Global patterns in the metacommunity structuring of lake macrophytes: regional variations and driving factors.

We studied community-environment relationships of lake macrophytes at two metacommunity scales using data from 16 regions across the world. More specifically, we examined (a) whether the lake macrophyte communities respond similar to key local environmental factors, major climate variables and lake spatial locations in each of the regions (i.e., within-region approach) and (b) how well can explained variability in the community-environment relationships across multiple lake macrophyte metacommunities be accounted for by elevation range, spatial extent, latitude, longitude, and age of the oldest lake within each metacommunity (i.e., across-region approach). In the within-region approach, we employed partial redundancy analyses together with variation partitioning to investigate the relative importance of local variables, climate variables, and spatial location on lake macrophytes among the study regions. In the across-region approach, we used adjusted R2 values of the variation partitioning to model the community-environment relationships across multiple metacommunities using linear regression and commonality analysis. We found that niche filtering related to local lake-level environmental conditions was the dominant force structuring macrophytes within metacommunities. However, our results also revealed that elevation range associated with climate (increasing temperature amplitude affecting macrophytes) and spatial location (likely due to dispersal limitation) was important for macrophytes based on the findings of the across-metacommunities analysis. These findings suggest that different determinants influence macrophyte metacommunities within different regions, thus showing context dependency. Moreover, our study emphasized that the use of a single metacommunity scale gives incomplete information on the environmental features explaining variation in macrophyte communities.

Racial differences in ischemic complications of pedicled versus free abdominal flaps for breast reconstruction.

PURPOSE: This study aims to determine the relationship between race and ischemic complications in women undergoing breast reconstruction with pedicled TRAM (pTRAM) and perforator flaps (DIEP). PATIENTS AND METHODS: A retrospective, cross-sectional study of women who underwent breast reconstruction utilizing either pTRAM or DIEP flaps from March 1, 2002 to September 1, 2012 was performed. Clinical and demographic variables, including race and ischemic complications (mastectomy flap necrosis, fat necrosis, partial abdominal flap necrosis, vascular compromise requiring reoperation), were examined. Fat necrosis was graded using a previously established scale (grade I = radiologically visible, II = palpable, III = palpable and visible, IV = symptomatic). RESULTS: Over the 10-year study period, adequate follow-up was available for 138 women (94 Caucasian, 36 African American) who underwent pTRAM or DIEP. Fat necrosis occurred more frequently in the pTRAM group (53.5% vs. 17.4%, P < 0.001). There was no statistically significant difference in partial flap necrosis or mastectomy flap necrosis between the 2 groups. The DIEP group had a higher rate of vascular compromise requiring reoperation (13% vs. 0, P = 0.003). In the pTRAM group, there was a higher rate of fat necrosis (77% vs. 45.6%, P < 0.001) and grade IV fat necrosis in African Americans (42.8% vs. 9.5%, P = 0.005). Rates of other ischemic complications were comparable between the 2 racial groups. In the DIEP group, ischemic complications were comparable between the 2 racial groups. After stratifying by flap type and race, we saw no differences in mastectomy flap necrosis (P = 0.0182). DISCUSSION: African Americans undergoing pTRAM flap are at higher risk for grade IV fat necrosis but not mastectomy flap necrosis or partial flap necrosis. This may be due to difficulty using physical examination to judge the vascular status of a pedicle flap that is known to undergo significant changes in vascular physiology following transfer. Intraoperative assessment of perfusion using new technologies may be useful in these higher risk patients.

Learning curves in abdominal wall reconstruction with components separation: one step closer toward improving outcomes and reducing complications.

