Is recovery from ankle sprains negatively affected by obesity?

OBJECTIVE: Ankle sprains are common injuries that may lead to long-term morbidity. Individuals with obesity are at increased risk for ankle sprains; however, prognostic associations between body mass index (BMI) and recovery are less well understood. This study investigated whether BMI status affects recovery from ankle sprains. METHODS: We included individuals≥16 years old with grade 1 or 2 ankle sprains who sought emergency department treatment in Kingston, Ontario, Canada. Height in centimeters and weight in kilograms were measured at baseline by using a height rod and a standard medical column scale, respectively. BMI was calculated and categorized as non-overweight,<25.0kg/m2; overweight, 25.0-29.9kg/m2; and obese,≥30kg/m2. Recovery was assessed at 1, 3 and 6 months post-injury by the Foot and Ankle Outcome Score (FAOS). Continuous FAOS and binary recovery status were compared by BMI group at each assessment using a repeated measures linear mixed effects model and logistic regression, respectively. RESULTS: In total, 504 individuals were recruited and 6-month follow-up data were collected for 80%. We observed no significant differences in recovery at 1 and 3 months post-injury. At 6 months, between 53% and 66% of the participants were considered to have recovered according to the FAOS. The mean difference in unadjusted FAOS between participants classified as obese and non-overweight was -23.02 (95% confidence interval, -38.99 to -7.05) but decreased after adjusting for confounders. The odds ratio for recovery was 0.60 (0.37-0.97) before adjustment and 0.74 (0.43-1.29) after adjustment. Six-month recovery was significantly lower for participants with obesity than non-overweight participants on the FAOS Pain and Function in Daily Living subscales but were not clinically meaningful. CONCLUSIONS: All BMI groups showed improvements from ankle sprain over time. However, at 6 months, a sizeable proportion of the participants had not fully recovered particularly among individuals classified as obese. The findings suggest that individuals with obesity may benefit from specialized interventions focused on symptom management and functional activity.

Osteogenic Potential of Human Umbilical Cord Mesenchymal Stem Cells on Coralline Hydroxyapatite/Calcium Carbonate Microparticles.

Coralline hydroxyapatite/calcium carbonate (CHACC) is a biodegradable and osteoconductive bone graft material with promising clinical performance. CHACC has been shown to support proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs) in vitro and demonstrated to work as a functional scaffold for bone formation in vivo. Umbilical cord matrix is a more accessible and abundant tissue source of MSCs, but its osteogenic capacity in comparison to human bone marrow when cultured on CHACC has not yet been demonstrated. In this study, we assessed the osteogenic differentiation capacity of human MSCs, isolated from bone marrow and umbilical cord matrix and characterised by flow cytometry, when cultured on 200-300 µm CHACC granules. The 3D cultures were characterised by brightfield and scanning electron microscopy (SEM). Osteogenic potential was assessed by immunocytochemistry and qPCR for key markers of bone differentiation (alkaline phosphatase, runx2, type I collagen, and osteocalcin). By day 1, the MSCs had enveloped the surface of the CHACC granules to form organoids, and by day 7, cells had proliferated to bridge nearby organoids. Extracellular matrix deposition and osteogenic differentiation were demonstrated by MSCs from both tissue sources at day 21. However, MSCs from bone marrow demonstrated superior osteogenic differentiation capability compared to those from umbilical cord matrix. In conclusion, it is possible to culture and induce osteogenic differentiation of umbilical cord matrix MSCs on CHACC. Further research is required to optimise the osteogenicity of umbilical cord matrix MSCs to release their full potential as a readily available, accessible, and abundant tissue source for bone tissue engineering.

An allogeneic 'off the shelf' therapeutic strategy for peripheral nerve tissue engineering using clinical grade human neural stem cells.

