Computational Screening for mAb Colloidal Stability with Coarse-Grained, Molecular-Scale Simulations.

Monoclonal antibodies (mAbs) are an important modality of protein therapeutics with broad applications for numerous diseases. However, colloidal instabilities occurring at high protein concentrations can limit the ability to develop stable, high-concentration liquid dosage forms that are required for patient-centric, device-mediated products. Therefore, it is advantageous to identify colloidally stable mAbs early in the discovery process to ensure that they are selected for development. Experimental screening for colloidal stability can be time- and resource-consuming and is most feasible at the later stages of drug development due to material requirements. Alternatively, computational approaches have emerging potential to provide efficient screening and focus developmental efforts on mAbs with the greatest developability potential, while providing mechanistic relationships for colloidal instability. In this work, coarse-grained, molecular-scale models were fine-tuned to screen for colloidal stability at amino-acid resolution. This model parameterization provides a framework to screen for mAb self-interactions and extrapolate to bulk solution behavior. This approach was applied to a wide array of mAbs under multiple buffer conditions, demonstrating the utility of the presented computational approach to augment early candidate screening and later formulation strategies for protein therapeutics.

Characterization of Heparin's Conformational Ensemble by Molecular Dynamics Simulations and Nuclear Magnetic Resonance Spectroscopy.

Heparin is a highly charged, polysulfated polysaccharide and serves as an anticoagulant. Heparin binds to multiple proteins throughout the body, suggesting a large range of potential therapeutic applications. Although its function has been characterized in multiple physiological contexts, heparin's solution conformational dynamics and structure-function relationships are not fully understood. Molecular dynamics (MD) simulations facilitate the analysis of a molecule's underlying conformational ensemble, which then provides important information necessary for understanding structure-function relationships. However, for MD simulations to afford meaningful results, they must both provide adequate sampling and accurately represent the energy properties of a molecule. The aim of this study is to compare heparin's conformational ensemble using two well-developed force fields for carbohydrates, known as GLYCAM06 and CHARMM36, using replica exchange molecular dynamics (REMD) simulations, and to validate these results with NMR experiments. The anticoagulant sequence, an ultra-low-molecular-weight heparin, known as Arixtra (fondaparinux, sodium), was simulated with both parameter sets. The results suggest that GLYCAM06 matches experimental nuclear magnetic resonance three-bond J-coupling values measured for Arixtra better than CHARMM36. In addition, NOESY and ROESY experiments suggest that Arixtra is very flexible in the sub-millisecond time scale and does not adopt a unique structure at 25 C. Moreover, GLYCAM06 affords a much more dynamic conformational ensemble for Arixtra than CHARMM36.

Regional distribution of body fat in relation to DNA methylation within the LPL, ADIPOQ and PPARγ promoters in subcutaneous adipose tissue.

Obesity may be related to differential DNA methylation and thus to differential expression of key genes in adipose tissue metabolism, such as LPL, ADIPOQ and PPARγ. Using subcutaneous adipose tissue (SAT) from 59 individuals of the European Prospective Investigation into Cancer and Nutrition-Potsdam study, we performed quantitative DNA methylation analysis within the promoters of LPL (LPL-CG1 and -CG2), ADIPOQ (ADIPOQ-CG1 and-CG2) and PPARγ (PPARγ-CG1). We then studied DNA methylation in relation to SAT gene expression, body composition measured using whole-body magnetic resonance imaging, body mass index (BMI), waist circumference (WC) and long-term changes in BMI and WC. For LPL-CG1 and LPL-CG2, higher methylation levels were associated with lower LPL expression, but with higher past WC gain. LPL-CG1 was also positively associated with BMI, WC, and visceral and subcutaneous fat mass. ADIPOQ-CG1 or -CG2 methylation exhibited no association with ADIPOQ expression or with anthropometric parameters. PPARγ-CG1 methylation was significantly higher in individuals with higher visceral fat mass. Among the investigated sites, LPL-CG1 methylation showed the strongest association with gene expression and regional body fat distribution, thereby possibly linking the degree of obesity with major metabolic processes in SAT.

Oleic acid phase behavior from molecular dynamics simulations.

