Changes in Pediatric Non-accidental Trauma Emergency Department Visits During and Following the COVID-19 Lockdown.

A level 1 pediatric trauma registry database was examined for all non-accidental trauma (NAT) emergency department visits between 2016 and 2021, and average injury severity score assigned to those patients with physical injuries over 2019-2021. There was a decline in NAT visits in 2020 (267) from prior years (343 visits average over 2016-2019), with subsequent increase in 2021 (548). Injury severity score increased (ISS) in 2020 (7.3) when compared to 2019 (5.71), with a decline in average ISS in 2021 (5.42). This data highlights the potential for missed abuse during closures with increased detection following reopening. Our data regarding ISS demonstrates the pediatric population is at risk of more severe abuse during times of familial stress. We need increased awareness that periods of vulnerability to NAT exist, as seen during the COVID-19 pandemic.

Routine Lower Extremity Screening Ultrasound Protocols in Trauma Patients Are Not Cost Effective.

BACKGROUND: Despite prophylaxis, deep vein thrombosis (DVT) and pulmonary embolism remain dreaded complications following traumatic injury and are associated with significant morbidity and mortality. Screening ultrasound (US) protocols have been employed in trauma centers for early detection of lower extremity (LE) deep venous thrombosis. We hypothesized that screening lower extremity venous duplex US would not prove cost effective in our trauma population who receives early pharmacologic prophylaxis. METHODS: Data was collected for one year on all adult trauma patients admitted to the trauma service from December 2019 to 2020. DVT screening US was obtained at 3 days after admission for patients with long bone or pelvic fracture, spinal cord injury, immobility, and/or spinal fracture requiring surgery. Screening US was obtained at 7 days for all others and repeated weekly until discharge. Data was retrospectively collected and analyzed. RESULTS: Exactly 1365 patients met inclusion criteria with median ISS 12 (IQR, 9-17), median age 56 (IQR, 36-73 years), and with majority blunt injuries (90.7%). A total of 1369 screening US were performed finding 27 DVTs (2%). The total cost of screening for the year analyzed amounted to over $270,000 with 50.7 screening US needed to detect 1 DVT. This resulted in an average screening cost of over $10,000 for the detection of a single DVT. DISCUSSION: In trauma patients receiving early pharmacologic prophylaxis, routine LE screening US protocols to detect LE DVT are not cost effective.

Use of Lean Six Sigma methodology shows reduction of inpatient waiting time for peripherally inserted central catheter placement.

AIM: The aim of this study was to assess the use of Lean Six Sigma methodology to improve the turnaround time (TAT) for inpatient peripherally inserted central catheter (PICC) placement. MATERIALS AND METHODS: Value stream mapping was used to analyse the workflow process for inpatient PICC placement and to divide it into its component parts. Unnecessary steps were eliminated and variation minimised in the remaining processes. The TAT for PICC line placement was recorded for the 6 months prior to implementation of changes, and subsequently, at the 6-month and 2-year follow-up points. RESULTS: Prior to implementing the changes, the mean TAT for PICC line placement was 3.74±3.28 days (95% confidence interval [CI]=3.3-4.17). Six months after implementation, the mean TAT was 1.89±1.82 days (95% CI=1.72-2.06, p<0.0001). The reduction was sustained such that at 2 years post-implementation the mean TAT was 1.88±1.87 days (95% CI=1.78-1.99, p<0.0001). This was achieved despite a 13.8% increase in overall interventional radiological activity. CONCLUSION: By applying Lean Six Sigma methodology to the complex multifactorial processes involved from ordering a PICC to its final insertion, it was possible to identify areas for improvement and to introduce simple, effective measures that resulted in a significant sustained decrease in the TAT without additional resources.

On the Failure Initiation in the Proximal Human Femur Under Simulated Sideways Fall.