Artificial tissues constructed from therapeutic cells offer a promising approach for improving the treatment of severe peripheral nerve injuries. In this study the effectiveness of using CTX0E03, a conditionally immortalised human neural stem cell line, as a source of allogeneic cells for constructing living artificial nerve repair tissue was tested. CTX0E03 cells were differentiated then combined with collagen to form engineered neural tissue (EngNT-CTX), stable aligned sheets of cellular hydrogel. EngNT-CTX sheets were delivered within collagen tubes to repair a 12 mm sciatic nerve injury model in athymic nude rats. Autologous nerve grafts (autografts) and empty tubes were used for comparison. After 8 weeks functional repair was assessed using electrophysiology. Further, detailed histological and electron microscopic analysis of the repaired nerves was performed. Results indicated that EngNT-CTX supported growth of neurites and vasculature through the injury site and facilitated reinnervation of the target muscle. These findings indicate for the first time that a clinically validated allogeneic neural stem cell line can be used to construct EngNT. This provides a potential 'off the shelf' tissue engineering solution for the treatment of nerve injury, overcoming the limitations associated with nerve autografts or the reliance on autologous cells for populating repair constructs.

Protocol adherence for continuously titrated interventions in randomized trials: an overview of the current methodology and case study.

BACKGROUND: The standard definition for protocol adherence is the proportion of all scheduled doses that are delivered. In clinical research, this definition has several limitations when evaluating protocol adherence in trials that study interventions requiring continuous titration. DISCUSSION: Building upon a specific case study, we analyzed a recent trial of a continuously titrated intervention to assess the impact of different definitions of protocol deviations on the interpretation of protocol adherence. The OVATION pilot trial was an open-label randomized controlled trial of higher (75-80 mmHg) versus lower (60-65 mmHg) mean arterial pressure (MAP) targets for vasopressor therapy in shock. In this trial, potential protocol deviations were defined as MAP values outside the targeted range for >4 consecutive hours during vasopressor therapy without synchronous and consistent adjustments of vasopressor doses. An adjudication committee reviewed each potential deviation to determine if it was clinically-justified or not. There are four reasons for this contextual measurement and reporting of protocol adherence. First, between-arm separation is a robust measure of adherence to complex protocols. Second, adherence assessed by protocol deviations varies in function of the definition of deviations and the frequency of measurements. Third, distinguishing clinically-justified vs. not clinically-justified protocol deviations acknowledges clinically sensible bedside decision-making and offers a clear terminology before the trial begins. Finally, multiple metrics exist to report protocol deviations, which provides different information but complementary information on protocol adherence. CONCLUSIONS: In trials of interventions requiring continuous titration, metrics used for defining protocol deviations have a considerable impact on the interpretation of protocol adherence. Definitions for protocol deviations should be prespecified and correlated with between-arm separation, if it can be measured.

Sham acupuncture is as efficacious as true acupuncture for the treatment of IBS: A randomized placebo controlled trial.

BACKGROUND: Irritable bowel syndrome (IBS) patients increasingly seek out acupuncture therapy to alleviate symptoms, but it is unclear whether the benefit is due to a treatment-specific effect or a placebo response. This study examined whether true acupuncture is superior to sham acupuncture in relieving IBS symptoms and whether benefits were linked to purported acupuncture mechanisms. METHODS: A double blind sham controlled acupuncture study was conducted with Rome I IBS patients receiving twice weekly true acupuncture for 4 weeks (n=43) or sham acupuncture (n=36). Patients returned at 12 weeks for a follow-up review. The primary endpoint of success as determined by whether patients met or exceeded their established goal for percentage symptom improvement. Questionnaires were completed for symptom severity scores, SF-36 and IBS-36 QOL tools, McGill pain score, and Pittsburg Sleep Quality Index. A subset of patients underwent barostat measurements of rectal sensation at baseline and 4 weeks. KEY RESULTS: A total of 53% in the true acupuncture group met their criteria for a successful treatment intervention, but this did not differ significantly from the sham group (42%). IBS symptom scores similarly improved in both groups. Scores also improved in the IBS-36, SF-36, and the Pittsburg Sleep Quality Index, but did not differ between groups. Rectal sensory thresholds were increased in both groups following treatment and pain scores decreased; however, these changes were similar between groups. CONCLUSIONS & INFERENCES: The lack of differences in symptom outcomes between sham and true treatment acupuncture suggests that acupuncture does not have a specific treatment effect in IBS.

