Influence of impaired selective motor control on gait in children with cerebral palsy.

PURPOSE: Spastic cerebral palsy (CP) is characterized by four neuromuscular deficits: weakness, short muscle-tendon unit, muscle spasticity and impaired selective motor control (SMC). We examined the influence of impaired SMC on gait in children with bilateral spastic CP. Delineating the influence of neuromuscular deficits on gait abnormalities can guide surgical and therapeutic interventions to reduce long-term debilitating effects of CP. METHODS: The relationship between impaired SMC and gait was assessed using multivariate linear regression analysis of Selective Control Assessment of the Lower Extremity (SCALE) in relation to stance phase knee flexion and temporal-spatial gait parameters calculated using 3D kinematics for 57 children with bilateral spastic CP, ages seven to 11 years. RESULTS: Mean SCALE values were 5.8 (0 to 10, sd 3.0) and 5.7 (0 to 10, sd 2.9) for right and left legs, respectively. Multivariate linear regression models, including right and left SCALE and height, significantly predicted right and left knee flexion at initial contact (R = 0.479, p = 0.003; R = 0.452, p = 0.007, respectively) and right and left knee flexion in midstance (R = 0.428, p = 0.013; R = 0.407, p = 0.022, respectively). The model significantly predicted right and left step length (R = 0.645, p = 0.000; R = 0.523, p = 0.001, respectively) and predicted gait velocity (R = 0.444, p = 0.008). The model including SCALE did not predict step width. CONCLUSION: Results indicate impaired SMC predicts increased knee flexion at initial contact, and reduces step length and velocity. Understanding the influence of impaired SMC on gait can inform decisions regarding therapy and surgery, such as hamstring lengthening. LEVEL OF EVIDENCE: Level II Retrospective Study.

Opsin evolution in the Ambulacraria.

Opsins--G-protein coupled receptors involved in photoreception--have been extensively studied in the animal kingdom. The present work provides new insights into opsin-based photoreception and photoreceptor cell evolution with a first analysis of opsin sequence data for a major deuterostome clade, the Ambulacraria. Systematic data analysis, including for the first time hemichordate opsin sequences and an expanded echinoderm dataset, led to a robust opsin phylogeny for this cornerstone superphylum. Multiple genomic and transcriptomic resources were surveyed to cover each class of Hemichordata and Echinodermata. In total, 119 ambulacrarian opsin sequences were found, 22 new sequences in hemichordates and 97 in echinoderms (including 67 new sequences). We framed the ambulacrarian opsin repertoire within eumetazoan diversity by including selected reference opsins from non-ambulacrarians. Our findings corroborate the presence of all major ancestral bilaterian opsin groups in Ambulacraria. Furthermore, we identified two opsin groups specific to echinoderms. In conclusion, a molecular phylogenetic framework for investigating light-perception and photobiological behaviors in marine deuterostomes has been obtained.

Phase I trial of olaparib in combination with cisplatin for the treatment of patients with advanced breast, ovarian and other solid tumors.

BACKGROUND: To establish the maximum tolerated dose, determine safety/tolerability and evaluate the pharmacokinetics and preliminary efficacy of olaparib in combination with cisplatin in patients with advanced solid tumors. PATIENTS AND METHODS: Patients aged ≥ 18 years with advanced solid tumors, who had progressed on standard treatment, were assigned to a treatment cohort and received oral olaparib [50-200 mg twice daily (bid); 21-day cycle] continuously or intermittently (days 1-5 or 1-10) in combination with cisplatin (60-75 mg/m(2) intravenously) on day 1 of each cycle. RESULTS: Dose-limiting toxicities (DLTs) of grade 3 neutropenia (cisplatin 75 mg/m(2) with continuous olaparib 100 mg bid or 200 mg bid; n = 1 each) and grade 3 lipase elevation (cisplatin 75 mg/m(2) with olaparib 100 mg bid days 1-10 or 50 mg bid days 1-5; n = 1 each) were reported. Olaparib and cisplatin doses were subsequently reduced to 50 mg bid days 1-5 and 60 mg/m(2), respectively; no DLTs were reported for patients receiving this regimen. The most frequent grade ≥ 3 adverse events were neutropenia (16.7%), anemia (9.3%) and leucopenia (9.3%). Thirty patients (55.6%) received colony-stimulating factors for hematologic support. The overall objective response rate was 41% for patients with measurable disease, and 43% and 71% among patients with a BRCA1/2 mutation who had ovarian and breast cancer, respectively. CONCLUSIONS: Olaparib in combination with cisplatin 75 mg/m(2) was not considered tolerable; intermittent olaparib (50 mg bid, days 1-5) with cisplatin 60 mg/m(2) improved tolerability. Promising antitumor activity in patients with germline BRCA1/2 mutations was observed and warrants further investigation.