The limitations of areal bone mineral density measurements for identifying at-risk individuals have led to the development of alternative screening methods for hip fracture risk including the use of geometrical measurements from the proximal femur and subject specific finite element analysis (FEA) for predicting femoral strength, based on quantitative CT data (qCT). However, these methods need more development to gain widespread clinical applications. This study had three aims: To investigate whether proximal femur geometrical parameters correlate with obtained femur peak force during the impact testing; to examine whether or not failure of the proximal femur initiates in the cancellous (trabecular) bone; and finally, to examine whether or not surface fracture initiates in the places where holes perforate the cortex of the proximal femur. We found that cortical thickness around the trochanteric-fossa is significantly correlated to the peak force obtained from simulated sideways falling (R 2 = 0.69) more so than femoral neck cortical thickness (R 2 = 0.15). Dynamic macro level FE simulations predicted that fracture generally initiates in the cancellous bone compartments. Moreover, our micro level FEA results indicated that surface holes may be involved in primary failure events.

Material mapping strategy to improve the predicted response of the proximal femur to a sideways fall impact.

Sideways falls are largely responsible for the highly prevalent osteoporotic hip fractures in today's society. These injuries are dynamic events, therefore dynamic FE models validated with dynamic ex vivo experiments provide a more realistic simulation than simple quasi-static analysis. Drop tower experiments using cadaveric specimens were used to identify the material mapping strategy that provided the most realistic mechanical response under impact loading. The present study tested the addition of compression-tension asymmetry, tensile bone damage, and cortical-specific strain rate dependency to the material mapping strategy of fifteen dynamic FE models of the proximal femur, and found improved correlations and reduced error for whole bone stiffness (R2 = 0.54, RSME = 0.87kN/mm) and absolute maximum force (R2 = 0.56, RSME =0.57kN), and a high correlation in impulse response (R2 = 0.82, RSME =12.38kg/s). Simulations using fully bonded nodes between the rigid bottom plate and PMMA cap supporting the femoral head had higher correlations and less error than simulations using a frictionless sliding at this contact surface. Strain rates over 100/s were observed in certain elements in the femoral neck and trochanter, indicating that additional research is required to better quantify the strain rate dependencies of both trabecular and cortical bone at these strain rates. These results represent the current benchmark in dynamic FE modeling of the proximal femur in sideways falls. Future work should also investigate improvements in experimental validation techniques by developing better displacement measurements and by enhancing the biofidelity of the impact loading wherever possible.

The Development of Technology for Effective Respiratory-Gated Irradiation Using an Image-Guided Small Animal Irradiator.

The development of image-guided small animal irradiators represents a significant improvement over standard irradiators by enabling preclinical studies to mimic radiotherapy in humans. The ability to deliver tightly collimated targeted beams, in conjunction with gantry or animal couch rotation, has the potential to maximize tumor dose while sparing normal tissues. However, the current commercial platforms do not incorporate respiratory gating, which is required for accurate and precise targeting in organs subject to respiration related motions that may be up to the order of 5 mm in mice. Therefore, a new treatment head assembly for the Xstrahl Small Animal Radiation Research Platform (SARRP) has been designed. This includes a fast X-ray shutter subsystem, a motorized beam hardening filter assembly, an integrated transmission ionization chamber to monitor beam delivery, a kinematically positioned removable beam collimator and a targeting laser exiting the center of the beam collimator. The X-ray shutter not only minimizes timing errors but also allows beam gating during imaging and treatment, with irradiation only taking place during the breathing cycle when tissue movement is minimal. The breathing related movement is monitored by measuring, using a synchronous detector/lock-in amplifier that processes diffuse reflectance light from a modulated light source. After thresholding of the resulting signal, delays are added around the inhalation/exhalation phases, enabling the "no movement" period to be isolated and to open the X-ray shutter. Irradiation can either be performed for a predetermined time of X-ray exposure, or through integration of a current from the transmission monitor ionization chamber (corrected locally for air density variations). The ability to successfully deliver respiratory-gated X-ray irradiations has been demonstrated by comparing movies obtained using planar X-ray imaging with and without respiratory gating, in addition to comparing dose profiles observed from a collimated beam on EBT3 radiochromic film mounted on the animal's chest. Altogether, the development of respiratory-gated irradiation facilitates improved dose delivery during animal movement and constitutes an important new tool for preclinical radiation studies. This approach is particularly well suited for irradiation of orthotopic tumors or other targets within the chest and abdomen where breathing related movement is significant.