Fatty acid aggregation is important for a number of diverse applications: from origins of life research to industrial applications to health and disease. Experiments have characterized the phase behavior of oleic acid mixtures, but the molecular details are complex and hard to probe with many experiments. Coarse-grained molecular dynamics computer simulations and free energy calculations are used to model oleic acid aggregation. From random dispersions, we observe the aggregation of oleic acid monomers into micelles, vesicles, and oil phases, depending on the protonation state of the oleic acid head groups. Worm-like micelles are observed when all the oleic acid molecules are deprotonated and negatively charged. Vesicles form spontaneously if significant amounts of both neutral and negative oleic acid are present. Oil phases form when all the fatty acids are protonated and neutral. This behavior qualitatively matches experimental observations of oleic acid aggregation. To explain the observed phase behavior, we use umbrella sampling free energy calculations to determine the stability of individual monomers in aggregates compared to water. We find that both neutral and negative oleic acid molecules prefer larger aggregates, but neutral monomers prefer negatively charged aggregates and negative monomers prefer neutral aggregates. Both neutral and negative monomers are most stable in a DOPC bilayer, with implications on fatty acid adsorption and cellular membrane evolution. Although the CG model qualitatively reproduces oleic acid phase behavior, we show that an updated polarizable water model is needed to more accurately predict the shift in pKa for oleic acid in model bilayers.

Characterization of the immersion properties of the peripheral membrane anchor of the FATC domain of the kinase "target of rapamycin" by NMR, oriented CD spectroscopy, and MD simulations.

The multidomain ser/thr kinase "target of rapamycin" (TOR) centrally controls eukaryotic growth and metabolism. The C-terminal FATC domain is important for TOR regulation and was suggested to directly mediate TOR-membrane interactions. Here, we present a detailed characterization of the membrane immersion properties of the oxidized and reduced yeast TOR1 FATC domain (2438-2470 = y1fatc). The immersion depth was characterized by NMR-monitored interaction studies with DPC micelles containing paramagnetically tagged 5- or 16-doxyl stearic acid (5-/16-SASL) and by analyzing the paramagnetic relaxation enhancement (PRE) from Mn(2+) in the solvent. Complementary MD-simulations of micellar systems in the absence and presence of protein showed that 5-/16-SASL can move in the micelle and that 16-SASL can bend such that the doxyl group is close to the headgroup region and not deep in the interior as commonly assumed. Based on oriented CD (OCD) data, the single α-helix of oxidized/reduced y1fatc has an angle to the membrane normal of ∼30-60°/∼35-65° in neutral and ∼5-35°/∼0-30° in negatively charged bilayers. The presented experimentally well-founded models help to better understand how this redox-sensitive peripheral membrane anchor may be part of a network of protein-protein and protein-membrane interactions regulating TOR localization at different cellular membranes. Moreover, the presented work provides a good methodological reference for the structural characterization of other peripherally membrane associating proteins.

Influence of dietary fat ingestion on asymmetrical dimethylarginine in lean and obese human subjects.

BACKGROUND AND AIMS: Asymmetrical dimethylarginine (ADMA) may contribute to hypertension and cardiovascular disease by decreasing NO formation. In diabetic patients, a high fat meal acutely increased plasma ADMA while impairing endothelial function. We hypothesized that chronic and acute increases in dietary fat intake augment ADMA also in lean and in obese subjects without diabetes. METHODS AND RESULTS: Seventeen lean and twelve obese volunteers were randomized to two weeks of isocaloric diets with approximately 20% or >40% calories from fat in a cross-over fashion. At the end of the high and low fat periods, volunteers received corresponding test meals. ADMA was measured by GC-MS/MS using a deuterated standard. Mean fasting plasma ADMA concentration was 0.52 (0.49-0.54; 95% CI) µmol/l in lean and 0.53 (0.50-0.55) µmol/l in obese subjects (p = 0.55). The two week high fat diet did not influence ADMA. Both test meals elicited a 6%increase in circulating ADMA in lean subjects. In obese subjects, plasma ADMA concentration did not change with the low fat meal, and decreased by approximately 4% with the high fat meal. CONCLUSION: Our findings challenge the idea that obesity and dietary fat intake have a major effect on plasma ADMA, at least in subjects without overt cardiovascular and metabolic disease. This finding is important with regard to dietary recommendations for weight loss. Overestimation of the influence of dietary fat intake and obesity on circulating ADMA in previous reports was most likely due to methodological issues concerning ADMA measurements.

Hypomyelination associated with bovine viral diarrhea virus type 2 infection in a longhorn calf.

A newborn Longhorn heifer calf presented with generalized tremors, muscle fasciculations, ataxia, and nystagmus. At necropsy, no gross central nervous system lesions were observed. Histologically, the brain and spinal cord had mild to moderate diffuse microgliosis and astrocytosis, minimal nonsuppurative encephalitis, and decreased myelin staining. Ultrastructural examination revealed thinning and absence of myelin sheaths. Various cell types were immunohistochemically positive for bovine viral diarrhea virus (BVDV). Noncytopathogenic BVDV was isolated from the brain and identified as BVDV type 2 by phylogenetic analysis. BVDV-induced hypomyelination is rare and analogous to lesions in neonates infected with border disease and classical swine fever viruses. This is the first documented case of hypomyelination in a calf specifically attributed to BVDV type 2 and the first description of the ultrastructural appearance of BVDV-induced hypomyelination.