Development of PK- and PBPK-based modeling tools for derivation of biomonitoring guidance values.

There are numerous programs ongoing to analyze environmental exposure of humans to xenobiotic chemicals via biomonitoring measurements (e.g.: EU ESBIO, COPHES; US CDC NHANES; Canadian Health Measures Survey). The goal of these projects is to determine relative trends in exposure to chemicals, across time and subpopulations. Due to the lack of data, there is often little information correlating biomarker concentrations with exposure levels and durations. As a result, it can be difficult to utilize biomonitoring data to evaluate if exposures adhere to or exceed hazard/exposure criteria such as the Derived No-Effect Level values under the EU REACH program, or Reference Dose/Concentration values of the US EPA. A tiered approach of simple, arithmetic pharmacokinetic (PK) models, as well as more standardized mean-value, physiologically-based (PBPK) models, have therefore been developed to estimate exposures from biomonitoring results. Both model types utilize a user-friendly Excel spreadsheet interface. QSPR estimations of chemical-specific parameters have been included, as well as accommodation of variations in urine production. Validation of each model's structure by simulations of published datasets and the impact of assumptions of major model parameters will be presented.

Transfer of gangliosides across the human placenta.

OBJECTIVES: Gangliosides are structural and functional glycosphingolipids, considered to have important roles in neuronal development in fetal and neonatal development and in memory formation. In this report, we have investigated the ability of bovine milk-derived gangliosides GM3 and GD3 to cross the human placenta. STUDY DESIGN: We have employed the ex-vivo model of dually-perfused isolated human placental lobules. RESULTS: There was significant uptake of both GD3 and GM3 from the maternal perfusate. There was significant increase of GM3 in the fetal side and a non-statistically significant trend for GD3 to increase on the fetal side. CONCLUSIONS: Hence an apparent preference for GM3 release into fetal circulation. We suggest that gangliosides consumed by the mother enter her circulation, can be transferred across the placenta and may be available to the developing fetus for building neural connections.

Efficacy of high-dose methotrexate, ifosfamide, etoposide and dexamethasone salvage therapy for recurrent or refractory childhood malignant lymphoma.

BACKGROUND: Children with recurrent or refractory malignant lymphoma generally have a poor prognosis. There is a need for new active drug combinations for this high-risk group of patients. PATIENTS AND METHODS: This study evaluated the activity and toxicity of the methotrexate, ifosfamide, etoposide and dexamethasone (MIED) regimen for childhood refractory/recurrent non-Hodgkin's lymphoma (NHL) or Hodgkin's lymphoma (HL). From 1991 through 2006, 62 children with refractory/recurrent NHL (n = 24) or HL (n = 38) received one to six cycles of MIED. Based on MIED response, intensification with hematopoietic stem cell transplantation (HSCT) was considered. RESULTS: There were 10 complete (CR) and 5 partial responses (PR) among the 24 children with NHL [combined response rate, 63%; 95% confidence interval (CI) 38% to 73%]. There were 13 CR and 18 PR among the 37 assessable children with HL (combined response rate, 84%; 95% CI, 68% to 94%). Although 59% courses were associated with grade IV neutropenia, treatment was well tolerated and without toxic deaths. CONCLUSIONS: MIED is an effective regimen for refractory/recurrent childhood malignant lymphoma, permitting a bridge to intensification therapy with HSCT.

Pharmacokinetics and bioavailability of diisopropanolamine (DIPA) in rats following intravenous or dermal application.

