Perfusion cell seeding on large porous PLA/calcium phosphate composite scaffolds in a perfusion bioreactor system under varying perfusion parameters.

A promising approach to bone tissue engineering lies in the use of perfusion bioreactors where cells are seeded and cultured on scaffolds under conditions of enhanced nutrient supply and removal of metabolic products. Fluid flow alterations can stimulate cell activity, making the engineering of tissue more efficient. Most bioreactor systems are used to culture cells on thin scaffold discs. In clinical use, however, bone substitutes of large dimensions are needed. In this study, MG63 osteoblast-like cells were seeded on large porous PLA/glass scaffolds with a custom developed perfusion bioreactor system. Cells were seeded by oscillating perfusion of cell suspension through the scaffolds. Applicable perfusion parameters for successful cell seeding were determined by varying fluid flow velocity and perfusion cycle number. After perfusion, cell seeding, the cell distribution, and cell seeding efficiency were determined. A fluid flow velocity of 5 mm/s had to be exceeded to achieve a uniform cell distribution throughout the scaffold interior. Cell seeding efficiencies of up to 50% were achieved. Results suggested that perfusion cycle number influenced cell seeding efficiency rather than fluid flow velocities. The cell seeding conducted is a promising basis for further long term cell culture studies in large porous scaffolds.

Low prevalence of HIV and other selected sexually transmitted infections in 2004 in pregnant women from Kinshasa, the Democratic Republic of the Congo.

This study examined the prevalence of HIV and other sexually transmitted infections (STIs) in pregnant women in Kinshasa, the Democratic Republic of the Congo (DRC). Between April and July 2004, antenatal attendees at two of the largest maternity clinics in Kinshasa were tested to identify HIV status, syphilis, Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). HIV seroprevalence was 1.9% in 2082 women. With PCR techniques, CT and NG infections were also uncommon in the first 529 women (1.7% and 0.4%, respectively). No active syphilis infection case was identified by Treponema pallidum haemagglutination assay (TPHA) and rapid plasma reagin test (RPR). A woman's risk of HIV infection was significantly associated with her reporting a male partner having had other female sexual partners (OR 2.7, 95% CI 1.2-6.2). The continuing low seroprevalence of HIV in pregnant women from Kinshasa was confirmed. Understanding factors associated with this phenomenon could help prevent a future HIV epidemic in low HIV transmission areas in Africa.

Plastidic trnFUUC pseudogenes in North American genus Boechera (Brassicaceae): mechanistic aspects of evolution.

The origin and maintenance of a plastidic tandem repeat next to the TRNF (UUC) gene were analyzed in the genus BOECHERA in a phylogenetic context and were compared to published analogous examples that emerged in parallel in the Asteraceae and Juncaceae, respectively. Although we identified some features common to these taxonomic groups with respect to structure and origin of the region, obvious differences were encountered, which argue against a specific mechanism or evolutionary principle underlying the parallel origin and maintenance of the TRNF-tandem repeats in those families. In contrast to the situation in the Asteraceae, no reciprocal recombinant repeat types have been observed in the Brassicaceae. Forty copy types, classified into three groups, were isolated from 103 chloroplast haplotypes of BOECHERA and it was demonstrated that they are composed of four subregions of various origins. We discuss various mutation mechanisms such as DNA replication slippage, and inter- and intrachromosomal recombination which were reported to mediate variation in copy numbers and other types of observed sequence length polymorphism. It is shown that the observed molecular structure of the tandem repeat region did not fully fit the particular patterns expected under a scenario of evolution including any of the known mechanisms. Nevertheless, it appeared that intermolecular unequal crossing-over is most likely the driving force in the evolution of this tandem repeat. However, it remains to be explained, why no reciprocal recombinant copy types have been observed. The reconstructed phylogenetic relationships among copies reflected different evolutionary scenarios as follows: (1) A single and ancient origin of copies pre-dates the radiation of BOECHERA. (2) Parallel expansion and shortening of the tandem repeat within different BOECHERA lineages. (3) Conservation of the first copy, as it was the only one present in all chloroplast haplotypes.

