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Understanding, predicting, and preventing pregnancy disorders have been a major research target. Nonetheless, the lack of progress is illustrated by research results related to preeclampsia and other hypertensive pregnancy disorders. These remain a major cause of maternal and infant mortality worldwide. There is a general consensus that the rate of progress toward understanding pregnancy disorders lags behind progress in other aspects of human health. In this presentation, we advance an explanation for this failure and suggest solutions. We propose that progress has been impeded by narrowly focused research training and limited imagination and innovation, resulting in the failure to think beyond conventional research approaches and analytical strategies. Investigations have been largely limited to hypothesis-generating approaches constrained by attempts to force poorly defined complex disorders into a single “unifying” hypothesis. Future progress could be accelerated by rethinking this approach. We advise taking advantage of innovative approaches that will generate new research strategies for investigating pregnancy abnormalities. Studies should begin before conception, assessing pregnancy longitudinally, before, during, and after pregnancy. Pregnancy disorders should be defined by pathophysiology rather than phenotype, and state of the art agnostic assessment of data should be adopted to generate new ideas. Taking advantage of new approaches mandates emphasizing innovation, inclusion of large datasets, and use of state of the art experimental and analytical techniques. A revolution in understanding pregnancy-associated disorders will depend on networks of scientists who are driven by an intense biological curiosity, a team spirit, and the tools to make new discoveries.

Volcanic eruption source parameters may be estimated from acoustic pressure recordings dominant at infrasonic frequencies ($$\le$$ ≤ 10−3 dB/m spectral energy transfer in the band 3–9 Hz for signals with amplitude on the order of several hundred Pa at 200–400 m range. The clarity of the nonlinear spectral signature increases with waveform amplitude, suggesting stronger nonlinear changes for greater source pressures. We observe similar results in application to synthetics generated through finite-difference wavefield simulations of nonlinear propagation, although limitations of the model complicate direct comparison to the observations. Our results provide quantitative evidence for nonlinear propagation that confirms previous interpretations made on the basis of qualitative observations of asymmetric waveforms.

The fetus largely depends on maternal supply and placental transport for its source of long-chain polyunsaturated fatty acids (LCPUFAs), which are essential for proper neurological and cardiovascular development. Pregnancy complications such as diabetes reduces neonatal LCPUFA supply, but little is known of how fatty acid delivery is affected by maternal body type or weight gain in uncomplicated pregnancies. In a cross-sectional study of maternal-neonatal pairs at term, we sought to determine the effect of gestational weight gain on neonatal LCPUFA supply. Forty maternal-neonatal pairs of uncomplicated (no gestational hypertension or diabetes) term pregnancies were recruited upon admission to Oregon Health & Science University Labor & Delivery for scheduled cesarean section. Maternal and umbilical cord plasma fatty acid profiles were measured using gas chromatography-mass spectrophotometry. First trimester weight gain was negatively correlated with maternal n-3 LCPUFA ( r = -0.80, P = .0002), and this was not affected by fetal sex. High maternal weight gain in the first trimester was negatively associated with cord n-3 polyunsaturated fatty acid levels ( r = -0.70, P = .03) and placental thickness ( r = -0.69, P = .03) in male, but not female, offspring. High maternal weight gain in the first trimester is associated with a thinner placenta and low levels of n-3 LCPUFA in male offspring. Further study is required to confirm that male offspring are at a higher risk of poor outcomes associated with high maternal weight gain early in pregnancy.