This study was conducted to determine the relative dermal bioavailability (absorption), distribution, metabolism, and excretion (ADME) of diisopropanolamine (DIPA), an alcohol amine used in a number of industrial and personal care products. Groups of 4 female Fischer 344 rats received either a single bolus i.v. dose of 19.0mg/kg (14)C-DIPA in water or a dermal application of 19.5mg/kg (14)C-DIPA in acetone to an area of 1cm(2) on the back and covered with a bandage. Time-course blood and excreta were collected and radioactivity determined. Urine was analyzed for DIPA and monoisopropanolamine (MIPA). Following i.v. administration, DIPA was rapidly cleared from the plasma and excreted into urine in a biexponential manner (t(1/2alpha), 0.4h; t(1/2beta), 2.9h). The levels of radioactivity in plasma dropped below the limit of detection 12h post-dosing. A total of 97+/-4% of the dose was actively excreted in urine by kidney, most ( approximately 71%) within 6h of dosing, virtually all as parent compound; renal clearance exceeded the glomerular filtration rate. Following dermal application, approximately 20% of the dose was absorbed in 48 h with the steady-state penetration rate of approximately 0.2%/h. Most (14.4%) of the applied radioactivity was excreted in urine at a relatively constant rate due to the presence of large amount of the (14)C-DIPA at the application site. Fecal elimination was <0.2% of the dose. The absorbed DIPA did not accumulate in tissues; only approximately 0.1% of the administered dose was found in liver and kidney. The absolute systemic dermal bioavailability (dose corrected AUC(dermal)/AUC(i.v.)) of (14)C-DIPA was 12%. The ADME of DIPA contrasts that of its diethanol analogue, diethanolamine, which displays a broad spectrum of toxicity in rats and mice. Toxicologically significant concentrations of DIPA are unlikely to be achieved in the systemic circulation and/or tissues as a result of repeated dermal application of products containing DIPA due to slow absorption from the skin, rapid unchanged elimination in urine, and majority of the products contain

Trends in the duration of school-day sleep among 10- to 15-year-old South Australians between 1985 and 2004.

AIM: To compare self-reported school-day sleep duration in 10- to 15-year-old South Australians between 1985 and 2004. METHODS: Data were collected from 10- to 15-year-old participants in the 1985 Australian Schools Health and Fitness Survey (n = 390) and the 2004 South Australian Physical Activity Survey (n = 510). Identical self-report questionnaires were administered in both surveys, providing data on school-day bed-time, wake-time and sleep duration; as well as age, gender and socioeconomic status (SES). Analysis of co-variance (ANCOVA), controlling for age and SES, was used to compare all sleep variables between surveys. RESULTS: Declines in sleep duration were found for both girls (28 min) and boys (33 min) between surveys. The reduction was more pronounced in 'lower SES' boys (44 min) than 'higher SES' boys (23 min). Boys reported later bed-time than girls in the 2004 survey, while no gender differences were apparent in the 1985 survey. CONCLUSION: Our findings suggest reductions in school-day sleep duration have occurred in Australian children and adolescents over the last 20 years, due largely to later bed-times. The physiological significance of these declines and mediating influences, such as SES, are yet to be explicated.

Purification and characterization of cytochrome c' from Neisseria meningitidis.

Cytochrome c', a c-type cytochrome with unique spectroscopic and magnetic properties, has been characterized in a variety of denitrifying and photosynthetic bacteria. Cytochrome c' has a role in defence and/or removal of NO but the mechanism of action is not clear. To examine the function of cytochrome c' from Neisseria meningitidis, the protein was purified after heterologous overexpression in Escherichia coli. The electronic spectra of the oxidized c' demonstrated a pH-dependent transition (over the pH range of 6-10) typical of known c'-type cytochromes. Interestingly, the form in which NO is supplied determines the redox state of the resultant haem-nitrosyl complex. Fe(III)-NO complexes were formed when Fe(II) or Fe(III) cytochrome c' was sparged with NO gas, whereas an Fe(II)-NO complex was generated when NO was supplied using DEA NONOate (diazeniumdiolate).

The influence of His94 and Pro149 in modulating the activity of V. cholerae DsbA.