[The influence of different local anaesthetics on the viability of preadipocytes]. / Vitalitätsunterschiede von Präadipozyten unter dem Einfluss verschiedener Lokalanästhetika.

BACKGROUND: Autogenous fat transfer with lipoinjection for soft-tissue augmentation is a commonly used technique in plastic surgery. The efficiency of this technique has often been discussed and authors still describe very different results after autologous fat transplantation. The purpose of our investigations was to evaluate the effect of the various local anaesthetics, used in the region of harvesting, on the viability of preadipocytes. MATERIAL AND METHOD: Preadipocytes were isolated from human subcutaneous adipose tissue samples and incubated with lidocaine, prilocaine, articaine + epinephrine, ropivacaine or our standardised tumescent solution for 30 minutes. NaCl solution (0.9 %) served as a control. Vitality was measured with FACS analysis. RESULTS: There are significant differences in the viability of the preadipocytes after incubation with the different substances. Viability ranged from > 90 % to less then 20 %. CONCLUSION: For the first time the influence of local anaesthesia on the viability of preadipocytes has been investigated. The significant differences between the substances could explain the varying results in autologous fat transplantation. Our results should help by choosing the right local anaesthesia for infiltrating the donor site.

Tyramine in the assessment of regional adrenergic function.

Regional adrenergic function is difficult to assess in humans. Tyramine given through a microdialysis probe may be a useful tool in this regard. However, tyramine data is hard to interpret given the drug's complex mode of action. We characterized the response to tyramine, isoproterenol, and dopamine in adipose tissue with microdialysis probes in normal subjects. We measured glycerol concentrations to follow changes in lipolysis and monitored tissue perfusion with ethanol dilution. During perfusion with tyramine, dialysate glycerol concentration increased dose-dependently from 83+/-8 microM at baseline to 181+/-18 microM at 3.5 mM tyramine (p<0.001) followed by a fall down to 121+/-9 microM at 35 mM tyramine (p<0.001). Propranolol almost completely blocked this response. A similar lipolytic response was not observed in isolated human adipocytes. Dopamine <35 microM did not replicate the tyramine-induced lipolysis; however, dopamine >35 microM potently inhibited lipolysis. We conclude that tyramine-induced lipolysis is explained by a pre-synaptic mechanism. Tyramine applied through a microdialysis probe in concentrations up to 3.5 mM can be used to assess pre- and post-synaptic mechanisms regulating lipid mobilization.

Adipose tissue and circulating endothelial cell specific molecule-1 in human obesity.

Adipocytes produce the endothelial-cell specific molecule-1 (ESM-1), which inhibits leukocyte adhesion and migration through the endothelium. This study investigates ESM-1 expression and regulation in human adipose tissue. Subcutaneous abdominal adipose tissue was obtained from seventy postmenopausal women. Fourteen women subsequently underwent non-pharmacological weight reduction. In vitro experiments were performed on adipocytes isolated from human mammary adipose tissue. We determined gene expression by TaqMan RT-PCR and measured ESM-1 levels in serum and cell culture medium by ELISA. Mature adipocytes produced ESM-1. ESM-1 gene expression was higher in adipocytes than in preadipocytes. Cortisol inhibited ESM-1 gene expression in preadipocytes. Insulin and cortisol inhibited adipocyte ESM-1 production in adipocytes. This inhibitory effect of insulin was attenuated by insulin resistance, as ESM-1 gene expression in subcutaneous adipose tissue was increased in obese, hyperinsulinemic women. In contrast, ESM-1 serum levels were reduced in obese women and inversely correlated to C-reactive protein levels. Five percent weight loss did not markedly change gene expression. Circulating ESM-1 levels increased significantly, albeit modestly. ESM-1 is actively produced by adipocytes. However, since ESM-1 adipocyte gene expression and circulating plasma levels are not correlated, other sources of ESM-1 may be more important. Circulating ESM-1 levels are reduced in the overweight and obese, consistent with the notion that ESM-1 may play some role in obesity-associated vascular disease.

Endothelial cell specific molecule-1--a newly identified protein in adipocytes.