Objectives: Occupational and residential noise exposure has been related to increased risk of cardiovascular disease. Alteration of serum lipid levels has been proposed as a possible causal pathway. The objective of this study was to investigate the relation between ambient and at-the-ear occupational noise exposure and serum levels of total cholesterol, low-density lipoprotein–cholesterol, high-density lipoprotein–cholesterol, and triglycerides when accounting for well-established predictors of lipid levels. Methods: This cross-sectional study included 424 industrial workers and 84 financial workers to obtain contrast in noise exposure levels. They provided a serum sample and wore portable dosimeters that every 5-s recorded ambient noise exposure levels during a 24-h period. We extracted measurements obtained during work and calculated the full-shift mean ambient noise level. For 331 workers who kept a diary on the use of a hearing protection device (HPD), we subtracted 10 dB from every noise recording obtained during HPD use and estimated the mean full-shift noise exposure level at the ear. Results: Mean ambient noise level was 79.9 dB (A) [range 55.0–98.9] and the mean estimated level at the ear 77.8 dB (A) [range 55.0–94.2]. Ambient and at-the-ear noise levels were strongly associated with increasing levels of triglycerides, cholesterol–HDL ratio, and decreasing levels of HDL–cholesterol, but only in unadjusted analyses that did not account for HPD use and other risk factors. Conclusion: No associations between ambient or at-the-ear occupational noise exposure and serum lipid levels were observed. This indicates that a causal pathway between occupational and residential noise exposure and cardiovascular disease does not include alteration of lipid levels.

The syncytiotrophoblast (SCT) at the maternal-fetal interface has been presumed to be the primary driver of placental metabolism, and the underlying progenitor cytotrophoblast cells (CTB) an insignificant contributor to placental metabolic activity. However, we now show that the metabolic rate of CTB is much greater than the SCT. The oxygen consumption and extracellular acidification rate, a measure of glycolysis, are both greater in CTB than in SCT in vitro (CTB: 96 ± 16 vs SCT: 46 ± 14 pmol O2 × min−1 × 100 ng DNA−1, p −1 × 100 ng DNA−1, p 2TMRosa, is higher in CTB than in SCT in culture and living explants. These data cast doubt on the previous supposition that the metabolic rate of the placenta is dominated by the SCT contribution. Moreover, differentiation into SCT leads to metabolic suppression. The normal suppression of metabolic activity during CTB differentiation to SCT is prevented with a p38 MAPK signaling inhibitor and epidermal growth factor co-treatment. We conclude that the undifferentiated CTB, in contrast to the SCT, is highly metabolically active, has a high level of fuel flexibility, and contributes substantially to global metabolism in the late gestation human placenta.

The aberrant activity of Ras homologous (Rho) family small GTPases (20 human members) has been implicated in cancer and other human diseases. However, in contrast to the direct mutational activation of Ras found in cancer and developmental disorders, Rho GTPases are activated most commonly in disease by indirect mechanisms. One prevalent mechanism involves aberrant Rho activation via the deregulated expression and/or activity of Rho family guanine nucleotide exchange factors (RhoGEFs). RhoGEFs promote formation of the active GTP-bound state of Rho GTPases. The largest family of RhoGEFs is comprised of the Dbl family RhoGEFs with 70 human members. The multitude of RhoGEFs that activate a single Rho GTPase reflects the very specific role of each RhoGEF in controlling distinct signaling mechanisms involved in Rho activation. In this review, we summarize the role of Dbl RhoGEFs in development and disease, with a focus on Ect2 (epithelial cell transforming squence 2), Tiam1 (T-cell lymphoma invasion and metastasis 1), Vav and P-Rex1/2 (PtdIns(3,4,5)P3 (phosphatidylinositol (3,4,5)-triphosphate)-dependent Rac exchanger).

As the main microtubule-organizing centre in animal cells, the centrosome has a fundamental role in cell function. Surrounding the centrioles, the pericentriolar material (PCM) provides a dynamic platform for nucleating microtubules. Although the importance of the PCM is established, its amorphous electron-dense nature has made it refractory to structural investigation. By using SIM and STORM subdiffraction-resolution microscopies to visualize proteins critical for centrosome maturation, we demonstrate that the PCM is organized into two main structural domains: a layer juxtaposed to the centriole wall, and proteins extending farther away from the centriole organized in a matrix. Analysis of Pericentrin-like protein (PLP) reveals that its carboxy terminus is positioned at the centriole wall, it radiates outwards into the matrix and is organized in clusters having quasi-nine-fold symmetry. By RNA-mediated interference (RNAi), we show that PLP fibrils are required for interphase recruitment and proper mitotic assembly of the PCMmatrix.