Morphology based anisotropic finite element models of the proximal femur validated with experimental data.

Finite element analysis (FEA) of bones scanned with Quantitative Computed Tomography (QCT) can improve early detection of osteoporosis. The accuracy of these models partially depends on the assigned material properties, but anisotropy of the trabecular bone cannot be fully captured due to insufficient resolution of QCT. The inclusion of anisotropy measured from high resolution peripheral QCT (HR-pQCT) could potentially improve QCT-based FEA of the femur, although no improvements have yet been demonstrated in previous experimental studies. This study analyzed the effects of adding anisotropy to clinical resolution femur models by constructing six sets of FE models (two isotropic and four anisotropic) for each specimen from a set of sixteen femurs that were experimentally tested in sideways fall loading with a strain gauge on the superior femoral neck. Two different modulus-density relationships were tested, both with and without anisotropy derived from mean intercept length analysis of HR-pQCT scans. Comparing iso- and anisotropic models to the experimental data resulted in nearly identical correlation and highly similar linear regressions for both whole bone stiffness and strain gauge measurements. Anisotropic models contained consistently greater principal compressive strains, approximately 14% in magnitude, in certain internal elements located in the femoral neck, greater trochanter, and femoral head. In summary, anisotropy had minimal impact on macroscopic measurements, but did alter internal strain behavior. This suggests that organ level QCT-based FE models measuring femoral stiffness have little to gain from the addition of anisotropy, but studies considering failure of internal structures should consider including anisotropy to their models.

The influence of the modulus-density relationship and the material mapping method on the simulated mechanical response of the proximal femur in side-ways fall loading configuration.

Contributing to slow advance of finite element (FE) simulations for hip fracture risk prediction, into clinical practice, could be a lack of consensus in the biomechanics community on how to map properties to the models. Thus, the aim of the present study was first, to systematically quantify the influence of the modulus-density relationship (E-ρ) and the material mapping method (MMM) on the predicted mechanical response of the proximal femur in a side-ways fall (SWF) loading configuration and second, to perform a model-to-model comparison of the predicted mechanical response within the femoral neck for all the specimens tested in the present study, using three different modelling techniques that have yielded good validation outcome in terms of surface strain prediction and whole bone response according to the literature. We found the outcome to be highly dependent on both the E-ρ relationship and the MMM. In addition, we found that the three modelling techniques that have resulted in good validation outcome in the literature yielded different principal strain prediction both on the surface as well as internally in the femoral neck region of the specimens modelled in the present study. We conclude that there exists a need to carry out a more comprehensive validation study for the SWF loading mode to identify which combination of MMMs and E-ρ relationship leads to the best match for whole bone and local mechanical response. The MMMs tested in the present study have been made publicly available at https://simtk.org/home/mitk-gem.

A Fixed-point Scheme for the Numerical Construction of Magnetohydrostatic Atmospheres in Three Dimensions.

Magnetohydrostatic models of the solar atmosphere are often based on idealized analytic solutions because the underlying equations are too difficult to solve in full generality. Numerical approaches, too, are often limited in scope and have tended to focus on the two-dimensional problem. In this article we develop a numerical method for solving the nonlinear magnetohydrostatic equations in three dimensions. Our method is a fixed-point iteration scheme that extends the method of Grad and Rubin (Proc. 2nd Int. Conf. on Peaceful Uses of Atomic Energy31, 190, 1958) to include a finite gravity force. We apply the method to a test case to demonstrate the method in general and our implementation in code in particular.

Comparison of explicit finite element and mechanical simulation of the proximal femur during dynamic drop-tower testing.