DsbA is the primary catalyst of disulfide bond formation in the periplasm of gram-negative bacteria. Numerous theoretical and experimental studies have been undertaken to determine the molecular mechanisms by which DsbA acts as a potent oxidant, whereas the homologous cytoplasmic protein, thioredoxin, acts as a reductant. Many of these studies have focused on the nature of the two residues that lie between the active-site cysteines. Although these are clearly important, they are not solely responsible for the differences in activity between these thiol-disulfide oxidoreductases. Q97 in the helical domain of E. coli DsbA has been implicated in influencing the redox potential of E. coli DsbA. In V. cholerae DsbA, the analogous residue is H94. In this study, the effect of H94 on the oxidase activity of DsbA is examined, along with the role of the conserved cis-proline residue P149. The DsbA mutant H94L shows a nearly fourfold increase in activity over the wild-type enzyme. To our knowledge, this is the first time an increase in the normal activity of a thiol-disulfide oxidoreductase has been reported. Potential reasons for this increase in activity are discussed.

Structural studies on phospho-CDK2/cyclin A bound to nitrate, a transition state analogue: implications for the protein kinase mechanism.

Eukaryotic protein kinases catalyze the phosphoryl transfer of the gamma-phosphate of ATP to the serine, threonine, or tyrosine residue of protein substrates. The catalytic mechanism of phospho-CDK2/cyclin A (pCDK2/cyclin A) has been probed with structural and kinetic studies using the trigonal NO(3)(-) ion, which can be viewed as a mimic of the metaphosphate transition state. The crystal structure of pCDK2/cyclin A in complex with Mg(2+)ADP, nitrate, and a heptapeptide substrate has been determined at 2.7 A. The nitrate ion is located between the beta-phosphate of ADP and the hydroxyl group of the serine residue of the substrate. In one molecule of the asymmetric unit, the nitrate is close to the beta-phosphate of ADP (distance from the nitrate nitrogen to the nearest beta-phosphate oxygen of 2.5 A), while in the other subunit, the nitrate is closer to the substrate serine (distance of 2.1 A). Kinetic studies demonstrate that nitrate is not an effective inhibitor of protein kinases, consistent with the structural results that show the nitrate ion makes few stabilizing interactions with CDK2 at the catalytic site. The binding of orthovanadate was also investigated as a mimic of a pentavalent phosphorane intermediate of an associative mechanism for phosphoryl transfer. No vanadate was observed bound in a 3.4 A resolution structure of pCDK2/cyclin A in the presence of Mg(2+)ADP, and vanadate did not inhibit the kinase reaction. The results support the notion that the protein kinase reaction proceeds through a mostly dissociative mechanism with a trigonal planar metaphosphate intermediate rather than an associative mechanism that involves a pentavalent phosphorane intermediate.

A (1-->3)-beta-D-linked heptasaccharide is the unit ligand for glucan pattern recognition receptors on human monocytes.

Glucans are fungal cell wall polysaccharides which stimulate innate immune responses. We determined the minimum unit ligand that would bind to glucan receptors on human U937 cells using laminarin-derived pentaose, hexaose, and heptaose glucan polymers. When U937 membranes were pretreated with the oligosaccharides and passed over a glucan surface, only the heptasaccharide inhibited the interaction of glucan with membrane receptors at a K(d) of 31 microM (95% CI 20-48 microM) and 100% inhibition. However, the glucan heptasaccharide did not stimulate U937 monocyte NFkappaB signaling, nor did it increase survival in a murine model of polymicrobial sepsis. Laminarin, a larger and more complex glucan polymer (M(w) = 7700 g/mol), only partially inhibited binding (61 +/- 4%) at a K(d) of 2.6 microM (99% CI 1.7-4.2 microM) with characteristics of a single binding site. These results indicate that a heptasaccharide is the smallest unit ligand recognized by macrophage glucan receptors. The data also indicate the presence of at least two glucan-binding sites on U937 cells and that the binding sites on human monocyte/macrophages can discriminate between glucan polymers. The heptasaccharide and laminarin were receptor antagonists, but they were not receptor agonists with respect to activation of NFkappaB-dependent signaling pathways or protection against experimental sepsis.

Plasma pharmacokinetics and cerebrospinal fluid penetration of thioguanine in children with acute lymphoblastic leukemia: a collaborative Pediatric Oncology Branch, NCI, and Children's Cancer Group study.