Vindication of Yb2Ti2O7 as a model exchange quantum spin ice.

We use numerical linked-cluster expansions to compute the specific heat C(T) and entropy S(T) of a quantum spin ice Hamiltonian for Yb2Ti2O7 using anisotropic exchange interactions, recently determined from inelastic neutron scattering measurements, and find good agreement with experimental calorimetric data. This vindicates Yb2Ti2O7 as a model quantum spin ice. We find that in the perturbative weak quantum regime, such a system has a ferrimagnetic ordered ground state, with two peaks in C(T): a Schottky anomaly signaling the paramagnetic to spin ice crossover, followed at a lower temperature by a sharp peak accompanying a first-order phase transition to the ordered state. We suggest that the two C(T) features observed in Yb2Ti2O7 are associated with the same physics. Spin excitations in this regime consist of weakly confined spinon-antispinon pairs. We anticipate that the conventional ground state with exotic quantum dynamics will prove a prevalent characteristic of many real quantum spin ice materials.

Randomized controlled trials in the era of molecular oncology: methodology, biomarkers, and end points.

BACKGROUND: We previously reported metrics of systemic therapy randomized controlled trials (RCTs) in breast cancer, colorectal cancer (CRC), and non-small-cell lung cancer (NSCLC) published 1975-2004. To evaluate trends in the era of targeted therapies (TT), we have repeated a similar analysis of RCTs published 2005-2009. METHODS: A search for phase III RCTs of systemic agents published in five major journals 2005-2009 was carried out. Trials were classified as TT if they involved any non-hormonal targeted agent. We extracted data regarding biomarker use. Integral biomarkers were defined as tests used to determine eligibility, stratification, or allocation. Descriptive statistics were used to analyze trends over time. RESULTS: One hundred and thirty-seven eligible RCTs were evaluated. Compared with 1995-2004, the number (17-27 RCTs/year) and size (median sample size 446-722, P < 0.001) of RCTs increased. The proportion of RCTs evaluating TT increased from 4% (7/167) to 29% (40/137) (P < 0.001). There was an increase in the proportion of trials with financial support from industry [57% (95/167) to 78% (107/137), P = 0.001]. Biomarkers were included in 58% (80/137) of RCTs; integral biomarkers were included in 36% (49/137) of trials. Among the 49 RCTs using integral biomarkers, 40 (82%) used HER2 and/or ER/PR status in studies of breast cancer. CONCLUSIONS: RCTs published in 2005-2009 are larger, more likely to evaluate TT, and be supported by industry. Biomarkers may be increasingly used, but the most common use relates to traditional use of ER/PR and evolving use of HER2 in breast cancer RCTs.

Utility of Gram stain in the clinical management of suspected ventilator-associated pneumonia. Secondary analysis of a multicenter randomized trial.

PURPOSE: Gram stains of endotracheal aspirates (EA) and bronchoalveolar lavages (BAL) may guide empiric antibiotic therapy in critically ill patients with suspected ventilator-associated pneumonia (VAP). Previous studies differ regarding the ability of the Gram stain to predict final culture results. The aim of the present study was to evaluate the relationship between EA or BAL Gram stains and final culture results in intensive care unit patients with a suspected VAP. MATERIAL AND METHODS: We retrospectively analyzed data from the Canadian multicenter VAP study to correlate EA or BAL Gram stain and final culture results. We categorized Gram stains as Gram positive (GP) and Gram negative (GN) if any GP or GN organisms respectively were seen on staining. Cultures were considered "positive" if they yielded pathogenic organisms on final results. RESULTS: Seven hundred forty patients were enrolled in the study; 35 did not have a Gram stain done leaving 350 BALs and 355 EAs from 705 patients. Pooling BAL and EA results, we found the overall agreement between Gram stain class and pathogenic bacteria culture results to be poor (kappa = 0.36; 95% CI, 0.31-0.40). Among specimens with Gram stains showing no organisms, 99 (30%) of 331 grew pathogenic organisms. Among specimens with Gram stains showing no GN organisms, 113 (25%) of 452 grew pathogenic GN organisms. Among specimens with Gram stains showing no GP organisms, 45 (11%) of 428 grew pathogenic GP organisms. CONCLUSIONS: Gram stains performed for clinically suspected VAP poorly predict the final culture result and thus have a limited role in guiding initial empiric antibiotic therapy in such patients.