Circulating fetal 3,3',5-tri-iodo-l-thyronine (T(3) ) is maintained at very low levels until a dramatic prepartum surge. 3,3',5-Tri-iodo-l-thyronine inhibits serum-stimulated proliferation in near-term ovine cardiomyocytes, but it is not known whether midgestation myocytes are also inhibited. Because early cessation of cardiomyocyte mitosis would result in an underendowed heart, we hypothesized that 0.67 gestation (100 of 145 days gestation) ovine cardiomyocytes would be insensitive to suppressive growth effects of T(3) . These younger cardiomyocytes were grown with T(3) in 10% serum-enriched media for 24 hours. Physiological (0.37, 0.75, and 1.5 nmol/L) concentrations of T(3) dramatically suppressed mitotic activity in cardiomyocytes (P.001). 3,3',5-Tri-iodo-l-thyronine stimulated phosphorylation of extracellular signal-regulated kinase and AKT (also known as Protein Kinase B [PKB]) signaling pathways. Nevertheless, the protein content of the cell cycle suppressor, p21, increased 2-fold (P.05), and promoter, cyclin D1, decreased by 50%. Contrary to our hypothesis, elevated levels of T(3) powerfully inhibit proliferation of midgestation fetal cardiomyocytes. Thus, midgestation maternal hyperthyroidism might lead to an underendowed fetal myocardium.

OBJECTIVE: The link between maternal under-nutrition and cardiovascular disease (CVD) in the offspring later in life is well recognized, but the impact of maternal over-nutrition on the offspring's cardiovascular function and subsequent risk for CVD later in life remains unclear. Here, we investigated the impact of maternal exposure to a high-fat/calorie diet (HFD) during pregnancy and early postnatal period on endothelial function of the offspring in a nonhuman primate model. METHODS: Offspring, naturally born to either a control (CTR) diet (14% fat calories) or a HFD (36% fat calories) consumption dam, were breast-fed until weaning at about 8 months of age. After weaning, the offspring were either maintained on the same diet (CTR/CTR, HFD/HFD), or underwent a diet switch (CTR/HFD, HFD/CTR). Blood samples and arterial tissues were collected at necropsy when the animals were about 13 months of age. RESULTS: HFD/HFD juveniles displayed an increased plasma insulin level and glucose-stimulated insulin secretion in comparison with CTR/CTR. In abdominal aorta, but not the renal artery, acetylcholine-induced vasorelaxation was decreased remarkably for HFD/HFD juveniles compared with CTR/CTR. HFD/HFD animals also showed a thicker intima wall and an abnormal vascular-morphology, concurrent with elevated expression levels of several markers related to vascular inflammation and fibrinolytic function. Diet-switching animals (HFD/CTR and CTR/HFD) displayed modest damage on the abdominal vessel. CONCLUSION: Our data indicate that maternal HFD exposure impairs offspring's endothelial function. Both early programming events and postweaning diet contribute to the abnormalities that could be reversed partially by diet intervention.

Canola, which is genetically modified (GM) for tolerance to glyphosate, has the potential to become established as a new glyphosate resistant weed, thus reducing the effectiveness of glyphosate. Volunteer from dormant canola seeds produced thousands of plants per hectare in the fourth year (2011) following a 2007 crop harvest. This occurred with no additional canola seed production since the 2007 harvest. Volunteer plants following harvests of annual crops are typically only a problem for the first year after harvest. In California, glyphosate is the core herbicide on over a million hectares of high value row, tree, and vine crops and new glyphosate resistant weeds reduce the effectiveness of glyphosate. The combination of dormant seed and herbicide resistance makes GM glyphosate-resistant canola a new and difficult California weed which was first observed in the winter of 2009.

