Sensitivity of the IQM and MatriXX detectors in megavolt photon beams

AimThis study focused on evaluating the sensitivity of integral quality monitoring (IQM^®) system and MatriXX detectors. These two detectors are recommended for radiotherapy pre-treatment quality assurance (QA).BackgroundIQM is a large wedged-shaped ionisation chamber mounted to the linear accelerator (linac) head in practice. MatriXX consists of an array of ionisation chambers also attached to the linac head.Materials and methodsIn this study, the dosimetric performance and sensitivity of MatriXX and IQM detectors were evaluated using the following characteristics: reproducibility, linearity, error detection capability and three-dimensional conformal radiotherapy (3D-CRT) plans of the head and neck, thorax and pelvic regions.ResultsThis study indicates that the signal responses of the large ionisation chamber device (IQM) and the small pixel array of ionisation chambers device (MatriXX) are reproducible, linear and sensitive to MLC positional errors, backup jaw positional errors and dose errors. The local percentage differences for dose errors of 1%, 2%, and 3% were, respectively, within 0.35–8.23%, 0.78–16.21%, and 1.10–24.41% for the IQM device. While for the MatriXX detector, the ranges were between 0.24–3.19, 0.57–6.43 and 0.81–12.95, respectively. Since IQM is essentially a double wedge-shaped large ionisation chamber, its reproducibility and detection capability are competitive to that of MatriXX. In addition, the sensitivity of the two QA systems increases with an increase in escalation percentage, and the signal responses are patient plan specific.ConclusionsThe two detectors response signals have good correlations and they are accurate for pre-treatment QA. Statistically, (P < 0.05) there is a significant difference between the IQM and MatriXX response to dose errors.     

A Deep Learning-based correction to EPID dosimetry for attenuation and scatter in the Unity MR-Linac system

PurposeEPID dosimetry in the Unity MR-Linac system allows for reconstruction of absolute dose distributions within the patient geometry. Dose reconstruction is accurate for the parts of the beam arriving at the EPID through the MRI central unattenuated region, free of gradient coils, resulting in a maximum field size of ~10 × 22 cm² at isocentre. The purpose of this study is to develop a Deep Learning-based method to improve the accuracy of 2D EPID reconstructed dose distributions outside this central region, accounting for the effects of the extra attenuation and scatter.MethodsA U-Net was trained to correct EPID dose images calculated at the isocenter inside a cylindrical phantom using the corresponding TPS dose images as ground truth for training. The model was evaluated using a 5-fold cross validation procedure. The clinical validity of the U-Net corrected dose images (the so-called DEEPID dose images) was assessed with in vivo verification data of 45 large rectum IMRT fields. The sensitivity of DEEPID to leaf bank position errors (±1.5 mm) and ±5% MU delivery errors was also tested.ResultsCompared to the TPS, in vivo 2D DEEPID dose images showed an average γ-pass rate of 90.2% (72.6%–99.4%) outside the central unattenuated region. Without DEEPID correction, this number was 44.5% (4.0%–78.4%). DEEPID correctly detected the introduced delivery errors.ConclusionsDEEPID allows for accurate dose reconstruction using the entire EPID image, thus enabling dosimetric verification for field sizes up to ~19 × 22 cm² at isocentre. The method can be used to detect clinically relevant errors.     

A Novel Region-Growing Based Semi-Automatic Segmentation Protocol for Three-Dimensional Condylar Reconstruction Using Cone Beam Computed Tomography (CBCT)

Objective

To present and validate a semi-automatic segmentation protocol to enable an accurate 3D reconstruction of the mandibular condyles using cone beam computed tomography (CBCT).

Materials and Methods

Approval from the regional medical ethics review board was obtained for this study. Bilateral mandibular condyles in ten CBCT datasets of patients were segmented using the currently proposed semi-automatic segmentation protocol. This segmentation protocol combined 3D region-growing and local thresholding algorithms. The segmentation of a total of twenty condyles was performed by two observers. The Dice-coefficient and distance map calculations were used to evaluate the accuracy and reproducibility of the segmented and 3D rendered condyles.