Effect of treating severe nasal obstruction on the severity of obstructive sleep apnoea.

An association between mouth breathing during sleep and increased propensity for upper airway collapse is well documented, but the effect of treatment for nasal obstruction on mouth breathing during sleep and simultaneous obstructive sleep apnoea (OSA) severity has not been described previously. A randomised single blind placebo- and sham-controlled crossover study of treatment (topical decongestant and external dilator strip) for nasal obstruction was carried out in 10 patients (nine males; mean+/-SEM 46+/-5 yrs) with nasal obstruction and OSA. All patients had normal acoustic pharyngometry. The effect of treatment on nasal resistance, mouth breathing during sleep and OSA severity was quantified. Treatment of nasal obstruction was associated with a dramatic and sustained reduction in nasal resistance and the oral fraction of ventilation during sleep (mean (95% confidence interval) absolute reduction in oral fraction 30% (12-49)). Improvements in sleep architecture were observed during active treatment, and there was a modest reduction in OSA severity (change in apnoea-hypopnoea index 12 (3-22)). In conclusion, treating nasal obstruction reduced mouth breathing during sleep and obstructive sleep apnoea severity, but did not effectively alleviate obstructive sleep apnoea.

A novel combination of two classic catalytic schemes.

The crystal structure of an alkaline Bacillus cellulase catalytic core, from glucoside hydrolase family 5, reveals a novel combination of the catalytic machinery of two classic textbook enzymes. The enzyme has the expected two glutamate residues in close proximity to one another in the active-site that are typical of retaining cellulases. However, the proton donor, glutamate 139 is also unexpectedly a member of a serine-histidine-glutamate catalytic triad, forming a novel combination of catalytic machineries. Structure and sequence analysis of glucoside hydrolase family 5 reveal that the triad is highly conserved, but with variations at the equivalent of the serine position. We speculate that the purpose of this novel catalytic triad is to control the protonation of the acid/base glutamate, facilitating the first step of the catalytic reaction, protonation of the substrate, by the proton donor glutamate. If correct, this will be a novel use for a catalytic triad.

Protein engineering of alpha-amylase for low pH performance.

Industrial-scale starch liquefaction is currently constrained to operating at pH 6.0 and above, as the enzyme used in the process, Bacillus licheniformis alpha-amylase, is unstable at lower pH under the conditions used. There is a need to develop an enzyme that can operate at lower pH. Recent progress has been made in engineering the B. licheniformis enzyme for improved industrial performance. The availability of crystal structures and subsequent analysis of improved variants, in a structural context, is revealing common factors and a rationale to make further improvements.

Role of isoleucine-164 at the active site of rubisco from Rhodospirillum rubrum.

Isoleucine-164 is in Van der Waals contact with two ligands (lysine-191 and aspartate-193) of the activator magnesium ion at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. To observe the effect of mutations in the second sphere of coordination of the metal ion, isoleucine-164 was replaced by threonine, asparagine, and aspartate. All the mutant enzymes obtained exhibit a low carboxylase activity. Ile164Asp has less than 0.1% of the wild-type carboxylase activity, Ile164Thr and Ile164Asn 6 and 1%, respectively. The mutations increase the Km(RuBP) and decrease the Kcat of the mutated enzymes. The Kcat/Km(RuBP) of Ile164Thr and Ile164Asn are 40- and 900-fold lower than wild-type, respectively. The alteration of the hydrophobic contacts between isoleucine-164 and the metal ion ligands modifies the binding of the magnesium ion and the stabilization of the 2-carboxy-arabinitol 1,5-bisphosphate and decreases the specificity factor, tau.

Refolding of barnase mutants and pro-barnase in the presence and absence of GroEL.