CCL23 [Ckβ8-1/myeloid progenitor inhibitory factor 1 (MPIF1)/macrophage inflammatory protein-3 (MIP3)], a member of the CC chemokine family, is involved in leukocyte trafficking, and implicated in inflammatory diseases. In the present study, we investigated the role of CCL23 in the development of human atherosclerosis, which is characterized by an inflammatory disease.CCL23 transcripts were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and CCL23 protein by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Expression of adhesion molecules was determined by flow cytometry, and matrix metalloproteinase-2 (MMP-2) levels by zymography.Proatherogenic factors such as oxidized low-density lipoprotein (oxLDL) and oxidative stress markedly enhanced CCL23 release from human THP-1 macrophages. CCL23 stimulated chemotaxis of human THP-1 monocytes in a dose-dependent manner and enhanced the expression of adhesion molecule CD11c, as well as release of MMP-2 from the THP-1 monocytes. Moreover, CCL23 expression at the mRNA level was significantly higher in human atherosclerotic lesions than in normal arteries, and CCL23 protein was co-expressed with CD68, a specific marker for macrophages. Circulating levels of plasma CCL23 were higher in atherosclerotic patients than in normal subjects.These findings suggest that CCL23 plays a role in the development of human atherosclerosis. CCL23 may be a useful target for the development of antiatherogenic agents.

An endogenous probe for CYP3A activity would be useful for early identification of in vivo cytochrome P450 (CYP) 3A4 inhibitors. The aim of this study was to determine whether formation clearance (CL(f)) of the sum of 6β-hydroxycortisol and 6β-hydroxycortisone is a useful probe of CYP3A4 inhibition in vivo. In human liver microsomes (HLMs), the formation of 6β-hydroxycortisol and 6β-hydroxycortisone was catalyzed by CYP3A4, and itraconazole inhibited these reactions with half maximal inhibitory concentration (IC(50))(,u) values of 3.1 nmol/l and 3.4 nmol/l, respectively. The in vivo IC(50,u) value of itraconazole for the combined CL(f) of 6β-hydroxycortisone and 6β-hydroxycortisol was 1.6 nmol/l. The greater inhibitory potency in vivo is probably due to circulating inhibitory itraconazole metabolites. The maximum in vivo inhibition was 59%, suggesting that f(m,CYP3A4) for cortisol and cortisone 6β-hydroxylation is ~60%. Given the significant decrease in CL(f) of 6β-hydroxycortisone and 6β-hydroxycortisol after 200-mg and 400-mg single doses of itraconazole, this endogenous probe can be used to detect moderate and potent CYP3A4 inhibition in vivo.

The photosynthetic bacterium, Rhodospirillum centenum, has a flexible life cycle that permits it to survive starvation as dormant cyst cells. Previous studies have identified some of the key regulators for encystment and demonstrated that the control of development is intricate. This complexity may arise from the need to integrate several environmental signals to mediate a switch from one mode of energy metabolism to another and to ensure that a transition to dormancy is initiated only when necessary. We searched for additional regulators of development by screening for encystment deficient strains after subjecting wild type R. centenum to mini-Tn5 mutagenesis. Analysis of "hypo-cyst" strains led to the identification of two genes that encode putative hybrid histidine kinases (cyd1 and cyd2). Cells with deletions of either gene fail to form cysts under conditions that normally induce development. Furthermore, the deletion strains exhibit altered swarming behavior suggesting that Cyd1 and Cyd2 affect behaviors utilized when the organism is attached to a substrate.