Results

The mean inter-observer Dice-coefficient was 0.98 (range [0.95–0.99]). An average 90^th percentile distance of 0.32 mm was found, indicating an excellent inter-observer similarity of the segmented and 3D rendered condyles. No systematic errors were observed in the currently proposed segmentation protocol.

Conclusion

The novel semi-automated segmentation protocol is an accurate and reproducible tool to segment and render condyles in 3D. The implementation of this protocol in the clinical practice allows the CBCT to be used as an imaging modality for the quantitative analysis of condylar morphology.     

High-Fat Diet-Induced Insulin Resistance Does Not Increase Plasma Anandamide Levels or Potentiate Anandamide Insulinotropic Effect in Isolated Canine Islets

BackgroundObesity has been associated with elevated plasma anandamide levels. In addition, anandamide has been shown to stimulate insulin secretion in vitro, suggesting that anandamide might be linked to hyperinsulinemia.ObjectiveTo determine whether high-fat diet-induced insulin resistance increases anandamide levels and potentiates the insulinotropic effect of anandamide in isolated pancreatic islets.ResultsProlonged fat feeding increased abdominal fat content by 81.3±21.6% (mean±S.E.M, P<0.01). In vivo insulin sensitivity decreased by 31.3±12.1% (P<0.05), concomitant with a decrease in plasma 2-arachidonoyl glycerol (from 39.1±5.2 to 15.7±2.0 nmol/L) but not anandamide, oleoyl ethanolamide, linoleoyl ethanolamide, or palmitoyl ethanolamide. In control-diet animals (body weight: 28.8±1.0 kg), islets incubated with anandamide had a higher basal and glucose-stimulated insulin secretion as compared with no treatment. Islets from fat-fed animals (34.5±1.3 kg; P<0.05 versus control) did not exhibit further potentiation of anandamide-induced insulin secretion as compared with control-diet animals. Glucagon but not somatostatin secretion in vitro was also increased in response to anandamide, but there was no difference between groups (P = 0.705). No differences in gene expression of CB1R or CB2R between groups were found.ConclusionsIn canines, high-fat diet-induced insulin resistance does not alter plasma anandamide levels or further potentiate the insulinotropic effect of anandamide in vitro.     

Independent human mesenchymal stromal cell–derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model

Background aimsExtracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow–derived MSCs, this study focused on improving the MSC-EV production for clinical application.MethodsIndependent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized.ResultsThe functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers.ConclusionsStandardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.     

Validation of a human-serum-based in vitro growth method for drug screening on juvenile development stages of Schistosoma mansoni

BackgroundSchistosomiasis affects over 200 million people worldwide but only praziquantel is available for treatment and control. Drug discovery is often based on phenotypic drug screening, involving different parasite stages retrieved from infected mice. Aiming to reduce animal use, we validated an in vitro growth method for juvenile Schistosoma mansoni for the purpose of drug sensitivity assays.Methodology/Principal findingsWe compared inter–batch variability of serum, worm size and organ development, gender distribution, and drug sensitivity between in vitro and in vivo grown worms over different life stages. In vitro developed S. mansoni in Hybridoma medium supplemented with 20% human serum were similar in size as in vivo worms until 28 days of incubation (males 1.4 ± 0.2 mm, females 1.1 ± 0.5 mm long). qPCR analysis revealed similar gender distribution both on newly transformed schistosomula and worms grown for 21 days. Worms developed in vitro and in vivo were similarly sensitive to praziquantel from 7 to 35 days of development with the exception of 21 days of development, where a slightly lower activity was observed for the in vitro grown worms (IC₅₀: 0.54 μM in vitro, 0.14 μM in vivo 72 hours post-incubation). The evaluation of five additional drugs revealed a similar sensitivity on worms developed for 21 days, with the exception of mefloquine, where we observed a 10-fold lower sensitivity on in vitro developed schistosomes when compared to in vivo grown (IC₅₀: 4.43 μM in vitro, 0.48 μM in vivo).ConclusionA large number of juvenile S. mansoni worms can be grown in vitro, which show similar drug sensitivity, gender distribution, size and morphology as the worms recovered from rodents, supporting the use of this method in drug screening efforts.     

