Deriving an optimal threshold of waist circumference for detecting cardiometabolic risk in sub-Saharan Africa.

BACKGROUND: Waist circumference (WC) thresholds derived from western populations continue to be used in sub-Saharan Africa (SSA) despite increasing evidence of ethnic variation in the association between adiposity and cardiometabolic disease and availability of data from African populations. We aimed to derive a SSA-specific optimal WC cut-point for identifying individuals at increased cardiometabolic risk. METHODS: We used individual level cross-sectional data on 24 181 participants aged ⩾15 years from 17 studies conducted between 1990 and 2014 in eight countries in SSA. Receiver operating characteristic curves were used to derive optimal WC cut-points for detecting the presence of at least two components of metabolic syndrome (MS), excluding WC. RESULTS: The optimal WC cut-point was 81.2 cm (95% CI 78.5-83.8 cm) and 81.0 cm (95% CI 79.2-82.8 cm) for men and women, respectively, with comparable accuracy in men and women. Sensitivity was higher in women (64%, 95% CI 63-65) than in men (53%, 95% CI 51-55), and increased with the prevalence of obesity. Having WC above the derived cut-point was associated with a twofold probability of having at least two components of MS (age-adjusted odds ratio 2.6, 95% CI 2.4-2.9, for men and 2.2, 95% CI 2.0-2.3, for women). CONCLUSION: The optimal WC cut-point for identifying men at increased cardiometabolic risk is lower (⩾81.2 cm) than current guidelines (⩾94.0 cm) recommend, and similar to that in women in SSA. Prospective studies are needed to confirm these cut-points based on cardiometabolic outcomes.International Journal of Obesity advance online publication, 31 October 2017; doi:10.1038/ijo.2017.240.

Most-probable-number rapid viability PCR method to detect viable spores of Bacillus anthracis in swab samples.

A comparison of Most-Probable-Number Rapid Viability (MPN RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs from a multi-center validation study was performed. The purpose of the study was to compare environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were typically within 1-log of the values from a plate count method for all three levels of spores tested (3.1x10(4), 400, and 40 spores sampled from surfaces with swabs) even in the presence of debris. The MPN method tended to overestimate the expected result, especially at lower spore levels. Blind negative samples were correctly identified using both methods showing a lack of cross contamination. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols, enhancing its utility for characterization and clearance following a biothreat agent release.

Rapid, high-throughput, culture-based PCR methods to analyze samples for viable spores of Bacillus anthracis and its surrogates.

To rapidly remediate facilities after a biothreat agent release, improved turnaround times are needed for sample analysis. Current methods to confirm the presence of a viable biothreat agent are limited by low sample throughput. We have developed a rapid-viability-polymerase chain reaction (RV-PCR) method to determine the presence of viable spores. The method combines high-throughput sample processing with 96-well PCR analysis, which measures a change in real-time, quantitative PCR response arising from increased target-cell populations during culturing. The method accurately detects 1 to 10 live spores in a high-dead spore background (10(6)). Field tests using approximately 1000 biological indicators, each containing 10(6) spores of the B. anthracis surrogate, Bacillus atrophaeus, exposed to seven lethal and sub-lethal chlorine dioxide levels showed no significant difference (p>0.05) between RV-PCR and standard culturing methods for detecting the percent survival of spores. RV-PCR results were obtained in <17 h compared to 7 days for the standard culturing method. High-throughput sample processing and RV-PCR protocols were also developed and tested for synthetic wipe samples containing reference dirt material. RV-PCR protocols allowed processing and accurate analysis of approximately100 dirty wipe samples (2''x2'' synthetic) containing approximately10 viable B. atrophaeus spores in <24 h. Quantitative RV-PCR protocols based on a Most-Probable-Number (MPN) statistical approach developed for B. anthracis Sterne resulted in more rapid turnaround times than those for traditional culturing and no significant difference in log colony-forming units compared to traditional viability analysis. Integration of RV-PCR assays with high-throughput protocols will allow the processing of 200 wipe samples per day per robot using commercially available automation.