Heart morphogenesis and growth are influenced by hemodynamic forces (wall shear stress and blood pressure) acting on the walls of the heart. Mechanisms by which hemodynamic forces affect heart development are not well understood, in part because of difficulties involved in measuring these forces in vivo. In this paper, we show how wall shear stress in the heart outflow tract (OFT) of chick embryos at an early developmental stage (HH18) are affected by changes in the geometry and motion of the OFT wall. In particular, we were interested in the effects of cardiac cushions, which are protrusions of the OFT wall toward the lumen and that are located where valves will later form. We developed idealized finite element models (FEM) of the chick OFT with and without cardiac cushions. Geometrical parameters used in these models were estimated from in vivo images obtained using optical coherence tomography (OCT) techniques. The FEMs showed significant reverse blood flow (backflow) in the OFT, consistent with experimental observations in the chick heart at HH18, and revealed that cardiac cushions decrease backflow. In addition, our FEMs showed that the spatial distribution of wall shear stress is affected by cardiac cushions, with larger absolute peak values observed at the cushions. Differences in mechanical stimuli (wall shear stress) that the cells in the cardiac cushions and elsewhere are subjected to may affect valve formation and heart development.

Androgen-withdrawal-induced apoptosis (AWIA) is deregulated in androgen refractory prostate cancer. Androgens have been shown to positively regulate expression of the antiapoptotic FADD-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP), and reduced FLIP expression precedes apoptosis after androgen withdrawal. Here, we show that FLIP protein expression is downregulated in castrated rats, while in LNCaP cells, androgens regulate FLIP in a manner that is dependent on phosphoinositol-3-kinase (PI3K) and Akt signaling. Specifically, treatment of LNCaP cells with LY294002, or expression of either PTEN or a non-phosphorylatable form of FOXO3a (FOXO3aTM), downregulates FLIP protein and mRNA. Conversely, treatment with androgens in the absence of PI3/Akt signaling, or following expression of FOXO3aTM, leads to increased FLIP expression. A FOXO3a binding site was identified in the FLIP promoter and shown necessary for the combined effects of androgens and FOXO3a on FLIP transcription. FOXO3a binds the androgen receptor, suggesting that the transcriptional synergy depends on an interaction between these proteins. Finally, LNCaP cells are sensitized to TRAIL-induced apoptosis by PTEN or LY294002, and rescued by androgens. FOXO3aTM also sensitizes cells to androgen-inhibited TRAIL apoptosis. Androgen rescue was diminished when either FOXO3a or FLIP was reduced by siRNA. These data support a role for FOXO3a in AWIA.

Background Genetically engineered mouse models are essential to the investigation of the molecular mechanisms underlying human prostate pathology and the effects of therapy on the diseased prostate. Serial in vivo volumetric imaging expands the scope and accuracy of experimental investigations of models of normal prostate physiology, benign prostatic hyperplasia and prostate cancer, which are otherwise limited by the anatomy of the mouse prostate. Moreover, accurate imaging of hyperplastic and tumorigenic prostates is now recognized as essential to rigorous pre-clinical trials of new therapies. Bioluminescent imaging has been widely used to determine prostate tumor size, but is semi-quantitative at best. Magnetic resonance imaging can determine prostate volume very accurately, but is expensive and has low throughput. We therefore sought to develop and implement a high throughput, low cost, and accurate serial imaging protocol for the mouse prostate. Methods We developed a high frequency ultrasound imaging technique employing 3D reconstruction that allows rapid and precise assessment of mouse prostate volume. Wild-type mouse prostates were examined (n = 4) for reproducible baseline imaging, and treatment effects on volume were compared, and blinded data analyzed for intra- and inter-operator assessments of reproducibility by correlation and for Bland-Altman analysis. Examples of benign prostatic hyperplasia mouse model prostate (n = 2) and mouse prostate implantation of orthotopic human prostate cancer tumor and its growth (n = 6) are also demonstrated. Results Serial measurement volume of the mouse prostate revealed that high frequency ultrasound was very precise. Following endocrine manipulation, regression and regrowth of the prostate could be monitored with very low intra- and interobserver variability. This technique was also valuable to monitor the development of prostate growth in a model of benign prostatic hyperplasia. Additionally, we demonstrate accurate ultrasound image-guided implantation of orthotopic tumor xenografts and monitoring of subsequent tumor growth from ~10 to ~750 mm3 volume. Discussion High frequency ultrasound imaging allows precise determination of normal, neoplastic and hyperplastic mouse prostate. Low cost and small image size allows incorporation of this imaging modality inside clean animal facilities, and thereby imaging of immunocompromised models. 3D reconstruction for volume determination is easily mastered, and both small and large relative changes in volume are accurately visualized. Ultrasound imaging does not rely on penetration of exogenous imaging agents, and so may therefore better measure poorly vascularized or necrotic diseased tissue, relative to bioluminescent imaging (IVIS). Conclusions Our method is precise and reproducible with very low inter- and intra-observer variability. Because it is non-invasive, mouse models of prostatic disease states can be imaged serially, reducing inter-animal variability, and enhancing the power to detect small volume changes following therapeutic intervention.

