting prenylated substrates, including a-factor mating pheromone in yeast and prelamin A in humans. Recently, Ste24 was shown to also cleave nonprenylated substrates. Reduced activity of the human ortholog, HsSte24, is linked to multiple disease states (laminopathies), including progerias and lipid disorders. Ste24 possesses a unique "α-barrel" structure consisting of seven transmembrane (TM) α-helices encircling a large intramembranous cavity (~14 000 Å3 ). The catalytic zinc, coordinated via a HExxH…E/H motif characteristic of gluzincin ZMPs, is positioned at one of the cavity's bases. The interrelationship between Ste24 as a gluzincin, a long-studied class of soluble ZMPs, and as a novel cavity-containing integral membrane protein protease has been minimally explored to date. Informed by homology to well-characterized soluble, gluzincin ZMPs, we develop a model of Ste24 that provides a conceptual framework for this enzyme family, suitable for development and interpretation of structure/function studies. The model consists of an interfacial, zinc-containing "ZMP Core" module surrounded by a "ZMP Accessory" module, both capped by a TM helical "α-barrel" module of as yet unknown function. Multiple sequence alignment of 58 Ste24 orthologs revealed 38 absolutely conserved residues, apportioned unequally among the ZMP Core (18), ZMP Accessory (13), and α-barrel (7) modules. This Tripartite Architecture representation of Ste24 provides a unified image of this enzyme family.

Phosphoramidon inhibits the integral membrane protein zinc metalloprotease ZMPSTE24.

The integral membrane protein zinc metalloprotease ZMPSTE24 possesses a completely novel structure, comprising seven long kinked transmembrane helices that encircle a voluminous 14 000 Å3 cavity within the membrane. Functionally conserved soluble zinc metalloprotease residues are contained within this cavity. As part of an effort to understand the structural and functional relationships between ZMPSTE24 and soluble zinc metalloproteases, the inhibition of ZMPSTE24 by phosphoramidon [N-(α-rhamnopyranosyl-oxyhydroxyphosphinyl)-Leu-Trp], a transition-state analog and competitive inhibitor of multiple soluble zinc metalloproteases, especially gluzincins, has been characterized functionally and structurally. The functional results, the determination of preliminary IC50 values by the use of an intramolecular quenched-fluorescence fluorogenic peptide assay, indicate that phosphoramidon inhibits ZMPSTE24 in a manner consistent with competitive inhibition. The structural results, a 3.85 Šresolution X-ray crystal structure of a ZMPSTE24-phosphoramidon complex, indicate that the overall binding mode observed between phosphoramidon and soluble gluzincins is conserved. Based on the structural data, a significantly lower potency than that observed for soluble gluzincins such as thermolysin and neprilysin is predicted. These results strongly suggest a close relationship between soluble gluzincins and the integral membrane protein zinc metalloprotease ZMPSTE24.

Acquisition of accurate data from intramolecular quenched fluorescence protease assays.

The Intramolecular Quenched Fluorescence (IQF) protease assay utilizes peptide substrates containing donor-quencher pairs that flank the scissile bond. Following protease cleavage, the dequenched donor emission of the product is subsequently measured. Inspection of the IQF literature indicates that rigorous treatment of systematic errors in observed fluorescence arising from inner-filter absorbance (IF) and non-specific intermolecular quenching (NSQ) is incompletely performed. As substrate and product concentrations vary during the time-course of enzyme activity, iterative solution of the kinetic rate equations is, generally, required to obtain the proper time-dependent correction to the initial velocity fluorescence data. Here, we demonstrate that, if the IQF assay is performed under conditions where IF and NSQ are approximately constant during the measurement of initial velocity for a given initial substrate concentration, then a simple correction as a function of initial substrate concentration can be derived and utilized to obtain accurate initial velocity data for analysis.

Ste24p Mediates Proteolysis of Both Isoprenylated and Non-prenylated Oligopeptides.

Rce1p and Ste24p are integral membrane proteins involved in the proteolytic maturation of isoprenylated proteins. Extensive published evidence indicates that Rce1p requires the isoprenyl moiety as an important substrate determinant. By contrast, we report that Ste24p can cleave both isoprenylated and non-prenylated substrates in vitro, indicating that the isoprenyl moiety is not required for substrate recognition. Steady-state enzyme kinetics are significantly different for prenylated versus non-prenylated substrates, strongly suggestive of a role for substrate-membrane interaction in protease function. Mass spectroscopy analyses identify a cleavage preference at bonds where P1' is aliphatic in both isoprenylated and non-prenylated substrates, although this is not necessarily predictive. The identified cleavage sites are not at a fixed distance position relative to the C terminus. In this study, the substrates cleaved by Ste24p are based on known isoprenylated proteins (i.e. K-Ras4b and the yeast a-factor mating pheromone) and non-prenylated biological peptides (Aß and insulin chains) that are known substrates of the M16A family of soluble zinc-dependent metalloproteases. These results establish that the substrate profile of Ste24p is broader than anticipated, being more similar to that of the M16A protease family than that of the Rce1p CAAX protease with which it has been functionally associated.