INTRODUCTION: Learning curves are characterized by incremental improvement of a process, through repetition and reduction in variability, but can be disrupted with the emergence of new techniques and technologies. Abdominal wall reconstruction continues to evolve, with the introduction of components separation in the 1990s and biologic mesh in the 2000s. As such, attempts at innovation may impact the success of reconstructive outcomes and yield a changing set of complications. The purpose of this project was to describe the paradigm shift that has occurred in abdominal wall reconstruction during the past 10 years, focusing on the incorporation of new materials and methods. METHODS: We reviewed 150 consecutive patients who underwent abdominal wall reconstruction of midline defects with components separation, from 2000 to 2010. Both univariate and multivariate logistic regression analyses were performed to identify risk factors for complications. Patients were stratified into the following periods: early (2000-2003), middle (2004-2006), and late (2007-2010). RESULTS: From 2000 to 2010, we performed 150 abdominal wall reconstructions with components separation [mean age, 50.2 years; body mass index (BMI), 30.4; size of defect, 357 cm; length of stay, 9.6 days; follow-up, 4.4 years]. Primary fascial closure was performed in 120 patients. Mesh was used in 114 patients in the following locations: overlay (n = 28), inlay (n = 30), underlay (n = 54), and unknown (n = 2). Complications occurred in a bimodal distribution, highest in 2001 (introduction of biologic mesh) and 2008 (conversion from underlay to overlay location). Age, sex, history of smoking, defect size, and length of stay were not associated with incidence of complications. Unadjusted risk factors for seroma (16.8%) were elevated BMI, of previous hernia repairs, use of overlay mesh, and late portion of the learning curve, with logistic regression supporting only late portion of the learning curve [odds ratio (OR), 4.3; 95% confidence interval (CI), 1.0-18.6] and BMI (OR, 1.17; 95% CI, 1.06-1.29). The only unadjusted risk factor for recurrence was location of mesh. Logistic regression, comparing underlay, inlay, and overlay mesh to no mesh, revealed that the use of underlay mesh predicted recurrence (OR, 3.0; 95% CI, 1.04-8.64). All P values were less than 0.05. CONCLUSIONS: The overall learning curve for a specific procedure, such as abdominal wall reconstruction, can be quite volatile, especially as innovative techniques and new technologies are introduced and incorporated into the surgeon's practice. Our current practice includes primary repair myofascial flap of the components separation and the use of biologic mesh as an overlay graft, anchored to the external oblique. This process of outcome improvement is not gradual but is often punctuated by periods of failure and redemption.

Comparative analysis of the Potter Tower and a new Track Sprayer for the application of residual sprays in the laboratory.

BACKGROUND: Efforts to evaluate the residual efficacy of new indoor residual spraying (IRS) formulations have identified limitations with the industry standard laboratory sprayer, the Potter Spray Tower (PT). Calibrating the PT can be time-consuming, and the dosing of surfaces may not be as accurate or uniform as previously assumed. METHODS: To address these limitations, the Micron Horizontal Track Sprayer with Spray Cabinet (TS) was developed to provide higher efficiency, ease of operation and deposition uniformity equal to or better than the PT. A series of studies were performed using a fluorescent tracer and three IRS formulations (Actellic® 300CS, K-Othrine WG250 and Suspend PolyZone) sprayed onto surfaces using either the PT or the TS. RESULTS: Deposition volumes could be accurately calibrated for both spray systems. However, the uniformity of spray deposits was higher for the TS compared to the PT. Less than 12% of the volume sprayed using the PT reaches the target surface, with the remaining 88% unaccounted for, presumably vented out of the fume hood or coating the internal surfaces of the tower. In contrast, the TS deposits most of the spray on the floor of the spray chamber, with the rest contained therein. The total sprayed surface area in one run of the TS is 1.2 m2, and the operational zone for spray target placement is 0.7 m2, meaning that 58% of the applied volume deposits onto the targets. The TS can treat multiple surfaces (18 standard 15 × 15 cm tiles) in a single application, whereas the PT treats one surface at a time and a maximum area of around 0.0225 m2. An assessment of the time taken to perform spraying, including the setup, calibration and cleaning, showed that the cost of application using the TS was around 25-35 × less per tile sprayed. Standard operating procedures (SOPs) for calibration and use of both the Potter Tower and Track Sprayer have been developed. CONCLUSIONS: Overall, the TS represents a significant improvement over the PT in terms of the efficiency and accuracy of IRS formulation applications onto test substrates and offers a useful additional tool for researchers and manufacturers wanting to screen new active ingredients or evaluate the efficacy of IRS or other sprayable formulations for insect control.