Protein structure similarity clustering and natural product structure as guiding principles for chemical genomics.

The majority of all proteins are modularly built from a limited set of approximately 1,000 structural domains. The knowledge of a common protein fold topology in the ligand-sensing cores of protein domains can be exploited for the design of small-molecule libraries in the development of inhibitors and ligands. Thus, a novel strategy of clustering protein domain cores based exclusively on structure similarity considerations (protein structure similarity clustering, PSSC) has been successfully applied to the development of small-molecule inhibitors of acetylcholinesterase and the 11beta-hydroxysteroid dehydrogenases based on the structure of a naturally occurring Cdc25 inhibitor. The efficiency of making use of the scaffolds of natural products as biologically prevalidated starting points for the design of compound libraries is further highlighted by the development of benzopyran-based FXR ligands.

Influence of tissue conductivity anisotropy on EEG/MEG field and return current computation in a realistic head model: a simulation and visualization study using high-resolution finite element modeling.

To achieve a deeper understanding of the brain, scientists, and clinicians use electroencephalography (EEG) and magnetoencephalography (MEG) inverse methods to reconstruct sources in the cortical sheet of the human brain. The influence of structural and electrical anisotropy in both the skull and the white matter on the EEG and MEG source reconstruction is not well understood. In this paper, we report on a study of the sensitivity to tissue anisotropy of the EEG/MEG forward problem for deep and superficial neocortical sources with differing orientation components in an anatomically accurate model of the human head. The goal of the study was to gain insight into the effect of anisotropy of skull and white matter conductivity through the visualization of field distributions, isopotential surfaces, and return current flow and through statistical error measures. One implicit premise of the study is that factors that affect the accuracy of the forward solution will have at least as strong an influence over solutions to the associated inverse problem. Major findings of the study include (1) anisotropic white matter conductivity causes return currents to flow in directions parallel to the white matter fiber tracts; (2) skull anisotropy has a smearing effect on the forward potential computation; and (3) the deeper a source lies and the more it is surrounded by anisotropic tissue, the larger the influence of this anisotropy on the resulting electric and magnetic fields. Therefore, for the EEG, the presence of tissue anisotropy both for the skull and white matter compartment substantially compromises the forward potential computation and as a consequence, the inverse source reconstruction. In contrast, for the MEG, only the anisotropy of the white matter compartment has a significant effect. Finally, return currents with high amplitudes were found in the highly conducting cerebrospinal fluid compartment, underscoring the need for accurate modeling of this space.

White matter asymmetry in the human brain: a diffusion tensor MRI study.

Language ability and handedness are likely to be associated with asymmetry of the cerebral cortex (grey matter) and connectivity (white matter). Grey matter asymmetry, most likely linked to language has been identified with voxel-based morphometry (VBM) using T(1)-weighted images. Differences in white matter obtained with this technique are less consistent, probably due to the relative insensitivity of the T(1) contrast to the ultrastructure of white matter. Furthermore, previous VBM studies failed to find differences related to handedness in either grey or white matter. We revisited these issues and investigated two independent groups of subjects with diffusion-tensor imaging (DTI) for asymmetries in white matter composition. Using voxel-based statistical analyses an asymmetry of the arcuate fascicle was observed, with higher fractional anisotropy in the left hemisphere. In addition, we show differences related to handedness in the white matter underneath the precentral gyrus contralateral to the dominant hand. Remarkably, these findings were very robust, even when investigating small groups of subjects. This highlights the sensitivity of DTI for white matter tissue differences, making it an ideal tool to study small patient populations.

Extensive chloroplast haplotype variation indicates Pleistocene hybridization and radiation of North American Arabis drummondii, A. x divaricarpa, and A. holboellii (Brassicaceae).