Elucidation of some Bax conformational changes through crystallization of an antibody-peptide complex.

The Bcl-2 family member Bax plays a critical role in apoptosis. In healthy resting cells, Bax resides in the cytoplasm and loosely attached to the mitochondrial membrane. Apoptotic stimuli induce Bax activation, which is characterized by translocation and multimerization on the mitochondrial membrane surface resulting in exposure of an amino terminal epitope recognized by the monoclonal antibody 6A7. To understand the structural changes that occur during Bax activation, we determined the crystal structure of a Bax peptide bound to the 6A7 Fab fragment to a resolution of 2.3 A. The structure reveals the conformation of the 6A7 peptide epitope on Bax in the activated form and elucidates the extensive structural changes that Bax must undergo during the conversion from its native to its activated conformation.

Synthesis of dinucleoside tetraphosphates in transfected cells by a firefly luciferase reporter gene.

The firefly luciferase gene is widely used as a reporter gene and its expression is generally considered to be non-toxic. In addition to its light-producing reaction, luciferase can synthesise dinucleoside polyphosphates, intracellular signalling molecules, in vitro. Here we show that COS-7 cells transfected with a luciferase expression vector accumulate up to 0.5 mM adenine-containing dinucleoside tetraphosphates (Ap4N) during the 24 h following luciferin addition. The optimal external concentration of luciferin was 0.4-0.6 mM. In agreement with its poor ability to synthesise adenine-containing dinucleoside triphosphates in vitro, the level of these compounds did not increase after transfection. Consequently, the results of experiments involving luciferase-mediated light production by live cells should now be viewed in the light of the possible effects of an increased intracellular Ap4N concentration on the properties of the system under investigation. This observation also points to a useful non-invasive procedure for the specific enhancement of intracellular Ap4N for studies directed at understanding the functions of these compounds.

RERG is a novel ras-related, estrogen-regulated and growth-inhibitory gene in breast cancer.

Using microarray analysis, we identified a unique ras superfamily gene, termed RERG (ras-related and estrogen-regulated growth inhibitor), whose expression was decreased or lost in a significant percentage of primary human breast tumors that show a poor clinical prognosis. Importantly, high RERG expression correlated with expression of a set of genes that define a breast tumor subtype that is estrogen receptor-positive and associated with a slow rate of tumor cell proliferation and a favorable prognosis for these cancer patients. RERG mRNA expression was induced rapidly in MCF-7 cells stimulated by beta-estradiol and repressed by tamoxifen treatment. Like Ras, RERG protein exhibited intrinsic GDP/GTP binding and GTP hydrolysis activity. Unlike Ras proteins, RERG lacks a known recognition signal for COOH-terminal prenylation and was localized primarily in the cytoplasm. Expression of RERG protein in MCF-7 breast carcinoma cells resulted in a significant inhibition of both anchorage-dependent and anchorage-independent growth in vitro and inhibited tumor formation in nude mice. These features of RERG are strikingly different from most Ras superfamily GTP-binding pro-teins and suggest that the loss of RERG expression may contribute to breast tumorigenesis.

Effect of distal fibular resection on ankle laxity.

Ten cadaver specimens were used to study the effect of distal fibular resection and ligamentous repair on ankle laxity. The distal fibula was resected sequentially at three levels. Talar tilt and anterior drawer were measured on stress radiographs after resection and then after ligament repair. Sequential resection of the distal fibula caused a progressive increase in talar tilt and anterior drawer at each cut level (p < 0.5), regardless of whether the ligaments were repaired or not. Ligament repair significantly decreased talar tilt at cuts above the ATFL insertion and decreased anterior drawer at cuts at the tibiotalar level, but these values did not approach control values.

Signaling mediated by the closely related mammalian Rho family GTPases TC10 and Cdc42 suggests distinct functional pathways.