Current screening techniques based on areal bone mineral density (aBMD) measurements are unable to identify the majority of people who sustain hip fractures. Biomechanical examination of such events may help determine what predisposes a hip to be susceptible to fracture. Recently, drop-tower simulations of in-vitro sideways falls have allowed the study of the mechanical response of the proximal human femur at realistic impact speeds. This technique has created an opportunity to validate explicit finite element (FE) models against dynamic test data. This study compared the outcomes of 15 human femoral specimens fractured using a drop tower with complementary specimen-specific explicit FE analysis. Correlation coefficient and root mean square error (RMSE) were found to be moderate for whole bone stiffness comparison (R(2)=0.3476 and 22.85% respectively). No correlation was found between experimentally and computationally predicted peak force, however, energy absorption comparison produced moderate correlation and RMSE (R(2)=0.4781 and 29.14% respectively). By comparing predicted strain maps to high speed video data we demonstrated the ability of the FE models to detect vulnerable portions of the bones. Based on our observations, we conclude that there exists a need to extend the current apparent level material models for bone to cover higher strain rates than previously tested experimentally.

Proximal femur elastic behaviour is the same in impact and constant displacement rate fall simulation.

Understanding proximal femur fracture may yield new targets for fracture prevention screening and treatment. The goal of this study was to characterize force-displacement and failure behaviours in the proximal femur between displacement control and impact loading fall simulations. Twenty-one human proximal femurs were tested in two ways, first to a sub-failure load at a constant displacement rate, then to fracture in an impact fall simulator. Comparisons of sub-failure energy and stiffness were made between the tests at the same compressive force. Additionally, the impact failure tests were compared with previous, constant displacement rate failure tests (at 2 and 100mm/s) in terms of energy, yield force, and stiffness. Loading and displacement rates were characterized and related to specimen stiffness in the impact tests. No differences were observed between the sub-failure constant displacement and impact tests in the aforementioned metrics. Comparisons between failure tests showed that the impact group had the lowest absorbed energy, 24% lower maximum force and 160% higher stiffness than the 100mm/s group (p<0.01 for all), but suffered from low statistical power to differentiate the donor age and specimen BMD. Loading and displacement rates for the specimens tested using impact varied during each test and between specimens and did not show appreciable viscoelasticity. These results indicate that constant displacement rate testing may help understand sub-failure mechanical behaviour, but may not elucidate failure behaviours. The differences between the impact and constant displacement rate fall simulations have important ramifications for interpreting the results of previous experiments.

Development of a balanced experimental-computational approach to understanding the mechanics of proximal femur fractures.

The majority of people who sustain hip fractures after a fall to the side would not have been identified using current screening techniques such as areal bone mineral density. Identifying them, however, is essential so that appropriate pharmacological or lifestyle interventions can be implemented. A protocol, demonstrated on a single specimen, is introduced, comprising the following components; in vitro biofidelic drop tower testing of a proximal femur; high-speed image analysis through digital image correlation; detailed accounting of the energy present during the drop tower test; organ level finite element simulations of the drop tower test; micro level finite element simulations of critical volumes of interest in the trabecular bone. Fracture in the femoral specimen initiated in the superior part of the neck. Measured fracture load was 3760N, compared to 4871N predicted based on the finite element analysis. Digital image correlation showed compressive surface strains as high as 7.1% prior to fracture. Voxel level results were consistent with high-speed video data and helped identify hidden local structural weaknesses. We found using a drop tower test protocol that a femoral neck fracture can be created with a fall velocity and energy representative of a sideways fall from standing. Additionally, we found that the nested explicit finite element method used allowed us to identify local structural weaknesses associated with femur fracture initiation.

Effect of increased fruit and vegetable consumption on bone turnover in older adults: a randomised controlled trial.