Arabis drummondii, A. holboellii and their hybrid A. x divaricarpa are widespread perennials of open habitats in North America. A phylogenetic analysis based on noncoding chloroplast DNA sequences (trnL intron and trnL/F intergenic spacer) resolved A. drummondii as a monophyletic taxon, but found A. holboellii to bear chloroplast haplotypes from highly diverged evolutionary lineages. This raised the question of a possible polyphyletic origin of A. holboellii. Arabis x divaricarpa was found to be of recent and polytopic origin, a result consistent with its presumed hybrid origin. One hundred and three chloroplast haplotypes were detected within 719 Arabis accessions investigated. The majority of chloroplast-types were estimated to have arisen prior to the Wisconsin glaciation. Phylogeographical analysis using nested clade analysis, suggested for A. holboellii (i). past fragmentation events, partitioning genetic variation in several instances between the Sierra Nevada, the Southern Rocky Mountains and the Colorado Plateau on the one hand and the Central to Northern Rockies of the United States and adjacent Cascades on the other; and for both parental species (ii). recolonization of major areas formerly covered by the Wisconsin glaciation by three haplotypes; and (iii). restricted gene flow indicating isolation by distance in areas south of the last glacial maximum. Arabis x divaricarpa is closely codistributed with its parental species and resampled their haplotypes. The highest genetic diversity was found in the Rocky Mountains from Idaho and Montana south to Utah and Colorado. This area was further hypothesized to have played a major role in the origin of both parental species and probably represented an important glacial refugium. However, evidence for glacial refugia was also found in arctic and boreal regions of Alaska and near the Great Lakes. In comparison to nuclear ribosomal internal transcribed spacer data, chloroplast DNA divergence was very high and evidently predated the origin of A. drummondii and possibly A. holboellii. Divergence of major chloroplast lineages dates back to the middle of the Pleistocene at least. Extensive hybridization is the most likely evolutionary factor working on A. holboellii to explain the revealed discrepancy in nuclear DNA and chloroplast DNA diversification.

Distortion-free diffusion tensor imaging of cranial nerves and of inferior temporal and orbitofrontal white matter.

The main impact of functional neuroimaging has been its ability to locate neuronal activity either directly (EEG, MEG) or through the hemodynamic response caused by neuronal activity (PET, fMRI). In the past decade functional neuroimaging has been extended to investigate how brain regions interact, using the concepts of functional and effective connectivity. These concepts are further strengthened by estimates of anatomical connectivity of the same subject. A tool to determine anatomical connectivity in vivo may be provided by diffusion tensor imaging (DTI) methods. These can be used to determine the orientation of fiber bundles in white matter on the basis of the diffusion characteristics of water. Commonly, DTI data are acquired using echo planar imaging which suffers from susceptibility artifacts in orbitofrontal and inferior temporal cortex. Here we demonstrate the use of an alternative pulse sequence, diffusion-weighted single-shot STEAM, for assessing fiber orientation in orbitofrontal cortex and the cranial nerves. The scope of DTI needs to be extended to these structures to investigate psychiatric disorders in which orbitofrontal pathology or temporo-frontal disconnection have been postulated.

Comparative genomics and regulatory evolution: conservation and function of the Chs and Apetala3 promoters.

DNA sequence variations of chalcone synthase (Chs) and Apetala3 gene promoters from 22 cruciferous plant species were analyzed to identify putative conserved regulatory elements. Our comparative approach confirmed the existence of numerous conserved sequences which may act as regulatory elements in both investigated promoters. To confirm the correct identification of a well-conserved UV-light-responsive promoter region, a subset of Chs promoter fragments were tested in Arabidopsis thaliana protoplasts. All promoters displayed similar light responsivenesses, indicating the general functional relevance of the conserved regulatory element. In addition to known regulatory elements, other highly conserved regions were detected which are likely to be of functional importance. Phylogenetic trees based on DNA sequences from both promoters (gene trees) were compared with the hypothesized phylogenetic relationships (species trees) of these taxa. The data derived from both promoter sequences were congruent with the phylogenies obtained from coding regions of other nuclear genes and from chloroplast DNA sequences. This indicates that promoter sequence evolution generally is reflective of species phylogeny. Our study also demonstrates the great value of comparative genomics and phylogenetics as a basis for functional analysis of promoter action and gene regulation.