Three mutants of barnase and a pro-barnase variant, which have a variety of different physical properties but the same overall protein structure, were analysed for their folding in the presence of the molecular chaperone GroEL. Mutants were chosen on the basis that changes in their refolding rate constants in solution are not correlated with the changes in their stability. All barnase variants fold considerably more slowly when bound to GroEL. However, barnase refolding on GroEL parallels that in solution: there is a linear relationship between the refolding rate constants, obtained for wild-type and all mutants of barnase, in the presence and absence of GroEL. Barnase is synthesized in vivo with a 13 amino acid pro-sequence attached to the N-terminus. The pro-sequence of pro-barnase is shown by NMR spectroscopy to be devoid of defined structure. The presence of this pro-sequence has no effect on the overall refolding rate constant or the activity of barnase. In the presence of GroEL, the refolding of pro-barnase is retarded relatively more strongly than that of wild-type and the mutant barnase proteins, suggesting that the pro-sequence provides additional binding sites for the chaperone.

Step-wise mutation of barnase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability.

We have chosen two members of the microbial RNase family, barnase and binase, which have 85% identity (17 substitutions and 1 deletion) and almost identical three-dimensional structure, to study the evolution of protein stability. The 17 residues that differ are scattered throughout the molecule. Each of the 17 differing residues has been mutated independently and the effect on protein stability analysed. Each point mutation has an effect on protein stability that ranges from +1.1 to -1.1 kcal mol-1. These changes in energy are additive. There is no clear correlation between the type of mutation and the effect on protein stability. A multiple mutant having six of the single mutations that increase the stability of barnase is 3.3 kcal mol-1 more stable than wild type and has the same activity. There could be selective pressure to maintain proteins at a certain stability and, consequently, mutations that decrease stability tend to be counterbalanced by stabilizing mutations. Alternatively, there could simply be pressure to maintain stability above a certain level, and any further increases in stability need not be maintained during evolution. These results suggest a simple way to improve the stability of proteins: choose two homologous proteins that have high similarity, mutate individually all of the residues that differ between the two, and combine the mutations that increase the stability in a multiple mutant.

Reaction of modified and unmodified tRNA(Tyr) substrates with tyrosyl-tRNA synthetase (Bacillus stearothermophilus).

Three species of tRNA(Tyr) have been examined as substrates for the transfer reaction of the tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus: Escherichia coli tRNA(Tyr), B. stearothermophilus tRNA(Tyr) expressed in E. coli, and B. stearothermophilus tRNA(Tyr) that has been transcribed in vitro. The binding of the first two substrates to TyrRS may be readily monitored by stopped-flow studies of tryptophan fluorescence to give the rate and equilibrium constants. The in vitro-transcribed tRNA(Tyr), which lacks the modified bases queuosine and 2-(methylthio)-N6-isopentenyladenosine in the anticodon loop, does not cause a significant change in tryptophan fluorescence upon binding. The three tRNA(Tyr) substrates exhibit very similar steady-state kinetics in the charging reaction. Pre-steady-state kinetics of the transfer reaction, monitored by stopped-flow measurements of the change in protein fluorescence on the addition of tRNA(Tyr) to the E.Tyr-AMP complex, show two exponential changes for the modified tRNA(Tyr) substrates. The first is that due to substrate binding. The second has an identical rate to the single change observed for the reaction with the in vitro-transcribed tRNA(Tyr) and to that monitored by quenched-flow measurements on the formation of Tyr-tRNA(Tyr). Hence, the transfer reaction can be observed by stopped-flow. The dissociation constants (KtRNA) of tRNA from the enzyme and rates of tyrosine transfer (k4) show that all three tRNA molecules are kinetically equivalent substrates for TyrRS. The value of k4 is also similar to that found for authentic tRNA(Tyr) from B. stearothermophilus.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of phenylalanine-327 in the closure of loop 6 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