PURPOSE: In preclinical studies, thioguanine (TG) has been shown to be more potent than the standard acute lymphoblastic leukemia (ALL) maintenance agent, mercaptopurine (MP), suggesting that TG may be more efficacious than MP in the treatment of childhood ALL. As part of a pilot trial in which TG was used in place of MP, we studied the plasma pharmacokinetics of oral TG and measured steady-state plasma and CSF TG concentrations during a continuous intravenous infusion (CIVI) in children with newly diagnosed standard-risk ALL. METHODS: Nine plasma samples were collected after each patient's first 60 mg/m2 oral TG dose during maintenance. CIVI TG (20 mg/m2/h over 24 h) was administered during the consolidation phase of therapy, and simultaneous plasma and CSF samples were collected near the end of the infusion. TG was measured by reverse-phase HPLC with ultraviolet detection. Erythrocyte TG nucleotide (TGN) concentrations were measured 7 days after a course of CIVI TG and prior to the start of each maintenance cycle. RESULTS: After oral TG (n = 35), the mean (+/- SD) peak plasma concentration was 0.46 +/- 0.68 microM and the AUC ranged from 0.18 to 9.5 microM.h (mean 1.5 microM.h). Mean steady-state plasma and CSF TG concentrations during CIVI (n = 33) were 2.7 and 0.5 microM, respectively. The mean (+/- SD) TG clearance was 935 +/- 463 ml/min per m2. Plasma TG concentrations did not correlate with erythrocyte TGN concentrations after oral or CIVI TG. The 8-OH-TG metabolite was detected in plasma and CSF. CONCLUSIONS: TG concentrations that are cytotoxic to human leukemia cell lines can be achieved in plasma after a 60 mg/m2 oral dose of TG and in plasma and CSF during CIVI of TG.

Performance of a recirculating wetland filter designed to remove particulate phosphorus for restoration of Lake Apopka (Florida, USA).

Operation of a 14-km2 wetland filter for removal of total phosphorus (TP) from lake water is part of the restoration program for hypereutrophic Lake Apopka, Florida. This system differs from most treatment wetlands because 1) water is recirculated back to the lake, and 2) the goal is removal of particulate phosphorus (P), the dominant form of P in Lake Apopka. The operational plan for the wetland is maximization of the rate rather than the efficiency of P removal. The St. Johns River Water Management District operated a 2-km2 pilot-scale wetland to examine the capacity of a wetland system to remove suspended solids and particulate nutrients from Lake Apopka. TP in the inflow from Lake Apopka ranged from about 0.12 to 0.23 mg l(-1), and hydraulic loading rate (HLR) varied from 6.5 to 42 m yr(-1). The performance of the pilot-scale wetland supported earlier predictions. Mass removal efficiencies for TP varied between about 30% and 67%. A first-order, area-based model indicated a rate constant for TP removal of 55 m yr(-1). We compared actual removal of P with model predictions and used modeled performance to examine optimal operational conditions. Correspondence between observed and modeled outflow TP was not good with constant variable values. Monte Carlo techniques used to introduce realistic stochastic variability improved the fit. The model was used to project a maximal rate of P removal of about 4 g P m(-2) yr(-1) at P loading 10-15 g P m(-2) yr(-1) (HLR 60-90 m yr(-1)). Data from the pilot wetland indicated that actual rates of P removal may prove to be higher. Further operation of the wetland at high hydraulic and P loading rates is necessary to verify or modify the application of the model.

Determining ecologically acceptable nutrient loads to natural wetlands for water quality improvement.

Natural wetlands often function as nutrient sinks, reducing nutrient inputs into lakes and streams. P loading from anthropogenic sources has significantly affected many natural wetlands. This paper describes a method to determine an acceptable P load to natural wetlands based on ecological principles. This approach can be used to determine how much P can be assimilated without diminishing species diversity and, thereby, sets a limit for cultural eutrophication of natural wetlands. The basis for determining an acceptable load is management of risk to species diversity by determination of the maximum area of a wetland that can be put at risk while preserving biodiversity of the overall wetland system. Two cases are distinguished: 1) simple-stress, where growth of the affected area immediately increases risks for species loss, and 2) subsidy-stress, where growth of the affected area first benefits then diminishes net species diversity.