Comparative evolutionary analysis of chalcone synthase and alcohol dehydrogenase loci in Arabidopsis, Arabis, and related genera (Brassicaceae).

We analyzed sequence variation for chalcone synthase (Chs) and alcohol dehydrogenase (Adh) loci in 28 species in the genera Arabidopsis and Arabis and related taxa from tribe Arabideae. Chs was single-copy in nearly all taxa examined, while Adh duplications were found in several species. Phylogenies constructed from both loci confirmed that the closest relatives of Arabidopsis thaliana include Arabidopsis lyrata, Arabidopsis petraea, and Arabidopsis halleri (formerly in the genus Cardaminopsis). Slightly more distant are the North American n = 7 Arabis (Boechera) species. The genus Arabis is polyphyletic-some unrelated species appear within this taxonomic classification, which has little phylogenetic meaning. Fossil pollen data were used to compute a synonymous substitution rate of 1.5 x 10 substitutions per site per year for both Chs and Adh. Arabidopsis thaliana diverged from its nearest relatives about 5 MYA, and from Brassica roughly 24 MYA. Independent molecular and fossil data from several sources all provide similar estimates of evolutionary timescale in the Brassicaceae.

A comparison of neonatal mortality risk prediction models in very low birth weight infants.

BACKGROUND: Risk-adjusted severity of illness is frequently used in clinical research and quality assessments. Although there are multiple methods designed for neonates, they have been infrequently compared and some have not been assessed in large samples of very low birth weight (VLBW; <1500 g) infants. OBJECTIVES: To test and compare published neonatal mortality prediction models, including Clinical Risk Index for Babies (CRIB), Score for Neonatal Acute Physiology (SNAP), SNAP-Perinatal Extension (SNAP-PE), Neonatal Therapeutic Interventions Scoring System, the National Institute of Child Health and Human Development (NICHD) network model, and other individual admission factors such as birth weight, low Apgar score (<7 at 5 minutes), and small for gestational age status in a cohort of VLBW infants from the Washington, DC area. METHODS: Data were collected on 476 VLBW infants admitted to 8 neonatal intensive care units between October 1994 and February 1997. The calibration (closeness of total observed deaths to the predicted total) of models with published coefficients (SNAP-PE, CRIB, and NICHD) was assessed using the standardized mortality ratio. Discrimination was quantified as the area under the curve (AUC) for the receiver operating characteristic curves. Calibrated models were derived for the current database using logistic regression techniques. Goodness-of-fit of predicted to observed probabilities of death was assessed with the Hosmer-Lemeshow goodness-of-fit test. RESULTS: The calibration of published algorithms applied to our data was poor. The standardized mortality ratios for the NICHD, CRIB, and SNAP-PE models were.65,.56, and.82, respectively. Discrimination of all the models was excellent (range:.863-.930). Surprisingly, birth weight performed much better than in previous analyses, with an AUC of.869. The best models using both 12- and 24-hour postadmission data, significantly outperformed the best model based on birth data only but were not significantly different from each other. The variables in the best model were birth weight, birth weight squared, low 5-minute Apgar score, and SNAP (AUC =.930). CONCLUSION: Published models for severity of illness overpredicted hospital mortality in this set of VLBW infants, indicating a need for frequent recalibration. Discrimination for these severity of illness scores remains excellent. Birth variables should be reevaluated as a method to control for severity of illness in predicting mortality.

Treatment refusal/attrition among adults randomly assigned to programs at a drug treatment campus: The New Jersey Substance Abuse Treatment Campus, Seacaucus, NJ.