Phenylalanine-327 of ribulosebisphosphate carboxylase/oxygenase (rubisco) from Rhodospirillum rubrum was mutated to tryptophan, leucine, valine, alanine, and glycine, and was also deleted. The least active mutant, the deletion mutant, exhibits less than 0.5% of the carboxylase activity of the wild-type enzyme. Steady-state kinetic analysis of F327-->Leu, Val, Ala, Gly mutant enzymes reveals that kcat and the CO2/O2 specificity are unchanged while Km(RuBP) (RuBP = ribulose 1,5-bisphosphate) is drastically increased. The mutant enzyme with the highest value for Km(RuBP),Phe327-->Gly, shows a 165-fold increase (1160 microM compared to 7 microM for the wild-type). The increase in Km(RuBP) suggests an alteration of the ratio kon/koff for RuBP. A longer hydrophobic lateral chain and/or the presence of an aromatic ring in the wild-type enzyme and the Phe327-->Trp mutant enzyme could explain a better packing of loop 6 in the closed conformation and thus a tighter binding of RuBP at the active site.

Barnase has subsites that give rise to large rate enhancements.

Barnase is found to have a series of subsites for binding its substrates that confers large rate enhancements. Ribonucleotide substrates of the type Zp0Gp1Xp2Y have been synthesized, where p is phosphate, X, Y, and Z are nucleosides, and G is guanosine. G occupies the primary specificity site. The most important subsite is for p2, followed by that for Y. There appears to be no subsite for the Z or p0 positions. Occupation of the subsite for p2 gives rise to a 1000-fold increase in kcat/Km, composed of a 100-fold increase in kcat and a 10-fold decrease in Km. The Y subsite gives rise to further 20-fold increase in kcat/Km. Rates approaching diffusion control for kcat/Km are observed. kcat for the dinucleotide monophosphate GpU = 0.55 s-1, and Km = 240 microM; this compares with 53 s-1 and 20 microM for GpUp, and 3.3 x 10(3) s-1 and 17 microM for GpApA (the best substrate tested). Cleavage occurs at the 3'-phosphate of guanosine in all cases. There are differences in base specificity at the two subsites for X and Y downstream of the scissile bond. The binding energies of different substrates have been analyzed using thermodynamic cycles. These show that the contributions of the X and Y sites are nonadditive.

Mutation of asparagine 111 of rubisco from Rhodospirillum rubrum alters the carboxylase/oxygenase specificity.

The conserved asparagine 111 of ribulose-1,5-bisphosphate carboxylase/oxygenase from the photosynthetic bacteria Rhodospirillum rubrum was identified as a candidate for a side-chain that might be involved in the carboxylase/oxygenase specificity. It was replaced by site-directed mutagenesis with aspartic acid, leucine, glutamine or glycine residues. The mutant enzymes exhibit a very low carboxylase activity compared with the wild-type enzyme. The values of Km(RuBP) and kcat for Asn111----Gly, the most active mutant, are 420 microM and 0.034 s-1, compared with 13 microM and 3.0 s-1 for wild-type. The mutation of Asn111----Gly causes a more than tenfold decrease in the CO2/O2 specificity factor, tau, tau Asn111----Gly = 0.56 and tau wild-type = 6.7. This is the first reported change in rubisco specificity by a single site-directed mutation alone and suggests a target for future protein engineering studies.

Expression of an antisense viral gene in transgenic tobacco confers resistance to the DNA virus tomato golden mosaic virus.

Transgenic tobacco plants carrying a genetic cassette including an antisense DNA sequence of the virally encoded AL1 gene of the geminivirus tomato golden mosaic virus (TGMV) were constructed; AL1 encodes a protein absolutely required for TGMV DNA replication. These genetic cassettes also contained, on the same transcription unit, a gene encoding hygromycin resistance, which allowed selection for concomitant expression of the antisense gene. In transgenic lines, RNA transcripts of the predicted size and strand specificity were detected in antisense plants and sense controls. After infection of plants with TGMV, by agroinoculation, the frequency of symptom development was very significantly reduced in a number of antisense lines and correlated, broadly, with the abundance of antisense RNA transcript and with a reduction in viral DNA harvested from infected leaf tissue. We used an in vitro assay to study viral DNA replication in the absence of cell-to-cell spread; no replication was seen in five of the six antisense lines studied, in contrast to controls.