UNLABELLED: Evidence suggests that increased fruit and vegetable (FV) intake may be associated with improved bone health, but there is limited evidence from intervention trials to support this. This 16-week study showed that increased FV consumption (five or more portions per day) does not have any effect on the markers of bone health in older adults. INTRODUCTION: Observational evidence suggests that increased FV consumption may be associated with improved bone health. However, there is lack of evidence from intervention trials to support this. This study examined the effect of increased FV consumption on bone markers among healthy, free-living older adults. METHODS: A randomised controlled trial was undertaken. Eighty-three participants aged 65-85 years, habitually consuming less than or equal to two portions of FV per day, were randomised to continue their normal diet or to consume five or more portions of FV per day for 16 weeks. FV were delivered to all participants each week, free of charge. Compliance was assessed at baseline and at 6, 12 and 16 weeks by diet histories and biomarkers of micronutrient status. Fasting serum bone markers (osteocalcin (OC) and C-terminal telopeptide of type 1 collagen (CTX)) were measured using enzyme-linked immunosorbent assay. RESULTS: Eighty-two participants completed the intervention. The five portions per day group showed a significantly greater change in daily FV consumption compared to the two portions per day group (p < 0.001), and this was reflected in significant increases in micronutrient status. No significant differences were evident in change in bone markers between the two portions per day group and the five portions per day group over the 16 weeks (geometric mean of week 16 to baseline ratio (95% confidence interval): OC-0.95 (0.89-1.02) and 1.04 (0.91-1.18), respectively, p = 0.25; CTX-1.06 (0.95-1.19) and 0.98 (0.90-1.06) respectively, p = 0.20). CONCLUSIONS: Increased FV consumption had no effect on bone markers in older adults. Larger intervention studies of longer duration are warranted to establish whether long-term FV consumption can benefit bone health.

Placental protein tyrosine nitration and MAPK in type 1 diabetic pre-eclampsia: Impact of antioxidant vitamin supplementation.

AIM: To examine the role of placental protein tyrosine nitration and p38-Mitogen-Activated Protein Kinase α (p38-MAPKα), Extra Cellular-Signal Regulated Kinase (ERK) and c-Jun NH2-Terminal Kinase (JNK) activity, in the pathogenesis of type 1 diabetic pre-eclampsia, and the putative modulation of these indices by maternal vitamin C and E supplementation. METHODS: Placental samples were obtained from a sub-cohort of the DAPIT trial: a randomised placebo-controlled trial of antioxidant supplementation to reduce pre-eclampsia in type 1 diabetic pregnancy. Placenta from placebo-treated: normotensive (NT) [n=17], gestational hypertension (GH) [n=7] and pre-eclampsia (PE) [n=6] and vitamin-treated: NT (n=20), GH (n=4) and PE (n=3) was analysed. Protein tyrosine nitration was assessed by immunohistochemistry in paraffin-embedded tissue. Catalytic activities of placental p38-MAPKα, ERK and JNK were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Nitrotyrosine immunostaining was present in placebo-treated NT, GH and PE placentae, with no significant difference observed between the groups. There was a non-significant trend towards decreased p38-MAPKα activity in PE vs NT control placentae. ERK and JNK were similar among the three outcome placebo groups and vitamin supplementation did not significantly alter their activity. CONCLUSION: Nitrotyrosine immunopositivity in normotensive diabetic placentae indicates some degree of tyrosine nitration in uncomplicated diabetic pregnancy, possibly due to inherent oxidative stress and peroxynitrite production. Our results suggest that p38-MAPKα, ERK and JNK are not directly involved in the pathogenesis of type 1 diabetic pre-eclampsia and are not modulated by vitamin-supplementation.

Pharmacokinetics of meloxicam in koalas (Phascolarctos cinereus) after intravenous, subcutaneous and oral administration.

The pharmacokinetic profile of meloxicam in clinically healthy koalas (n = 15) was investigated. Single doses of meloxicam were administered intravenously (i.v.) (0.4 mg/kg; n = 5), subcutaneously (s.c.) (0.2 mg/kg; n = 1) or orally (0.2 mg/kg; n = 3), and multiple doses were administered to two groups of koalas via the oral or s.c. routes (n = 3 for both routes) with a loading dose of 0.2 mg/kg for day 1 followed by 0.1 mg/kg s.i.d for a further 3 days. Plasma meloxicam concentrations were quantified by high-performance liquid chromatography. Following i.v. administration, meloxicam exhibited a rapid clearance (CL) of 0.44 ± 0.20 (SD) L/h/kg, a volume of distribution at terminal phase (Vz ) of 0.72 ± 0.22 L/kg and a volume of distribution at steady state (Vss ) of 0.22 ± 0.12 L/kg. Median plasma terminal half-life (t(1/2)) was 1.19 h (range 0.71-1.62 h). Following oral administration either from single or repeated doses, only maximum peak plasma concentration (C(max) 0.013 ± 0.001 and 0.014 ± 0.001 µg/mL, respectively) was measurable [limit of quantitation (LOQ) >0.01 µg/mL] between 4-8 h. Oral bioavailability was negligible in koalas. Plasma protein binding of meloxicam was ~98%. Three meloxicam metabolites were detected in plasma with one identified as the 5-hydroxy methyl derivative. This study demonstrated that koalas exhibited rapid CL and extremely poor oral bioavailability compared with other eutherian species. Accordingly, the currently recommended dose regimen of meloxicam for this species appears inadequate.