The New Jersey Substance Abuse Treatment Campus was funded to assess the feasibility of establishing a new model for delivering substance abuse treatment services and to serve as a research laboratory for conducting comparative evaluations of those services. The 350-bed campus was designed to improve treatment effectiveness by providing special services needed by underserved populations, and reduce treatment costs by serving large numbers of clients, centralizing services, and sharing facilities. First-time clients who met preliminary eligibility requirements during phone screening were randomly assigned to therapeutic community and chemical dependency programs. We used data collected on 1,573 adults who were ultimately accepted for admission to analyze treatment refusals and attrition during the 25 days after admission. Only 6.4% of the clients refused admission when informed of their treatment assignment. Planned duration of the residential phase of treatment, gender, and language spoken (English/Spanish) interacted with one another and differentially predicted treatment refusal/attrition. These findings may be useful for understanding treatment refusal and attrition in substance abuse treatment programs.

Therapeutic efficacy of oral lactobacillus preparation for antibiotic-associated enteritis in guinea pigs.

Enteritis is a potential complication of antimicrobial agent use, particularly in certain species of rodents. The organism most frequently implicated in this disease is Clostridium difficile. Anecdotal information suggests that administration of yogurt or other Lactobacillus-containing products in conjunction with antimicrobial agents will prevent or minimize the effects of antibiotic-associated enteritis. We wanted to determine whether a single subcutaneous injection of clindamycin phosphate could induce enteritis in guinea pigs and whether a commercial Lactobacillus preparation would ameliorate the clinical effects of antibiotic administration in these animals. Juvenile male guinea pigs were divided into three treatment groups. Group 1 guinea pigs (n=8) received a single saline injection followed by an oral Lactobacillus preparation twice daily; group 2 (n=8) received a single antibiotic injection followed by an oral Lactobacillus preparation twice daily; group 3 (n=8) received a single antibiotic injection. Attitude, body temperature, body weight, and feed and water consumption were recorded for each guinea pig 7 days prior to and after treatment. Fecal samples were collected and necropsies performed on each guinea pig at the time of euthanasia. C. difficile and other enteric pathogens were not isolated from any group before or after treatment, although some guinea pigs receiving the antibiotic developed enteritis. There were no significant clinical differences between guinea pigs receiving antibiotics with the oral Lactobacillus preparation, and those receiving antibiotics alone. The results of this study suggest that a single injection of clindamycin phosphate can induce enteritis in guinea pigs and that oral administration of a Lactobacillus-containing product is ineffective in preventing clinical disease in guinea pigs administered clindamycin phosphate.

A randomized controlled trial of smoking cessation intervention in pregnancy in an academic clinic.

To evaluate the effectiveness of a physician-based intervention to promote smoking cessation during pregnancy, we conducted this randomized controlled trial in the resident-staffed prenatal clinics at the University of North Carolina Women's Hospital . Two hundred fifty prenatal patients who smoked were enrolled at their first visit and randomly assigned to the intervention or the usual-care group. Resident physicians provided self-help materials to intervention subjects and used a script to set goals with them at each prenatal visit. Subjects who set quit dates were contacted by volunteer cessation counselors. To verify smoking status, subjects provided a self-report and breath carbon monoxide (CO) sample at each visit. Controls were similarly assessed at enrollment and at three additional predetermined intervals. Twenty percent of intervention subjects and 10% of controls reported cessation, which was verified by CO level (P = .052). Fifty-one percent of subjects reduced their consumption by half or more, compared with 30% of controls (P = .002). The intervention is effective in promoting smoking cessation and reduction. In addition, this technique is inexpensive, readily accepted by staff, and efficient.

Equal effectiveness of very-low-intensity anticoagulation and standard low-intensity anticoagulation: a pilot study.