High Population Frequency of GNRHR p.Q106R in Malta: An Evaluation of Fertility and Hormone Profiles in Heterozygotes.

Context: The gonadotropin-releasing hormone receptor variant GNRHR p.Q106R (rs104893836) in homozygosity, compound heterozygosity, or single heterozygosity is often reported as the causative variant in idiopathic hypogonadotropic hypogonadism (IHH) patients with GnRH deficiency. Genotyping of a Maltese newborn cord-blood collection yielded a minor allele frequency (MAF) 10 times higher (MAF = 0.029; n = 493) than that of the global population (MAF = 0.003). Objective: To determine whether GNRHR p.Q106R in heterozygosity influences profiles of endogenous hormones belonging to the hypothalamic-pituitary axis and the onset of puberty and fertility in adult men (n = 739) and women (n = 239). Design Setting and Participants: Analysis of questionnaire data relating to puberty and fertility, genotyping of the GNRHR p.Q106R variant, and hormone profiling of a highly phenotyped Maltese adult cohort from the Maltese Acute Myocardial Infarction Study. Main Outcome and Results: Out of 978 adults, 43 GNRHR p.Q106R heterozygotes (26 men and 17 women) were identified. Hormone levels and fertility for all heterozygotes are within normal parameters except for TSH, which was lower in men 50 years or older. Conclusion: Hormone data and baseline fertility characteristics of GNRHR p.Q106R heterozygotes are comparable to those of homozygous wild-type individuals who have no reproductive problems. The heterozygous genotype alone does not impair the levels of investigated gonadotropins and sex steroid hormones or affect fertility. GNRHR p.Q106R heterozygotes who exhibit IHH characteristics must have at least another variant, probably in a different IHH gene, that drives pathogenicity. We also conclude that GNRHR p.Q106R is likely a founder variant due to its overrepresentation and prevalence in the island population of Malta.

Incidence and management of adverse events after the use of laser therapies for the treatment of hypertrophic burn scars.

INTRODUCTION: Hypertrophic burn scars may generate significant morbidity, due to intense pruritus, persistent dysesthesias, and contracture. Although treatment with pulsed dye laser and fractional CO2 laser may improve symptoms, incidence of secondary wound complications is not well known. We examined the adverse event profile of laser therapies for the treatment of hypertrophic burn scars. METHODS: We performed a descriptive, retrospective, 6-month study of all patients who underwent laser therapies, at an accredited regional burn center, to improve the vascularity, texture, thickness, and stiffness of symptomatic burn scars. Data regarding skin type, mechanism, area treated, and laser parameters were collected. Main outcome measures included pigmentation changes, blistering, rash, infection. χ analysis and Student t test were used to evaluate associations between variables. RESULTS: A total of 95 patients underwent 163 treatment sessions (mean, 2.7 sessions/patient) with pulsed dye laser (71%), CO2 laser (22%), and other lasers (7%). Forty-one adverse events were recorded: hyperpigmentation (2%), hypopigmentation (12%), mild blistering (27%), pain (37%), rash (7%), fever (10%), and infection (2%). Patients with scald burns were more likely to develop blistering, rash, and fever after treatment (all P < 0.05). Higher Fitzpatrick skin type was associated with hypopigmentation and blistering, whereas CO2 laser was associated with increased postoperative pain (all P < 0.05) CONCLUSIONS: Despite the frequent occurrence of pain and mild blistering after laser treatment of hypertrophic burn scars, major adverse effects were exceedingly rare, with improvement noted in all patients. Patients with higher Fitzpatrick skin types must be handled with care, to avoid complications of blistering and hypopigmentation.