Factors associated with serum/plasma concentrations of vitamins A, C, E and carotenoids in older people throughout Europe: the EUREYE study.

PURPOSE: To report on plasma/serum levels of antioxidant vitamin and carotenoids in older adults resident in multiple countries in Europe and examine relationships with potential modifiers. METHODS: Population-based cross-sectional European Eye Study in 7 centres from northern to southern Europe. In total, 4,133 participants aged 65 years or over, collected by random sampling, were recruited. Questionnaires relating to diet, lifestyle and medical history were administered. Non-fasting blood samples were analysed in a single laboratory for vitamins A, C and E and a panel of carotenoids. Associations were analysed by bootstrapped multivariable regression analysis. RESULTS: Centre and season influenced the serum and plasma concentrations of all antioxidant vitamins and carotenoids. Gender, BMI, smoking, age, education, alcohol consumption and supplement use were also significantly associated with some, but not all, of the antioxidant vitamins and carotenoids examined. The proportion of variance explained ranged from 4.8 % for retinol to 25.2 % for zeaxanthin. CONCLUSIONS: In older people, antioxidant vitamin and carotenoid status varies by centre and season, but is also associated with other behavioural and lifestyle variables. Studies aiming to demonstrate an association between antioxidant vitamins and carotenoid status and chronic disease risk should consider these potential confounders.

Effect of lycopene supplementation on insulin-like growth factor-1 and insulin-like growth factor binding protein-3: a double-blind, placebo-controlled trial.

OBJECTIVE: Studies have suggested a link between lycopene and insulin-like growth factor-1 (IGF-1). The aim of this study was to test the effect of lycopene supplementation on IGF-1 and binding protein-3 (IGFBP-3) status in healthy male volunteers. DESIGN, SETTING, SUBJECTS AND INTERVENTION: This was a 4 week randomized, double-blind, placebo-controlled study of lycopene supplementation (15 mg/day) in healthy male volunteers (n=20). Fasting blood samples were collected at baseline and after 4 weeks. Samples were analysed for lycopene by high-performance liquid chromatography (HPLC) and IGF-1 and IGFBP-3 by enzyme-linked immunosorbent assay (ELISA). Changes in end points from baseline were compared in those who received placebo versus those who received the lycopene supplement. RESULTS: Median change in lycopene from baseline (post-supplement - baseline) was higher in subjects in the intervention than those on placebo (lycopene group 0.29 (0.09, 0.46); placebo group 0.03 (-0.11, 0.08) micromol/l; median (25th, 75th percentiles), P<0.01). There was no difference in median change in IGF-1 concentrations (lycopene group -0.6 (-2.6, 1.9); placebo group -1.15 (-2.88, 0.95) nmol/l, P=0.52), or median change in IGFBP-3 concentrations (lycopene group 245 (-109, 484); placebo group 101 (-34, 234) nmol/l, P=0.55) between intervention and control groups. Change in lycopene concentration was associated with the change in IGFBP-3 in the intervention group (r=0.78; P=0.008; n=10). CONCLUSIONS: Lycopene supplementation in healthy male subjects has no effect on IGF-1 or IGFBP-3 concentrations in a healthy male population. However, the association between change in lycopene concentration and change in IGFBP-3 in the intervention group suggests a potential effect of lycopene supplementation on IGFBP-3.