Background We examined whether self-reported employee health status data can improve the performance of administrative data-based models for predicting future high health costs, and develop a predictive model for predicting new high cost individuals. Methods This retrospective cohort study used data from 8,917 Safeway employees self-insured by Safeway during 2008 and 2009. We created models using step-wise multivariable logistic regression starting with health services use data, then socio-demographic data, and finally adding the self-reported health status data to the model. Results Adding self-reported health data to the baseline model that included only administrative data (health services use and demographic variables; c-statistic = 0.63) increased the model” predictive power (c-statistic = 0.70). Risk factors associated with being a new high cost individual in 2009 were: 1) had one or more ED visits in 2008 (adjusted OR: 1.87, 95 % CI: 1.52, 2.30), 2) had one or more hospitalizations in 2008 (adjusted OR: 1.95, 95 % CI: 1.38, 2.77), 3) being female (adjusted OR: 1.34, 95 % CI: 1.16, 1.55), 4) increasing age (compared with age 18-35, adjusted OR for 36-49 years: 1.28; 95 % CI: 1.03, 1.60; adjusted OR for 50-64 years: 1.92, 95 % CI: 1.55, 2.39; adjusted OR for 65+ years: 3.75, 95 % CI: 2.67, 2.23), 5) the presence of self-reported depression (adjusted OR: 1.53, 95 % CI: 1.29, 1.81), 6) chronic pain (adjusted OR: 2.22, 95 % CI: 1.81, 2.72), 7) diabetes (adjusted OR: 1.73, 95 % CI: 1.35, 2.23), 8) high blood pressure (adjusted OR: 1.42, 95 % CI: 1.21, 1.67), and 9) above average BMI (adjusted OR: 1.20, 95 % CI: 1.04, 1.38). Discussion The comparison of the models between the full sample and the sample without theprevious high cost members indicated significant differences in the predictors. This has importantimplications for models using only the health service use (administrative data) given that the past high costis significantly correlated with future high cost and often drive the predictive models. Conclusions Self-reported health data improved the ability of our model to identify individuals at risk for being high cost beyond what was possible with administrative data alone.

Guanine nucleotide-exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to diverse extracellular stimuli, and ultimately regulate numerous cellular responses such as proliferation, differentiation and movement. With 69 distinct homologues, Dbl-related GEFs represent the largest family of direct activators of Rho GTPases in humans, and they activate Rho GTPases within particular spatio-temporal contexts. The failure to do so can have significant consequences and is reflected in the aberrant function of Dbl-family GEFs in some human diseases.

Over the years, many authors have tried to describe, conceptualize, and visualize the instructional design or development processes via a variety of process models. Most descriptions imply a rather homogeneous view of design, depicting it as an overall problem-solving process following general phases such as analysis, design and development, implementation, and evaluation (ADDIE). However, researchers who have investigated how instructional designers actually work suggest that the process is much more heterogeneous and diverse than these ADDIE models suggest. This study collected case study data from 24 instructional designers in six different settings; they were identified as experts by their peers. The design processes they used for a specific project were compared to four different paradigms created from the literature. The four paradigms and their underlying theoretical foundations are described and illustrated. Detailed results are reported, and reasons that designers did or did not use a particular paradigm are considered.