The mammalian Rho family GTPases TC10 and Cdc42 share many properties. Activated forms of both proteins stimulate transcription mediated by nuclear factor kappaB, serum response factor, and the cyclin D1 promoter; activate c-Jun NH2-terminal kinase; cooperate with activated Raf to transform NIH-3T3 cells; and, by a mechanism independent of all of these effects, induce filopodia formation. In contrast, previously reported differences between TC10 and Cdc42 are not striking. We now present studies of TC10 and Cdc42 in cell culture that reveal clear functional differences: (a) wild-type TC10 localizes predominantly to the plasma membrane and less extensively to a perinuclear membranous compartment, whereas wild-type Cdc42 localizes predominantly to this compartment and less extensively to the plasma membrane; (b) expression of Rho guanine nucleotide dissociation inhibitor alpha results in a redistribution of wild-type Cdc42 to the cytosol but has no effect on the plasma membrane localization of wild-type TC10; (c) TC10 fails to rescue a Saccharomyces cerevisiae cdc42 mutation, unlike mammalian Cdc42; (d) dominant negative Cdc42, but not dominant negative TC10, inhibits neurite outgrowth in PC12 cells stimulated by nerve growth factor; and (e) activation of nuclear factor kappaB-dependent transcription by Cdc42, but not by TC10, is inhibited by sodium salicylate. These findings point to distinct pathways in which TC10 and Cdc42 may act and distinct modes of regulation of these proteins.

Effect of perhexiline and oxfenicine on myocardial function and metabolism during low-flow ischemia/reperfusion in the isolated rat heart.

Perhexiline is a potent prophylactic anti-anginal agent that has been shown to inhibit myocardial utilization of long-chain fatty acids and to inhibit the mitochondrial enzyme carnitine palmitoyltransferase (CPT)-1. We compared the hemodynamic and biochemical effects of perhexiline (0.5 and 2.0 microM) and of another CPT-1 inhibitor, oxfenicine (0.5 mM), in Langendorff-perfused rat hearts subjected to 60 min of low-flow ischemia (95% flow reduction) followed by 30 min of reperfusion. Both perhexiline (2 microM only) and oxfenicine attenuated (p < 0.003, p < 0.0002, respectively) increases in diastolic tension during ischemia, without significant effects on developed tension, or on cardiac function during reperfusion. Myocardial concentrations of long-chain acylcarnitines (LCAC), products of CPT-1 action, were decreased (p < 0.05) by oxfenicine, unaffected by 2 microM perhexiline, and increased slightly by 0.5 microM perhexiline. Perhexiline, but not the active metabolite of oxfenicine, also inhibited cardiac CPT-2 with similar IC50 and Emax, although lower Hill slope, compared with CPT-1. Oxfenicine, but not perhexiline, reduced concentrations of the endogenous CPT-1 inhibitor, malonyl-CoA. Perhexiline, but not oxfenicine, inhibited myocardial release of lactate during normal flow. We conclude that (a) perhexiline protects against diastolic dysfunction during ischemia in this model, independent of major changes in LCAC accumulation and (b) this may result from simultaneous effects of perhexiline on myocardial CPT-1 and CPT-2.

The Fhit tumor suppressor protein regulates the intracellular concentration of diadenosine triphosphate but not diadenosine tetraphosphate.

To determine the role of the FHIT tumor suppressor gene product, a diadenosine 5',5'''-P1,P3-triphosphate (Ap3A) hydrolase, in the regulation of the concentration of Ap3A and diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) in vivo, the levels of the adenosine(5')triphospho(5')nucleoside (Ap3N) and adenosine(5')tetraphospho(5')nucleoside (Ap4N) families were measured by luminometry in a number of human cell lines and correlated with the expression of Fhit determined by immunoblotting. Fhit-positive cells had no Ap3N or a very low level of Ap3N, whereas most Fhit-negative cells had Ap3N in the range 0.2-0.9 pmol/10(6) cells. Ap4N (mean value, 0.17 pmol/10(6) cells) did not correlate with Fhit expression. The results suggest that Fhit efficiently metabolizes Ap3A and Ap3N but not Ap4A or Ap4N in vivo.