The influence of the tertiary bronchi on dynamic lung deformation.

Finite element models of thoracic injury often treat the lung as a bulk homogeneous and isotropic material, which reduces the computational costs associated with such investigations. Ignoring the heterogeneous structure of the lung may be computationally expedient, but this simplification may inadvertently fail to capture the true lung strain dynamics. In the present work, a series of direct impact experiments were performed on porcine lungs, inflated to a relevant expiratory pressure, and monitored using high-speed X-ray imaging. The lungs were instrumented with radiopaque markers within the parenchyma and tertiary bronchi to monitor the resulting deformation mechanics. The deformation mechanics demonstrate a high degree of strain localization related to the structural heterogeneity of the lung. The relative motion of the tertiary bronchi was measured during the impact event, and used to estimate the parenchyma tissue strains in the inter-bronchial regions. These were shown to exceed the trans-lobe strains by a factor 3 to 5 times higher in their tensile, compressive, and shear strain responses. Our results demonstrate that the lung parenchyma and bronchial tissues form a heterogeneous structure with a substantial stiffness differential that cannot be appropriately modelled as a homogeneous and isotropic monolithic mass without loss of accuracy and predictive relevance.

Genetic diversity in three invasive clonal aquatic species in New Zealand.

BACKGROUND: Elodea canadensis, Egeria densa and Lagarosiphon major are dioecious clonal species which are invasive in New Zealand and other regions. Unlike many other invasive species, the genetic variation in New Zealand is very limited. Clonal reproduction is often considered an evolutionary dead end, even though a certain amount of genetic divergence may arise due to somatic mutations. The successful growth and establishment of invasive clonal species may be explained not by adaptability but by pre-existing ecological traits that prove advantageous in the new environment. We studied the genetic diversity and population structure in the North Island of New Zealand using AFLPs and related the findings to the number of introductions and the evolution that has occurred in the introduced area. RESULTS: Low levels of genetic diversity were found in all three species and appeared to be due to highly homogeneous founding gene pools. Elodea canadensis was introduced in 1868, and its populations showed more genetic structure than those of the more recently introduced of E. densa (1946) and L. major (1950). Elodea canadensis and L. major, however, had similar phylogeographic patterns, in spite of the difference in time since introduction. CONCLUSIONS: The presence of a certain level of geographically correlated genetic structure in the absence of sexual reproduction, and in spite of random human dispersal of vegetative propagules, can be reasonably attributed to post-dispersal somatic mutations. Direct evidence of such evolutionary events is, however, still insufficient.

Invasion strategies in clonal aquatic plants: are phenotypic differences caused by phenotypic plasticity or local adaptation?

BACKGROUND AND AIMS: The successful spread of invasive plants in new environments is often linked to multiple introductions and a diverse gene pool that facilitates local adaptation to variable environmental conditions. For clonal plants, however, phenotypic plasticity may be equally important. Here the primary adaptive strategy in three non-native, clonally reproducing macrophytes (Egeria densa, Elodea canadensis and Lagarosiphon major) in New Zealand freshwaters were examined and an attempt was made to link observed differences in plant morphology to local variation in habitat conditions. METHODS: Field populations with a large phenotypic variety were sampled in a range of lakes and streams with different chemical and physical properties. The phenotypic plasticity of the species before and after cultivation was studied in a common garden growth experiment, and the genetic diversity of these same populations was also quantified. KEY RESULTS: For all three species, greater variation in plant characteristics was found before they were grown in standardized conditions. Moreover, field populations displayed remarkably little genetic variation and there was little interaction between habitat conditions and plant morphological characteristics. CONCLUSIONS: The results indicate that at the current stage of spread into New Zealand, the primary adaptive strategy of these three invasive macrophytes is phenotypic plasticity. However, while limited, the possibility that genetic diversity between populations may facilitate ecotypic differentiation in the future cannot be excluded. These results thus indicate that invasive clonal aquatic plants adapt to new introduced areas by phenotypic plasticity. Inorganic carbon, nitrogen and phosphorous were important in controlling plant size of E. canadensis and L. major, but no other relationships between plant characteristics and habitat conditions were apparent. This implies that within-species differences in plant size can be explained by local nutrient conditions. All together this strongly suggests that invasive clonal aquatic plants adapt to a wide range of habitats in introduced areas by phenotypic plasticity rather than local adaptation.