Computational fluid dynamic (CFD) experimentation provides a unique medium for detailed examination of flow through complex embryonic heart structures. The purpose of this investigation was to demonstrate that streaming blood flow patterns exist in the early embryonic heart and that fluid surface stresses change significantly with anomalous alterations in fetal heart lumen shape. Stages 10 and 11 early human embryo hearts were digitized as calibrated two-dimensional (2D) cross-sectional sequential images. A 3D surface was constructed from the stacking of these 2D images. CFD flow solutions were obtained (steady and pulsatile flow). Particle traces were placed in the inlet and outlet portions of these two stages. Sections of the embryonic heart were artificially reshaped. CFD flow solutions were obtained and surface stress changes analyzed. Streaming was shown to exist, with particles released on one or the other side of the cardiac lumen tending not to cross over and mix with particles released from the opposite side of the cardiac lumen. Shear stress changes (stage 10) occur in the altered lumens. Streaming exists in steady and pulsatile flow scenarios in the embryonic heart models. There are differences in local shear stress distributions with surface shape anomalies of the fetal heart lumen. These observations may help shed light on the potential role of fluid dynamic factors in determining patterns of abnormal heart development.

This article critiques the instructional design tools described in the special issue of Educational Technology Research and Development, Volume 50 Number 4, “Computer-Based Tools for Instructional Design.” Although focusing on four tools, (a) digital management systems, (b) CASCADE, (c) knowledge management systems, and (d) ADAPTIT, the author also comments on the introductory article (van Merrienboer and Martens), and compares the four described tools with earlier models.

Objective Several reports indicate that fluvoxamine decreases the clearance of cytochrome P4501A2 (CYP1A2) substrates. This study compared in vitro and in vivo inhibition potencies of fluvoxamine toward CYP1A2 with an approach based on inhibition constants (Ki) determined in vitro and in vivo. Methods In vitro inhibition constant values were determined with human liver microsomes and complementary deoxyribonucleic acid-expressed CYP1A2 (supersomes). Fluvoxamine in vivo inhibition constants (Kiiv) for CYP1A2 were obtained from an investigation of single-dose theophylline (250 mg) disposition in 9 healthy volunteers receiving steady-state (9 days) fluvoxamine at 3 doses (0, 25, or 75 mg/d) in a randomized crossover design. Results In vitro Ki values based on total inhibitor concentrations were 177 ±56 nmol/L, 121 ±21 nmol/L, and 52 ±13 nmol/L in human liver microsomes with 1 mg/ml protein and 0.5 mg/ml protein and in supersomes with 0.3 mg/ml protein, respectively. The corresponding in vitro Ki values based on unbound fluvoxamine concentrations were 35 nmol/L, 36 nmol/L, and 36 nmol/L. The ratio of 1-methyluric acid formation clearances (control/inhibited) in 8 subjects was positively correlated with fluvoxamine concentration (r2 = 0.87; P < .001) with an intercept near 1. Mean values for Kiiv based on total and unbound plasma concentrations at steady state were 25.3 nmol/L (range, 14–39 nmol/L) and 3.6 nmol/L (range, 2.4–5.9 nmol/L), respectively. Conclusion Comparison of in vitro and in vivo Ki values based on unbound fluvoxamine concentrations suggests that fluvoxamine inhibition potency is approximately 10 times greater in vivo than in vitro. Clinical Pharmacology & Therapeutics (2001) 70, 415–424; doi: 10.1016/S0009-9236(01)62844-3

Rho GTPases are activated by a family of guanine nucleotide exchange factors (GEFs) known as Dbl family proteins. The structural basis for how GEFs recognize and activate Rho GTPases is presently ill defined. Here, we utilized the crystal structure of the DH/PH domains of the Rac-specific GEF Tiam1 in complex with Rac1 to determine the structural elements of Rac1 that regulate the specificity of this interaction. We show that residues in the Rac1 beta2-beta3 region are critical for Tiam1 recognition. Additionally, we determined that a single Rac1-to-Cdc42 mutation (W56F) was sufficient to abolish Rac1 sensitivity to Tiam1 and allow recognition by the Cdc42-specific DH/PH domains of Intersectin while not impairing Rac1 downstream activities. Our findings identified unique GEF specificity determinants in Rac1 and provide important insights into the mechanism of DH/PH selection of GTPase targets.