Mutants of Escherichia coli heat-labile toxin act as effective mucosal adjuvants for nasal delivery of an acellular pertussis vaccine: differential effects of the nontoxic AB complex and enzyme activity on Th1 and Th2 cells.

Mucosal delivery of vaccines is dependent on the identification of safe and effective adjuvants that can enhance the immunogenicity of protein antigens administered by nasal or oral routes. In this study we demonstrate that two mutants of Escherichia coli heat-labile toxin (LT), LTK63, which lacks ADP-ribosylating activity, and LTR72, which has partial enzyme activity, act as potent mucosal adjuvants for the nasal delivery of an acellular pertussis (Pa) vaccine. Both LTK63 and LTR72 enhanced antigen-specific serum immunoglobulin G (IgG), secretory IgA, and local and systemic T-cell responses. Furthermore, using the murine respiratory challenge model for infection with Bordetella pertussis, we demonstrated that a nasally delivered diphtheria, tetanus, and acellular pertussis (DTPa) combination vaccine formulated with LTK63 as an adjuvant conferred a high level of protection, equivalent to that generated with a parenterally delivered DTPa vaccine formulated with alum. This study also provides significant new information on the roles of the binding and enzyme components of LT in the modulation of Th1 and Th2 responses. LTK63, which lacks enzyme activity, promoted T-cell responses with a mixed Th1-Th2 profile, but LTR72, which retains partial enzyme activity, and the wild-type toxin, especially at low dose, induced a more polarized Th2-type response and very high IgA and IgG antibody titers. Our findings suggest that the nontoxic AB complex has broad adjuvant activity for T-cell responses and that the ADP-ribosyltransferase activity of the A subunit also appears to modulate cytokine production, but its effect on T-cell subtypes, as well as enhancing, may be selectively suppressive.

Cellular functions of TC10, a Rho family GTPase: regulation of morphology, signal transduction and cell growth.

The small Ras-related GTPase, TC10, has been classified on the basis of sequence homology to be a member of the Rho family. This family, which includes the Rho, Rac and CDC42 subfamilies, has been shown to regulate a variety of apparently diverse cellular processes such as actin cytoskeletal organization, mitogen-activated protein kinase (MAPK) cascades, cell cycle progression and transformation. In order to begin a study of TC10 biological function, we expressed wild type and various mutant forms of this protein in mammalian cells and investigated both the intracellular localization of the expressed proteins and their abilities to stimulate known Rho family-associated processes. Wild type TC10 was located predominantly in the cell membrane (apparently in the same regions as actin filaments), GTPase defective (75L) and GTP-binding defective (31N) mutants were located predominantly in cytoplasmic perinuclear regions, and a deletion mutant lacking the carboxyl terminal residues required for post-translational prenylation was located predominantly in the nucleus. The GTPase defective (constitutively active) TC10 mutant: (1) stimulated the formation of long filopodia; (2) activated c-Jun amino terminal kinase (JNK); (3) activated serum response factor (SRF)-dependent transcription; (4) activated NF-kappaB-dependent transcription; and (5) synergized with an activated Raf-kinase (Raf-CAAX) to transform NIH3T3 cells. In addition, wild type TC10 function is required for full H-Ras transforming potential. We demonstrate that an intact effector domain and carboxyl terminal prenylation signal are required for proper TC10 function and that TC10 signals to at least two separable downstream target pathways. In addition, TC10 interacted with the actin-binding and filament-forming protein, profilin, in both a two-hybrid cDNA library screen, and an in vitro binding assay. Taken together, these data support a classification of TC10 as a member of the Rho family, and in particular, suggest that TC10 functions to regulate cellular signaling to the actin cytoskeleton and processes associated with cell growth.