Shortened dental arch and body mass index in adults 45-65 years of age: results from National Health and Nutrition Examination Survey 2005-2008.

OBJECTIVE: Many people have dental arches with unrestored edentulous areas posterior to natural teeth. One dental pattern is the shortened dental arch (SDA). As a result of the lack of teeth, individuals with an SDA may eat a restricted diet, including soft, highly processed foods. Such diets may increase the risk of being overweight or of obesity. We examined whether there was an association between SDA and body mass index (BMI) in adults 45-65 years of age. METHODS: The data for this study were US National Health and Nutrition Examination Survey (NHANES) 2005-2008 merged files. There were 5,773 eligible participants. The data were examined for frequencies, and the results were analysed using the chi-square test and logistic regression. RESULTS: There were 69.3% participants with a shortened mandibular arch and a BMI of ≥25 compared with 71.8% of participants who had a complete mandibular dental arch and a BMI of ≥25 (P=0.7246). There were 70.6% of participants with a shortened maxillary arch and a BMI of ≥25 compared with 71.9% of participants who had a complete maxillary dental arch and a BMI of ≥25 (P=0.8859). The adjusted odds ratio for shortened mandibular dental arch was 0.70 (95% CI: 0.46-1.08) for a BMI of ≥25 as compared with individuals with a BMI<25. The adjusted odds ratio for shortened maxillary dental arch was 1.06 (95% CI: 0.63-1.78) as compared with individuals with a BMI<25. CONCLUSIONS: The research hypothesis that an SDA was related to higher BMI, and the corollary that restored or complete dentition had better odds of a lower BMI, were not supported.

Integral membrane proteins and free electron lasers - a compatible couple indeed!

Several structures of membrane transport proteins in complex with mechanistically-relevant ligands, determined by serial femtosecond crystallography of microcrystals at an X-ray free-electron source source, are presented. These results, including investigation of approaches to data quality assessment and refinement from low-redundancy data, indicate the feasibility of using this approach for ligand screening.

Comorbid depression/anxiety and teeth removed: Behavioral Risk Factor Surveillance System 2010.

OBJECTIVE: The purpose of this study was to examine the association between participants (i) who reported having had clinical diagnoses of depression and anxiety with 6+ teeth removed and (ii) who reported having had clinical diagnoses of depression and anxiety with edentulism. METHODS: The Behavioral Risk Factor Surveillance System (BRFSS) Survey 2010 was used for the study. Analyses involved using SAS 9.3® to determine variable frequencies, Rao-Scott chi-square bivariate analyses, and Proc Surveylogistic for the logistic regressions on complex survey designs. Participants eligibility included being 18 years or older and having complete data on depression, anxiety, and number of teeth removed. RESULTS: There were 76 292 eligible participants; 13.4% reported an anxiety diagnosis, 16.7% reported a depression diagnosis, and 8.6% reported comorbid depression and anxiety. The adjusted logistic regression models were significant for anxiety and depression alone and in combination for 6+ teeth removed (AOR: anxiety 1.23; 95% CI: 1.10, 1.38; P = 0.0773; AOR: depression 1.23; 95% CI: 1.10, 1.37; P = 0.0275; P < 0.0001; and AOR: comorbid depression and anxiety 1.30; 95% CI: 1.14, 1.49; P = 0.0001). However, the adjusted models with edentulism as the outcome failed to reach significance. CONCLUSIONS: Comorbid depression and anxiety are associated independently with 6+ teeth removed compared with 0-5 teeth removed in a national study conducted in United States. Comorbid depression and anxiety were not shown to be associated with edentulism as compared with any teeth present.

A critical evaluation of in silico methods for detection of membrane protein intrinsic disorder.

Intrinsically disordered regions in proteins possess important biological roles including transcriptional regulation, molecular recognition, and provision of sites for posttranslational modification. In three-dimensional crystallization of both soluble and membrane proteins, identification and removal of disordered regions is often necessary for obtaining crystals possessing sufficient long-range order for structure determination. Disordered regions can be identified experimentally, with techniques such as limited proteolysis coupled with mass spectrometry, or computationally, by using disorder prediction programs, of which many are available. Although these programs use various methods to predict disorder from a protein's primary sequence, they all were developed using information derived from soluble protein structures. Therefore, their performance and accuracy when applied to integral membrane proteins remained an open question. We evaluated the performance of 13 disorder prediction programs on a dataset containing 343 membrane proteins, and upon subdatasets containing only α-helical or ß-barrel proteins. These programs were ranked using multiple metrics, including metrics specifically created for membrane proteins. Analysis of these data shows a clear distinction between programs that accurately predict disordered regions in membrane proteins and programs which perform poorly, and allows for the robust integration of in silico disorder prediction into our PSI:Biology membrane protein structural genomics pipeline.