Expression of the endothelial cell-specific molecule (ESM)-1 was originally identified in lung and kidney endothelial cells, where its expression is regulated by cytokines. In vitro, ESM-1 interferes with the molecular mechanisms of immune cell migration by binding to adhesion molecules. In this study, we have explored the expression of ESM-1 in isolated human adipocytes and in rat adipose tissue depots. Human primary adipocytes were cultivated after collagenase digestion and used for in vitro incubation studies. Adipocytes were also isolated from different fat depots of Sprague-Dawley rats. Gene expression was quantified by TaqMan RT-PCR using specific human and rat ESM-1 primers. The cellular localisation of ESM-1 was determined by confocal microscopy using a specific antibody. ESM-1 expression in human adipocytes was stimulated by phorbol ester, an activator of protein kinase C, and by retinoic acid, an activator of nuclear receptors. The maximum increase in gene expression was 3.2-fold after 72 h treatment with phorbol ester and 4.6-fold after 72 h treatment with retinoic acid. The highest expression was found in subcutaneous rat adipose tissue - two-fold compared to epididymal and six-fold compared to intrascapular brown adipose tissue. As obesity is related to systemic inflammation (examplified by increased circulating levels of C-reactive protein and interleukin-6), the formation of ESM-1 in adipocytes and its activation by protein kinase C may play a role in the regulation of inflammatory processes.

Hybrid nanorod-polymer solar cells.

We demonstrate that semiconductor nanorods can be used to fabricate readily processed and efficient hybrid solar cells together with polymers. By controlling nanorod length, we can change the distance on which electrons are transported directly through the thin film device. Tuning the band gap by altering the nanorod radius enabled us to optimize the overlap between the absorption spectrum of the cell and the solar emission spectrum. A photovoltaic device consisting of 7-nanometer by 60-nanometer CdSe nanorods and the conjugated polymer poly-3(hexylthiophene) was assembled from solution with an external quantum efficiency of over 54% and a monochromatic power conversion efficiency of 6.9% under 0.1 milliwatt per square centimeter illumination at 515 nanometers. Under Air Mass (A.M.) 1.5 Global solar conditions, we obtained a power conversion efficiency of 1.7%.

Validation of endogenous controls for gene expression studies in human adipocytes and preadipocytes.

Quantitative gene expression protocols require adequate controls to monitor intersample variation. Quantitative approaches to describe relative changes in gene expression use endogenous controls--"housekeeping" genes. Given the low amounts of mRNA in fat cells, RT-PCR is the method of choice, and housekeeping genes are widely used as endogenous controls. However, literature reports suggest changes in gene expression of typical housekeeping genes (e. g. GAPDH, beta-actin, 18S rRNA) upon hormonal stimulation or during adipogenic differentiation. Thus, we tested the influence of 6 hormones and adipogenic differentiation on gene expression levels of 11 commonly used housekeeping genes in primary cultured mature human adipocytes and preadipocytes. Using the TaqMan RT-PCR technique and "Human Endogenous Control Assays" (PE Biosystems), we found several housekeeping genes with at least twice the difference in expression levels between stimulated and unstimulated cells (such as acidic ribosomal protein, beta-actin, beta(2)-microglobulin and beta-glucuronidase). Only GAPDH and transferrin receptor gene expression levels did not change under any of the stimuli tested, thus appeared best suited for gene expression studies in human adipose cells across a wide range of experimental settings.

Extraction of total RNA from adipocytes.

RNA isolation from adipocytes presents with several technical problems and yields unacceptable results when following standard protocols. Here, we will describe additional steps and modifications necessary for the use of different RNA isolation protocols in terms of RNA yield, RNA quality and preparation time. Using five times the recommended quantity of lysis buffer, incubating the lysate at 37 degrees C, repeatedly passing the lysate through a cannula, and centrifugation to remove the lipid layer are essential additional steps when working with adipocytes. With these modifications, isolation of total RNA resulted in an average yield of 12-30 microg total RNA from 2 x 10(6) cells. Preparation times were similar for all but the CsCl gradient method. The purest RNA was obtained by spin-column purification, whereas acid phenol-chloroform methods yielded the highest amounts of total RNA. CsCl gradient ultracentrifugation is suggested for situations where DNase I digestion is impractical.

Experimental evaluation of a cell module for hybrid liver support.