Engineered mutants in the switch II loop of Ran define the contribution made by key residues to the interaction with nuclear transport factor 2 (NTF2) and the role of this interaction in nuclear protein import.

Nuclear protein import requires a precisely choreographed series of interactions between nuclear pore components and soluble factors such as importin-beta, Ran, and nuclear transport factor 2 (NTF2). We used the crystal structure of the GDPRan-NTF2 complex to design mutants in the switch II loop of Ran to probe the contribution of Lys71, Phe72 and Arg76 to this interaction. X-ray crystallography showed that the F72Y, F72W and R76E mutations did not introduce major structural changes into the mutant Ran. The GDP-bound form of the switch II mutants showed no detectable binding to NTF2, providing direct evidence that salt bridges involving Lys71 and Arg76 and burying Phe72 are all crucial for the interaction between Ran and NTF2. Nuclear protein accumulation in digitonin-permeabilzed cells was impaired with Ran mutants deficient in NTF2 binding, confirming that the NTF2-Ran interaction is required for efficient transport. We used mutants of the yeast Ran homologue Gsp1p to investigate the effect of the F72Y and R76E mutations in vivo. Although neither mutant was viable when integrated into the genome as a single copy, yeast mildly overexpressing the Gsp1p mutant corresponding Ran F72Y on a centromeric plasmid were viable, confirming that this mutant retained the essential properties of wild-type Ran. However, yeast expressing the Gsp1p mutant corresponding to R76E to comparable levels were not viable, although strains overexpressing the mutant to higher levels using an episomal 2micrometers plasmid were viable, indicating that the R76E mutation may also have interfered with other interactions made by Gsp1p.

Risk of sural nerve injury with intramedullary screw fixation of fifth metatarsal fractures: a cadaver study.

The risk of injury to the sural nerve and its branches during operative procedures performed on the lateral foot and ankle is well recognized; however, there have been no anatomic studies demonstrating the proximity of the sural nerve branches to the head of an intramedullary screw used for fixation of fractures of the proximal fifth metatarsal. Dissection of 10 cadaver specimens, after insertion of 4.5-mm screws, demonstrated that the screw head was within 2 mm of the dorsolateral branch of the sural nerve in five specimens and within 3 mm of eight specimens. Irritation of or injury to the nerve during screw insertion may explain the persistence of pain after screw removal in some patients. Furthermore, patients could sustain injury to the sural nerve at the time of screw removal. Careful surgical technique, including the use of drill guides and tissue protectors, may help lessen the risk of sural nerve injury and subsequent neuroma formation.

Selective downregulation of neutrophils by a phosphatidic acid generation inhibitor in a porcine sepsis model.

Effects of lisofylline (1-(5-R-hydroxyhexyl)-3,7-dimethylxanthine), a functional inhibitor of phosphatidic acid (PA) generation derived from de novo synthesis, on neutrophil function were examined in a porcine sepsis model. Hanford minipigs (18-25 kg) were randomly separated into six groups of six animals each: (1) saline control group; (2) sepsis control group, infused with Pseudomonas aeruginosa (1 x 10(6) colony-forming units/kg/min) for 2 h; (3) lisofylline control group, given a 25 mg/kg bolus of lisofylline 30 min prior to time zero, followed by a continuous infusion of 10 mg/kg/h throughout the study; (4) lisofylline pretreatment sepsis group, given lisofylline 30 min prior to sepsis, (5) lisofylline 1-h post-treatment sepsis group, and (6) lisofylline 2-h post-treatment sepsis group. All animals were studied for 6 h. Neutrophils were isolated at -0.5, 2, and 6 h. In the pretreatment and 1-h post-treatment groups, sepsis-induced neutrophil attachment to fibronectin was significantly attenuated. Sepsis-enhanced phagocytic activity was significantly reduced in the lisofylline pretreatment sepsis group, but not in the post-treatment groups. No treatment affected phorbol 12-myristate 13-acetate-induced chemiluminescence and basal filamentous actin content, which increased in sepsis, and cap formation, which declined in sepsis. Sepsis caused neutropenia, pretreatment produced neutrophilia, and 1-h post-treatment caused the neutropenia to recover to control levels. Interestingly, toward the end of the 6-h period, the neutrophil count was higher in the lisofylline control group than in the saline control groups. Thus, the inhibition of PA generation from de novo synthesis during sepsis not only can selectively downregulate some neutrophil functions but can also reverse neutropenia.