We compared the efficacy of very-low-intensity oral anticoagulation (OA) with that of the recommended standard low-intensity oral anticoagulation, using international normalized ratios (INRs). We enrolled 101 patients into a pilot study--51 patients in the very-low-intensity anticoagulation arm (INR 1.4 to 2.0) and 50 in the standard low-intensity anticoagulation arm (INR 2.0 to 3.0). They were monitored for thrombotic/embolic and hemorrhagic complications for an average follow-up of 1.5 years. Two thrombotic/embolic events occurred in the very-low-intensity group; no thrombotic/embolic events occurred in the standard low-intensity group. No major bleeding occurred in the very-low-intensity group; one major hemorrhagic event occurred in the standard low-intensity group. These findings did not achieve a statistically significant difference in major complications between the two groups. It appears that very-low-intensity OA (INR 1.4 to 2.0) is as effective in preventing thromboses as standard low-intensity OA (INR 2.0 to 3.0).

Induction of labour with intravaginal misoprostol in the second and third trimesters of pregnancy.

OBJECTIVE: To confirm the effectiveness of misoprostol as a labour-induction agent. DESIGN AND SETTING: One hundred and ten consecutive second- and third-trimester hospital patients. Patients with intra-uterine deaths (group A) received 100 micrograms misoprostol 4-hourly and those with live fetuses (group B) 50 micrograms misoprostol 4-hourly until labour commenced. SUBJECTS: Forty-eight patients in group A (group A1 second trimester 27, group A2 third trimester 21); 62 mainly hypertensive patients in group B. OUTCOME MEASURES: These were the amount of misoprostol required to induce labour; duration of induction and labour; success and completeness of vaginal delivery; neonatal outcome; and cost. RESULTS: In group A1, labour was successfully induced in 21/27 (77.8%) patients with 157.4 micrograms misoprostol; and in 19/21 (90.5%) patients in group A2 with 128.9 micrograms misoprostol. Cost per successful induction was R0.55 and R0.44 respectively. Mean induction times were 13.2 hours and 13.4 hours respectively. All patients delivered vaginally but incompletely in 7/21 group A1 and 1/19 group A2 patients. In group A2, the mean duration of labour was 5.97 hours. In group B induction was successful in 51/62 (82%) with 95.1 micrograms of misoprostol; the mean cost was R0,32. Twelve out of 51 (23%) received oxytocin and 44/51 delivered vaginally. Mean duration of induction was 11.4 hours and of labour 5.4 hours. Two babies had low Apgar scores. There were two stillbirths (perinatal mortality rate 39.2/1000), both apparently unrelated to misoprostol. CONCLUSIONS: Misoprostol is an effective, easy to use, apparently safe and cheap drug for the induction of labour.

Influence of nitric oxide on the hemodynamic response to hemorrhage in conscious rabbits.

We investigated the role of nitric oxide, an endothelium-derived relaxing factor, in the hemodynamic response to acute hemorrhage in conscious rabbits. Chronically instrumented rabbits were treated with the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) or vehicle and hemorrhaged until mean arterial pressure fell below 40 mmHg. Control animals were treated with L-NAME or vehicle but not subjected to hemorrhage. L-NAME increased mean arterial pressure and decreased heart rate in control animals. Hindquarters and mesenteric blood flow velocity and conductance were reduced by L-NAME. Nitric oxide synthase inhibition also produced significant changes in the hemodynamic response to hypotensive hemorrhage. Mean arterial pressure was higher and regional vascular conductances were lower throughout hemorrhage and during recovery. L-NAME treatment significantly (but in some cases, subtly) altered the characteristic pattern of changes in vascular conductance associated with acute hypotensive hemorrhage and recovery. Similar experiments with other arginine analogues or phenylephrine infusion showed that L-NAME's effects during hemorrhage were due to nitric oxide synthase inhibition. We conclude that nitric oxide plays a role in the hemodynamic response to acute hemorrhage in the rabbit and is essential for the full expression of the vasodilation associated with hypotensive hemorrhage.