CTLA4Ig delivered by high-capacity adenoviral vector induces stable expression of dystrophin in mdx mouse muscle.

Adenoviral (Ad) vector-mediated gene delivery of normal, full-length dystrophin to skeletal muscle provides a promising strategy for the treatment of Duchenne muscular dystrophy (DMD), an X-linked recessive, dystrophin-deficient muscle disease. Studies in animal models suggest that successful DMD gene therapy by Ad vector-mediated gene transfer would be precluded by cellular and humoral immune responses induced by vector capsid and transgene proteins. To address the immunity induced by Ad vector-mediated dystrophin gene delivery to dystrophic muscle, we developed high-capacity adenoviral (HC-Ad) vectors expressing mouse dystrophin driven by the muscle creatine kinase promoter (AdmDys) and mCTLA4Ig (AdmCTLA4Ig) individually, or together from one vector (AdmCTLA4Ig/mDys). We found stable expression of dystrophin protein in the tibialis anterior muscles of mdx mice, coinjected with AdmCTLA4Ig and AdmDys, or injected alone with AdmCTLA4Ig/mDys, whereas the expression of dystrophin protein in the control group coinjected with AdmDys and an empty vector decreased by at least 50% between 2 and 8 weeks after administration. Additionally, we observed reductions in Ad vector-induced Th1 and Th2 cytokines, Ad vector-specific cytotoxic T lymphocyte activation and neutralizing anti-Ad antibodies in both experimental groups that received a mCTLA4Ig-expressing vector as compared to the control group. This study demonstrates that the coexpression of mCTLA4Ig and dystrophin in skeletal muscle provided by HC-Ad vector-mediated gene transfer can provide stable expression of dystrophin in immunocompetent, adult mdx mouse muscle and applies a potentially powerful strategy to overcome adaptive immunity induced by Ad vector-mediated dystrophin gene delivery toward the ultimate goal of treatment for DMD.

A focused ultrasoft x-ray microbeam for targeting cells individually with submicrometer accuracy.

The application of microbeams is providing new insights into the actions of radiation at the cell and tissue levels. So far, this has been achieved exclusively through the use of collimated charged particles. One alternative is to use ultrasoft X rays, focused by X-ray diffractive optics. We have developed a unique facility that uses 0.2-0.8-mm-diameter zone plates to focus ultrasoft X rays to a beam of less than 1 microm diameter. The zone plate images characteristic K-shell X rays of carbon or aluminum, generated by focusing a beam of 5-10 keV electrons onto the appropriate target. By reflecting the X rays off a grazing-incidence mirror, the contaminating bremsstrahlung radiation is reduced to 2%. The focused X rays are then aimed at selected subcellular targets using rapid automated cell-finding and alignment procedures; up to 3000 cells per hour can be irradiated individually using this arrangement.

The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells.

To fully understand genome function, the linear genome map must be integrated with a spatial map of chromosomes in the nucleus. Distinct nuclear addresses for a few human chromosomes have been described. Previously we have demonstrated that the gene-rich human chromosome 19 is located in a more central position in the nucleus than the similarly sized, but gene-poor, chromosome 18. To determine whether these two chromosomes are a paradigm for the organization of chromatin in the human nucleus, we have now analysed the nuclear organization of every human chromosome in diploid lymphoblasts and primary fibroblasts. We find that the most gene-rich chromosomes concentrate at the centre of the nucleus, whereas the more gene-poor chromosomes are located towards the nuclear periphery. In contrast, we find no significant relationship between chromosome size and position within the nucleus. Proteins of the nuclear membrane or lamina are candidates for molecules that might anchor regions of the genome at the nuclear periphery and it has been suggested that disruption of this organization may play a role in some disease pathologies. We show that the intranuclear organization of chromosomes is not altered in cells that lack the integral nuclear membrane protein emerin, from an individual with X-linked Emery--Dreifuss muscular dystrophy. This suggests that emerin is not necessary for localizing chromosomes at the nuclear periphery and that the muscular dystrophy phenotype in such individuals is not due to grossly altered nuclear organization of chromatin.