Inhibition of long-chain fatty acid metabolism does not affect platelet aggregation responses.

A number of anti-anginal agents (perhexiline, amiodarone, trimetazidine) have been shown to inhibit myocardial carnitine palmitoyltransferase-1, which controls access of long-chain fatty acids to mitochondrial sites of beta-oxidation. In view of clinical data suggesting that perhexiline improves symptomatic status in unstable angina pectoris, and the known role of mitochondrial beta-oxidation in platelet metabolism, we compared the platelet carnitine palmitoyltransferase-1 inhibitory and putative anti-aggregatory effects of perhexiline, amiodarone and trimetazidine with those of specific carnitine palmitoyltransferase-1 inhibitors: etomoxir and hydroxyphenylglyoxylate in both normal subjects and patients with stable angina. All of the compounds examined inhibited platelet carnitine palmitoyltransferase-1 activity; rank order of potency etomoxir > malonyl-CoA > hydroxyphenylglyoxylate > amiodarone > or = perhexiline > trimetazidine. However, only perhexiline, amiodarone and trimetazidine inhibited platelet aggregation. We conclude that (a) the carnitine palmitoyltransferase-1 inhibitors perhexiline, amiodarone and trimetazidine exert significant anti-aggregatory effects which may be therapeutically relevant and, (b) these effects are independent of carnitine palmitoyltransferase-1 inhibition.

Biomechanical consequences of sequential plantar fascia release.

Plantar fascia release has long been a mainstay in the surgical treatment of persistent heel pain, although its effects on the biomechanics of the foot are not well understood. With the use of cadaver specimens and digitized computer programs, the changes in the medial and lateral columns of the foot and in the transverse arch were evaluated after sequential sectioning of the plantar fascia. Complete release of the plantar fascia caused a severe drop in the medial and lateral columns of the foot, compared with release of only the medial third. Equinus rotation of the calcaneus and a drop in the cuboid indicate that strain of the plantar calcaneocuboid joint capsule and ligament is a likely cause of lateral midfoot pain after complete plantar fascia release.

A T42A Ran mutation: differential interactions with effectors and regulators, and defect in nuclear protein import.

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran.GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran.GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin beta (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

Quantitative but not qualitative variation in MHC class II alters CD4 interaction and influences T cell repertoire formation.

The influence of the interaction between CD4 and MHC class II molecules on selection of the T cell repertoire was studied in transgenic mice expressing human or human/mouse hybrid MHC class II beta chains. Either wild-type DR beta chains (DR1 beta) or hybrid beta chains comprising the beta1 domain of DR and the beta2, transmembrane, and intracytoplasmic domains of I-E (DRbeta 1Ebeta2) were introduced into and expressed in transgenic mice as a heterodimer with endogenous I-E alpha. Mice expressing low levels of DR1beta:I-E alpha or those expressing low or higher levels of the hybrid DRbeta 1Ebeta2:I-E alpha were studied. Immunization with a suboptimal dose of influenza nucleoprotein peptide exposed a fivefold lower frequency of DR-restricted, peptide-specific, IL-2-secreting T cells in the mice with low-level expression of DRbeta1 Ebeta2:I-E alpha when compared to mice expressing the same molecule at higher levels. The frequency in DRbeta wild-type mice was only twofold lower than that measured in mice with comparable levels of expression of DRbeta 1Ebeta2. These results suggest that positive selection is sensitive to quantitative variation in MHC class II density, unmasked when antigen is limiting, but is relatively insensitive to qualitative variation in the MHC class II: CD4 interaction.