Global patterns and determinants of lake macrophyte taxonomic, functional and phylogenetic beta diversity.

Documenting the patterns of biological diversity on Earth has always been a central challenge in macroecology and biogeography. However, for the diverse group of freshwater plants, such research program is still in its infancy. Here, we examined global variation in taxonomic, functional and phylogenetic beta diversity patterns of lake macrophytes using regional data from six continents. A data set of ca. 480 lake macrophyte community observations, together with climatic, geographical and environmental variables, was compiled across 16 regions worldwide. We (a) built the very first phylogeny comprising most freshwater plant lineages; (b) exploited a wide array of functional traits that are important to macrophyte autoecology or that relate to lake ecosystem functioning; (c) assessed if different large-scale beta diversity patterns show a clear latitudinal gradient from the equator to the poles using null models; and (d) employed evolutionary and regression models to first identify the degree to which the studied functional traits show a phylogenetic signal, and then to estimate community-environment relationships at multiple spatial scales. Our results supported the notion that ecological niches evolved independently of phylogeny in macrophyte lineages worldwide. We also showed that taxonomic and phylogenetic beta diversity followed the typical global trend with higher diversity in the tropics. In addition, we were able to confirm that species, multi-trait and lineage compositions were first and foremost structured by climatic conditions at relatively broad spatial scales. Perhaps more importantly, we showed that large-scale processes along latitudinal and elevational gradients have left a strong footprint in the current diversity patterns and community-environment relationships in lake macrophytes. Overall, our results stress the need for an integrative approach to macroecology, biogeography and conservation biology, combining multiple diversity facets at different spatial scales.

Pelvic fractures in children.

Although rare, pelvic fractures in children have significant morbidity and mortality. No specific guidelines have been developed for the management of these injuries. We reviewed all trauma patients of age 16 years or younger with pelvic fractures treated at our Level I trauma center over the past 12 years. Of 1008 patients with pelvic fractures, 74 were children. Early hemodynamic instability was seen in 14 per cent of cases. Blood transfusions were required in 26 per cent of cases, angiography in 3 per cent of cases, operations for associated injuries in 46 per cent of cases, operative pelvic fracture fixation in 18 per cent of cases, and intensive care unit care in 58 per cent of cases. Mortality was 5 per cent, mostly from hemorrhage and multiple complex injuries. We conclude that pelvic fractures in children are associated with a high frequency of pelvic bleeding and associated injuries that often require operative interventions and intensive care unit care.

The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine.

Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine.

High body mass index and short- and long-term renal allograft survival in adults.