Structure of the integral membrane protein CAAX protease Ste24p.

Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a-factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.

Hygiene self-care of older adults in West Virginia: effects of gender.

PURPOSE: This study investigated whether oral hygiene self-care behavior differs between genders in older adults in Appalachia, a geographic area with significant oral health concerns. Identify ing the practices of older adults may provide valuable information for designing interventions and improving overall oral health outcomes. METHODS: As part of a larger, on-going study on cognition and oral health in later life in Appalachia, a sample of dentate, older adults without dementia aged 70 and above (n=245, 86 men and 159 women) received an oral assessment by either a dentist or dental hygienist. Psychometricians assessed cognition using a standardized battery of neuropsychological tests. They also administered the General Oral Health Assessment Index and conducted structured interviews concerning diet, oral hygiene practices, oral health, social support, income and years of education Results: Over 80% of women (n=128) and 52.3% of men (n=45) reported brushing their teeth twice daily. Multivariate logistic regression analysis was conducted, controlling for socioeconomic status, social support (i.e., frequency of contacting friends and relatives), general oral health assessment items, number of decayed, missing and filled surfaces, plaque index and having regular dental visits. The results showed that women reported more frequent tooth brushing than their male counterparts (OR=4.04, 95% CI:1.93,8.42). CONCLUSION: Older women in West Virginia had significantly better oral hygiene practices than older men, particularly regarding tooth brushing. Interventions are needed to improve older men's dental hygiene behaviors to improve overall oral health outcomes.

The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal.

Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (enterokinase, factor Xa, human rhinovirus 3C protease, SUMOstar, tobacco etch virus protease, and thrombin) by use of a panel of 94 individual detergents. Thrombin activity was insensitive to the entire panel of detergents, thus suggesting it as the optimal choice for use with membrane proteins. Enterokinase and factor Xa were only affected by a small number of detergents, making them good choices as well.

Do modern conflicts create different medical needs?

PURPOSES: This study involved analysis of (a) type of injuries and medical services used by military casualties in 3 conflicts and (b) medical needs of military and civilian casualties from the 2006 conflict. BASIC PROCEDURES: Military casualties from 3 conflicts and military and civilian casualties from the 2006 conflict were analyzed. Casualties were compared in relation to type of injury, length of stay (LOS), and operating room use (ORU). MAIN FINDINGS: Hospital LOS of soldiers in many departments decreased while increasing in intensive care unit (ICU). Type of injuries sustained by civilians and military casualties differed significantly. More civilian casualties required admission to ICU. Civilian ORU was higher for orthopedic and otolaryngological procedures. PRINCIPAL CONCLUSIONS: Civilians are less well protected during military conflicts, therefore, more susceptible to certain kind of injuries. Civilian and military casualties during conflicts have different medical needs. Civilians have higher morbidity than soldiers, which result in an increased need for treatment in ICU.

Multivariate statistical analysis of deformation momenta relating anatomical shape to neuropsychological measures.

The purpose of this study is to characterize the neuroanatomical variations observed in neurological disorders such as dementia. We do a global statistical analysis of brain anatomy and identify the relevant shape deformation patterns that explain corresponding variations in clinical neuropsychological measures. The motivation is to model the inherent relation between anatomical shape and clinical measures and evaluate its statistical significance. We use Partial Least Squares for the multivariate statistical analysis of the deformation momenta under the Large Deformation Diffeomorphic framework. The statistical methodology extracts pertinent directions in the momenta space and the clinical response space in terms of latent variables. We report the results of this analysis on 313 subjects from the Mild Cognitive Impairment group in the Alzheimer's Disease Neuroimaging Initiative (ADNI).

Recruitment of rural and cognitively impaired older adults for dental research.

The recruitment of community-dwelling older adults, particularly those with cognitive impairment and those residing in rural areas, has been consistently challenging for researchers, especially in the dental field. This study reports on recruitment experiences from an ongoing study investigating the association between oral health and cognitive status in later life. Multiple recruitment strategies, including educational presentations and traveling to participants' homes, were used to enroll rural elderly participants with various levels of cognitive function. In general, multipronged, proactive recruitment strategies were more effective than traditional, passive methods in reaching participants with varying degrees of cognitive impairment. The outcome of this study suggests that successful recruitment of such populations involves gaining the support of staff at relevant community organizations, informing community members (including older adults and their family members) of the project and the importance of oral health, and making data collection sites accessible for older adults.

Conformational exchange in a membrane transport protein is altered in protein crystals.

Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B(12). Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.