Identification of factors contributing to increased length of stay in two diagnosis related groups.

This paper discusses a study conducted to identify factors that contributed to increased length of stay for two diagnosis related groups (DRGs) and their consequential impact on nursing salaries. The study shows that three separate clusters of cost drivers (DRG-related, nurse-related, and patient-related) contributed to increased length of stay for DRG 177 (chronic obstructive airways disease) and DRG 367 (cholecystectomy without exploration of the common bile duct). It was not possible to establish a link between length of stay and nursing salaries due to lack of relevant data. The results of the study can be used to raise professional awareness to the difficulties encountered by nurse managers in controlling length of stay when there are substantial numbers of different DRGs in acute care wards. The results can also be used as a basis for conducting larger studies into DRGs with higher than expected lengths of stay.

Catalytic mechanism of phosphorylase kinase probed by mutational studies.

The contributions to catalysis of the conserved catalytic aspartate (Asp149) in the phosphorylase kinase catalytic subunit (PhK; residues 1-298) have been studied by kinetic and crystallographic methods. Kinetic studies in solvents of different viscosity show that PhK, like cyclic AMP dependent protein kinase, exhibits a mechanism in which the chemical step of phosphoryl transfer is fast and the rate-limiting step is release of the products, ADP and phosphoprotein, and possibly viscosity-dependent conformational changes. Site-directed mutagenesis of Asp149 to Ala and Asn resulted in enzymes with a small increase in K(m) for glycogen phosphorylase b (GPb) and ATP substrates and dramatic decreases in k(cat) (1.3 x 10(4) for Asp149Ala and 4.7 x 10(3) for Asp149Asn mutants, respectively). Viscosometric kinetic measurements with the Asp149Asn mutant showed a reduction in the rate-limiting step for release of products by 4.5 x 10(3) and a significant decrease (possibly as great as 2.2 x 10(3)) in the rate constant characterizing the chemical step. The date combined with the crystallographic evidence for the ternary PhK-AMPPNP-peptide complex [Lowe et al. (1997) EMBO J. 6, 6646-6658] provide powerful support for the role of the carboxyl of Asp149 in binding and orientation of the substrate and in catalysis of phosphoryl transfer. The constitutively active subunit PhK has a glutamate (Glu182) residue in the activation segment, in place of a phosphorylatable serine, threonine, or tyrosine residue in other protein kinases that are activated by phosphorylation. Site-directed mutagenesis of Glu182 and other residues involved in a hydrogen bond network resulted in mutant proteins (Glu182Ser, Arg148Ala, and Tyr206Phe) with decreased catalytic efficiency (approximate average decrease in k(cat)/K(m) by 20-fold). The crystal structure of the mutant Glu182Ser at 2.6 A resolution showed a phosphate dianion about 2.6 A from the position previously occupied by the carboxylate of Glu182. There was no change in tertiary structure from the native protein, but the activation segment in the region C-terminal to residue 182 showed increased disorder, indicating that correct localization of the activation segment is necessary in order to recognize and present the protein substrate for catalysis.

Thioguanine administered as a continuous intravenous infusion to pediatric patients is metabolized to the novel metabolite 8-hydroxy-thioguanine.

Thiopurine antimetabolites have been in clinical use for more than 40 years, yet the metabolism of thiopurines remains only partially understood. Data from our previous pediatric phase 1 trial of continuous i.v. infusion of thioguanine (CIVI-TG) suggested that TG was eliminated by saturable mechanism, with conversion of the drug to an unknown metabolite. In this study we have identified this metabolite as 8-hydroxy-thioguanine (8-OH-TG). The metabolite coeluted with the 8-OH-TG standard on HPLC and had an identical UV spectrum, with a lambda(max) of 350 nm. On mass spectroscopy, the positive ion, single quad scan of 8-OH-TG yielded a protonated molecular ion at 184 Da and contained diagnostic ions at m/z 167, 156, 142, and 125 Da. Incubation of TG in vitro with partially purified aldehyde oxidase resulted in 8-OH-TG formation. 8-OH-TG is the predominant circulating metabolite found in patients receiving CIVI-TG and is likely generated by the action of aldehyde oxidase.