BACKGROUND: The effect of recipient obesity on kidney allograft survival remains enigmatic. The purpose of this study was to evaluate the effect of donor and recipient body mass index on graft survival. METHODS: Retrospective study of 193 consecutive, adult renal transplants, with at least six months follow-up (mean 24+/-14.1 months). Patients were divided into two groups based upon body mass index (BMI), [weight (kg)/height (m)]: normal (<30.0, n=137) and obese (> or =30.0, n=56). Endpoints were graft loss, defined as either total loss of graft function (return to dialysis) or patient death with a functional graft. Unadjusted and adjusted multivariate analysis techniques, including Kaplan-Meier and Cox proportional hazards regression were used. RESULTS.: Individuals with a BMI > or =30 were not more likely to experience graft loss (O.R. 0.93, 95% C.I. 0.50, 1.72). Rates of acute rejection were not increased in obese recipients. While mortality was not increased in the BMI > 30 group, morbidity, especially surgical, had an increased incidence. The ratio of recipient to donor BMI did not influence graft survival. CONCLUSION: Obese recipients (BMI > or =30.0) were not at increased risk for graft failure. Additionally, matching donor and recipient BMI's would not appear to substantially improve transplant outcome. Obese recipients do have increased posttransplant morbidity and risk all the known health consequences associated with obesity. Careful evaluation and clinical management of obese patients allows for successful kidney transplantation with results equivalent to normal BMI patients.

Association of UGT1A1 polymorphism with prevalence and age at onset of cholelithiasis in sickle cell anemia.

BACKGROUND AND OBJECTIVES: High levels of erythrocyte destruction in sickle cell anemia (SCA) result in chronic hyperbilirubinemia, with cholelithiasis occurring in a subset of patients. We investigated whether susceptibility to cholelithiasis in SCA was associated with the promoter polymorphism of the 5?-diphosphate-glucuronosyltransferase 1A1 (UGT1A1) gene encoding a key enzyme in bilirubin catabolism. DESIGN AND METHODS: We determined the frequencies of UGT1A1 promoter alleles in 171 SCA children and 153 SCA adults regularly followed for a number of years at the Guadeloupe sickle cell center. These patients had undergone liver/biliary tree ultrasound scans every year. We analyzed the relationships between the various UGT1A1 promoter alleles and hemoglobin levels, steady-state total and unconjugated bilirubin concentrations and the frequency of cholelithiasis. RESULTS: In both children and adults, (TA)6 was less frequent and (TA)7 more frequent in patients with cholelithiasis than in those without this condition. Total and unconjugated bilirubin levels and the frequency of cholelithiasis were significantly higher in patients with (TA)7/(TA)7 and (TA)7/(TA)8 genotypes than in those with other genotypes. Those homozygous for (TA)6 or carrying at least one (TA)5 allele had the lowest total and unconjugated bilirubin levels and were least likely to have cholelithiasis. Patients with (TA)6/(TA)7 and (TA)6/(TA)8 genotypes presented intermediate values. Kaplan-Meier analysis of cholelithiasis-free survival in children demonstrated an early age-at-onset for cholelithiasis in patients with (TA)7/(TA)7 and (TA)7/(TA)8 genotypes. INTERPRETATIONS AND CONCLUSIONS: This study shows that the UGT1A1 gene promoter polymorphism is a major genetic risk factor modifying the frequency and age-at-onset of cholelithiasis in SCA patients.

Clinical outcomes and disease progression among patients coinfected with HIV and human T lymphotropic virus types 1 and 2.

The goal of this study was to investigate clinical outcomes and survival probabilities among persons coinfected with human immunodeficiency virus (HIV) and human T lymphotropic viruses types 1 and 2 (HTLV-I/II). A nonconcurrent cohort study of 1033 HIV-infected individuals was also conducted. Sixty-two patients were coinfected with HTLV-I, and 141 patients were coinfected with HTLV-II. HTLV-I/II coinfection was highly associated with African-American race/ethnicity, age of >36 years, higher CD4(+) T cell count at baseline and over time, and history of injection drug use. Coinfected patients were more likely to have neurologic complications, thrombocytopenia, respiratory and urinary tract infections, and hepatitis C. Despite having higher CD4(+) T cell counts over time, there was no difference in the incidence of opportunistic infections. Progression to both acquired immunodeficiency syndrome (AIDS; adjusted hazard ratio [aHR], 0.50; 95% confidence interval [CI], 0.25-0.98) and death (aHR, 0.57, 95% CI, 0.37-0.89) were slower among HTLV-II-coinfected patients, compared with time-entry- and CD4(+) T cell count-matched control subjects. In conclusion, HIV-HTLV-I/II coinfection may result in improved survival and delayed progression to AIDS, but this happens at the expense of an increased frequency of other of clinical complications.