Purpose. The intestinal metabolism of some CYP3A substrates canbe altered profoundly by co-administration of the potent inhibitor,ketoconazole. The present research was conducted to test the hypothesisthat, unlike the inhibition kinetics observed with isolated microsomes,inhibition of CYP3A4 by ketoconazole in an intestinal cell monolayeris time-dependent and slowly reversible.

Two isolates of Cryptococcus neoformans were previously described as being highly divergent in their level of capsule synthesis in vivo and in their virulence for mice. The highly virulent isolate (NU-2) produced more capsule than a weakly virulent isolate (184A) in vitro under tissue culture conditions and in vivo. This investigation was done to determine if there were differences between the two isolates in other factors that might also contribute to virulence. Growth rate was not a factor as NU-2 grew more slowly than 184A. Based on PCR fingerprinting the two isolates were genetically different providing an opportunity to examine differences in multiple virulence traits. Quantitative analysis revealed that NU-2 expressed significantly more melanin and mannitol than did 184A. Although the isolates expressed the same capsular chemotype, NU-2 produced an additional structure reporter group (SRG) under tissue culture conditions that was not present when grown in glucose salts/urea/basal medium (GSU). Capsular polysaccharide SRGs of 184A were unaffected by shifting the growth conditions from GSU to tissue culture conditions. Our results suggest that pathogenesis of a C. neoformans strain is dictated by the quantitative expression of the strain's combined virulence traits. Regulators of the expression of these genes may be playing key roles in virulence.

Novel patterns of teaching/learning have emerged from faculty and students who use our three teaching/learning theaters at the University of Maryland, College Park. These fully-equipped electronic classrooms have been used by 74 faculty in 264 semester-long courses since the fall of 1991 with largely enthusiastic reception by both faculty and students. The designers of the teaching/learning theaters sought to provide a technologically rich environment and a support staff so that faculty could concentrate on changing the traditional lecture from its unidirectional information flow to a more collaborative activity. As faculty have evolved their personal styles in using the electronic classrooms, novel patterns of teaching/learning have emerged. In addition to enhanced lectures, we identified three common patterns: (a) active individual learning, (b) small-group collaborative learning, and (c) entire-class collaborative learning.

The Rho family of small GTPases has attracted considerable research interest over the past 5 years. During this time, we have witnessed a remarkable increase in our knowledge of the biochemistry and biology of these Ras-related proteins. Thus, Rho family proteins have begun to rival, if not overshadow, interest in their more celebrated cousins, the Ras oncogene proteins. The fascination in Rho family proteins is fueled primarily by two major observations. First, like Ras, Rho family proteins serve as guanine nucleotide-regulated binary switches that control signaling pathways that in turn regulate diverse cellular processes. Rho family proteins are key components in cellular processes that control the organization of the actin cytoskeleton, activate kinase cascades, regulate gene expression, regulate membrane trafficking, promote growth transformation and induce apoptosis. Second, at least five Rho family proteins have been implicated as critical regulators of oncogenic Ras transformation. Thus, it is suspected that Rho family proteins contribute significantly to the aberrant growth properties of Ras-transformed cells. Rho family proteins are also critical mediators of the transforming actions of other transforming proteins and include Dbl family oncogene proteins, G protein-coupled receptors and G protein alpha subunits. Thus, Rho family proteins may be key components for the transforming actions of diverse oncogene proteins. Major aims of Rho family protein studies are to define the molecular mechanism by which Rho family proteins regulate such a diverse spectrum of cellular behavior. These efforts may reveal novel targets for the development of anti-Ras and anti-cancer drugs.