Aim of the study was to evaluate a hybrid liver support system in a porcine model of acute liver failure, after hepatectomy. Pigs with a body weight of 70+/-18 kg underwent total hepatectomy and porto-cavo-caval shunting as well as ligation of the bile duct and the hepatic artery. Control animals were connected to the system (including capillary membrane plasma separation) containing a four compartment bioreactor with integral oxygenation and decentralized mass exchange but without liver cells. The treatment group received hybrid liver support with the same system including 370+/-42 g primary isolated porcine parenchymal liver cells in co-culture with hepatocyte nursing cells, tissue engineered to liver- like structures at high density. Treatment started after complete recovery from anesthesia and was performed continuously. A positive influence on peripheral vascular resistance and a reduced need of catecholamine dosage was observed in the treatment group. Hybrid liver support with a cell module upscaled for clinical application significantly prolonged survival time in animals after hepatectomy with the longest survival being 26 hours in the control group an 57 hours in the treatment group.

Suppression of tumorigenicity in breast cancer cells by the microfilament protein profilin 1.

Differential display screening was used to reveal differential gene expression between the tumorigenic breast cancer cell line CAL51 and nontumorigenic microcell hybrids obtained after transfer of human chromosome 17 into CAL51. The human profilin 1 (PFN1) gene was found overexpressed in the microcell hybrid clones compared with the parental line, which displayed a low profilin 1 level. A comparison between several different tumorigenic breast cancer cell lines with nontumorigenic lines showed consistently lower profilin 1 levels in the tumor cells. Transfection of PFN1 cDNA into CAL51 cells raised the profilin 1 level, had a prominent effect on cell growth, cytoskeletal organization and spreading, and suppressed tumorigenicity of the stable, PFN1-overexpressing cell clones in nude mice. Immunohistochemical analysis revealed intermediate and low levels of profilin 1 in different human breast cancers. These results suggest profilin 1 as a suppressor of the tumorigenic phenotype of breast cancer cells.

The effect of relaxation therapy on preterm labor outcomes.

OBJECTIVE: To examine the effect of relaxation on preterm labor outcome. DESIGN: Quasi-experimental, with women who experienced preterm labor randomly assigned to a control or experimental group. The experimental group was to do a daily relaxation exercise. A third group was added to the study: women who were originally assigned to the relaxation group but were unable to adhere to the daily practice. Final data were analyzed for three groups: control (n = 40), experimental (n = 44), and nonadherent (n = 23) participants. SETTING: Women were referred to the study from physician offices and a hospital-based obstetric triage clinic in the Northwest. PARTICIPANTS: Total sample was comprised of 107 women with singleton gestations, documented contractions with cervical change, and intact membranes. INTERVENTIONS: The experimental group was instructed in a progressive relaxation exercise. The participants were given tapes of the exercise and instructed to do it daily. OUTCOME MEASURES: Study outcomes included gestational age at birth, rate of pregnancy prolongation, and birth weight. RESULTS: The outcome variables were analyzed using analysis of covariance, with the preterm labor risk score entered as a covariate to compensate statistically for group differences. A positive response to the relaxation intervention was found: The experimental group had significantly longer gestations and larger newborns when compared to the control and nonadherent groups. CONCLUSIONS: Relaxation therapy made a difference in preterm labor outcome. Women who practiced relaxation had larger newborns, longer gestations, and higher rates of pregnancy prolongation. Given the low cost of the intervention, it should be offered to all women at risk for preterm labor.

Mammalian protein homologous to VAT-1 of Torpedo californica: isolation from Ehrlich ascites tumor cells, biochemical characterization, and organization of its gene.

Recently, interest has focused on the human gene encoding the putative protein homologous to VAT-1, the major protein of the synaptic vesicles of the electric organ of the Pacific electric ray Torpedo californica, after it has been localized on chromosome locus 17q21 in a region encompassing the breast cancer gene BRCA1. Chromosomal instability in this region is implicated in inherited predisposition for breast and ovarian cancer. Here we describe isolation and biochemical characterization of a mammalian 48 kDa protein homologous to the VAT-1 protein of Torpedo californica. This VAT-1 homolog was isolated from a murine breast cancer cell line (Ehrlich ascites tumor) and identified by sequencing of cleavage peptides. The isolated VAT-1 homolog protein displays an ATPase activity and exists in two isoforms with isoelectric points of 5.7 and 5.8. cDNA was prepared from Ehrlich ascites tumor cells, and the murine VAT-1 homolog sequence was amplified by polymerase chain reaction and partially sequenced. The known part of the murine and the human translated sequences share 97% identity. By Northern blots, the size of the VAT-1 homolog mRNA in both murine and human (T47D) breast cancer cells was determined to be 2.8 kb. Based on the presented data, a modified gene structure of the human VAT-1 homolog with an extended exon 1 is proposed. VAT-1 and the mammalian VAT-1 homolog form a subgroup within the protein superfamily of medium-chain dehydrogenases/reductases.