Enhancement of seed phytosterol levels by expression of an N-terminal truncated Hevea brasiliensis (rubber tree) 3-hydroxy-3-methylglutaryl-CoA reductase.

Dietary intake of phytosterols (plant sterols) has been shown to be effective in reducing blood cholesterol levels, thereby reducing the risk of cardiovascular disease. Phytosterols are most commonly sourced from vegetable oils, where they are present as minor components. We report here the generation of transgenic tobacco seeds substantially enhanced in phytosterol content by the expression of a modified form of one of the key sterol biosynthetic enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). The constitutive expression of an N-terminal truncated Hevea brasiliensis HMGR (t-HMGR), lacking the membrane binding domain, enhanced seed HMGR activities by 11-fold, leading to increases in total seed sterol of 2.4-fold. Seed-specific expression of t-HMGR enhanced total seed sterol levels by 3.2-fold, to 1.36% dry weight or 3.25% of oil. 4-desmethylsterols were increased by 2.2-fold, whilst certain sterol biosynthetic intermediates, in particular cycloartenol and 24-ethylidene lophenol, also accumulated. The additional sterol in seed tissue was present in the form of fatty acid esters. Constitutive expression of t-HMGR increased leaf phytosterol sterol levels by 10-fold, representing 1.8% dry weight, and the sterol was sequestered, in acyl ester form, as cytoplasmic 'oil droplets'. These studies establish HMGR as a key enzyme controlling overall flux into the sterol biosynthesis pathway in seed tissue, but the accumulation of certain intermediates suggests additional slow steps in the pathway. The expression of an N-truncated HMGR activity has generated novel phytosterol-enriched raw materials that may provide the basis of new sourcing opportunities for this important class of cholesterol-lowering actives.

Sensitization of apoptotically-resistant breast carcinoma cells to TNF and TRAIL by inhibition of p38 mitogen-activated protein kinase signaling.

The mitogen-activated protein kinase (MAPK) cascade is a critical component in the regulation of cell survival and proliferation decisions. In breast carcinoma cells, activation of the p38-MAPK member of this family occurs in response to pro-inflammatory cytokines and cellular stress. The involvement of p38-MAPK in the activation of the transcription factor, NF-kappaB, suggests a potential role and mechanism for regulation of cell survival and drug resistance. Generation of the resistant MCF-7 variant (MCF-7TN-R) was achieved by prolonged exposure of MCF-7N cells to increasing concentrations of TNF. Differences in MAPK activation and function in the MCF-7 cell variants were determined. The role of the p38-MAPK pathway in regulation of resistance was determined using pharmacological (SB 203580) or molecular [Dominant Inhibitory (DI)-p38] inhibition. The effect of p38 inhibition on NF-kappaB transcriptional activation was analyzed. As compared to the sensitive MCF-7N parent cell line, the MCF-7TN-R cell line displayed significant resistance to TNF- and TRAIL-induced cell death. Analysis of the expression and phosphorylation of members of the MAPK family revealed an increased basal activation of p38 in the MCF-7TN-R variant. The p38-mediated phosphorylation and transcriptional activity were suppressed by pharmacologic inhibition with SB 230580. Treatment of MCF-7TN-R cells with SB partially restored sensitivity to TNF-induced cell death. In addition, use of a DI-p38 construct with or without the addition to TNF induced cell death, thus restoring TNF-sensitivity to these cells. The ability of p38 inhibition to restore apoptotic sensitivity was correlated with suppression of the TNF-induced cell survival pathway, NF-kappaB. The increased activation of p38-MAPK in MCF-7TN-R cells demonstrates that this signaling pathway through activation of NF-kappaB is an important route for control of resistance to cell death in breast carcinoma. Molecular and pharmacological inhibition of p38-MAPK signaling may represent a mechanism for sensitizing cancer cells to chemotherapeutic regimens and restoration of apoptotic signaling.

The placental-umbilical unit in sickle cell disease pregnancy: a model for studying in vivo functional adjustments to hypoxia in humans.

The placental-umbilical unit in sickle cell disease (SCD) pregnancy was used to explore hypoxia in vivo, an important factor in the pathophysiology of this disease. Gross examination and microscopic analysis of the placentas, taken immediately after delivery, indicate good concordance between maturity and term as controls, but higher frequency of vascular injuries such as excess syncytial knots, excess fibrin deposits, congestion and villous necroses. Unexpectedly, neither leukocyte recruitment nor alteration in extraplacental membrane was observed, suggesting the absence of inflammation. Additionally, interleukin (IL)-6 and IL-8 concentrations, measured by enzyme-linked immunosorbent assay (ELISA), were similar in the placental maternal blood from controls and SCD. There were also no significant differences found in IL-6 vein blood concentrations between controls and SCD, IL-8 being not detected. Immunostaining of umbilical vein endothelium in SCD pregnancies showed redistribution of PECAM-1 (CD31), von Willebrand factor (vWF), and P-selectin to the cell surface, controls exhibiting the classical pattern. Staining quantification indicated increases in vWF (+36.2%; P=.006) and vascular endothelial growth factor (VEGF) expression (+96.0%; P=.006) over control, but a reduction in endothelial nitric oxide synthase (eNOS) (-45.5%; P=.029). These results document, for the first time, direct functional adjustments in response to hypoxia in human in vivo. The mechanism for these changes has not been clearly established, but it may reflect increased tolerance to SCD hypoxic conditions and hypoxia in general.

Regulation of estrogen-mediated cell survival and proliferation by p160 coactivators.

BACKGROUND: Estrogen receptor (ER) activity is dependent on coactivator (CoA) proteins. The role of CoA-ER interactions in breast cancer apoptosis remains unexplored. METHODS: Expression vectors for the p160 CoA genes NCOA-1, NCOA-2, or NCOA-3 were transiently transfected into MCF-7 cells. Cell survival was determined by viability and clonogenic survival assays. Effects of CoA expression on estrogen (E2) signaling were determined by estrogen response element (ERE)-luciferase reporter-gene assay. Clonogenic and reporter-gene survival assays were used to examine the molecular inhibition of CoA function (dominant inhibitory [DI]-decoy-CoA) on cell survival. Statistical significance was established at the P < .05 level. RESULTS: Overexpression of NCOA-1, NCOA-2, and NCOA-3 enhanced E2-mediated gene expression by 3.17 +/- 0.51-, 2.33 +/- 0.8-, and 3.65 +/- 0.65-fold, respectively, and enhanced cell survival by suppressing tumor necrosis factor alpha (TNF-alpha)-induced cell death from 80.23% +/- 2.66% viability to 101.5% +/- 8.9%, 86.9% +/- 9.9%, and 95.7% +/- 8.5% viability, respectively. NCOA-1 enhancement of cell survival occurred via suppression of TNF-alpha-induced apoptosis as confirmed by viability and morphologic evaluation. Clonogenic survival and E2-stimulated colony formation in MCF-7 cells were suppressed by expression of DI-decoy-NCOA-1 and DI-decoy-NCOA-3 to 34.4% +/- 7.4% and 54% +/- 5.4% of vector control, but not DI-decoy-NCOA-2. CONCLUSIONS: Overexpression of NCOA-1 and NCOA-3 exerted potent survival effects in breast carcinoma cells. Use of DI-CoA constructs enhanced TNF-alpha-induced cell death and abrogated E2-induced survival. Inhibition of CoA proteins represents a mechanism for enhancing sensitivity therapies in breast carcinoma.