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol ζ) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol ζ alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol ζ. Pol ζ is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol ζ is substantially lower than that of homologous B family pols α, δ and . Pol ζ is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol ζ in vitro is consistent with Pol ζ-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol ζ contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.     

Stimulation of D-loop formation by polypurine/polypyrimidine sequences

Most of the approaches used to correct gene mutations in mammalian cells involve the targeting of short nucleotide molecules to homologous chromosomal sequences and the replacement of resident sequences via homologous recombination and mismatch repair. The limited efficiency and inconsistent reproducibility of these techniques are major constraints to their use in gene therapy. One of the main problems is that it is impossible to obtain reproducible results when the targeted gene loci differ. We investigated the effects of flanking sequences on homologous recombination by means of an in vitro assay of the efficiency of oligonucleotide targeting to its homologous sequence on a large duplex molecule in a reaction catalysed by the Escherichia coli RecA protein. We demonstrated that polypurine·polypyrimidine tracts (PPTs) in duplex DNA strongly stimulate the formation of D-loops with short oligodeoxynucleotides. This result was reproduced with various PPT sequences and oligonucleotides. The stimulatory effect was observed at loci as far as 4000 bp from the PPT. The formation of complexes between the oligonucleotide and the duplex molecule depended on the extent of sequence similarity between the two DNAs and the presence of the RecA protein. The stimulatory effect was inhibited by excess RecA and restored by adding heterologous DNA. We suggest that PPT sequences induce conformational changes in duplex DNA, leading to the aggregation of molecules, facilitating homology searches. We com pared, in vivo, the efficiency of the oligonucleotide-mediated correction of a URA3 chromosomal mutation for sequences with and without a PPT sequence in the vicinity. Consistent with our in vitro results, the efficiency of correction was eight times higher in the presence of the PPT sequence.     

Passive marker tracking via phase-only cross correlation (POCC) for MR-guided needle interventions: Initial in vivo experience

PurposeIn this work, a passive tracking sequence employing a phase-only cross correlation (POCC) algorithm was studied with a focus on the in vivo applicability of the technique. Therefore, MR-guided needle interventions were performed in a phantom and two animal experiments.MethodsThe targeting accuracy was quantified in an agarose phantom with 15 fiducials. For each fiducial, the distance between needle trajectory and target point was measured. In a first animal experiment at 3 T, the prostate of a pig was punctured under POCC guidance. Second, POCC-based tracking was performed during a laser-induced thermal therapy procedure in peripheral porcine muscle tissue at 1.5 T.ResultsIn the phantom experiment, the 15 fiducials were penetrated with a mean accuracy of 1.5 ± 0.9 mm (mean duration for one puncture about 2 min). In the first animal experiment, the center of the pig's right prostatic lobe was accurately punctured within 15 min. In the second, targeting and insertion of the needle could be performed within 5 min and a thermal lesion was successfully created.ConclusionOur initial experience with the POCC-based tracking sequence indicates that this technique has the potential as an accurate and versatile tool for in vivo MR-guided needle interventions.     

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.     

Segmentation d’images de lésions cérébrales primitives en TEP FET : influence du volume de travail

Purpose(1) Evaluate the reproducibility of segmentation methods depending on the preselection region for tumour volume determination on ¹⁸F-fluoro-ethyl-tyrosine (FET) PET. (2) Evaluate the intra and inter-operator reproducibility of the manual delineation. (3) Compare this delineation with the segmentation methods.Materials and methodsEighteen FET PET of patients with glioblastoma were analysed. Preselection regions were determined prior to any segmentation. Two physicians delineated the tumour volume manually. The tumour volume was also delineated with a threshold method (40 and 70% of SUV_max), and a random walk based method. Pearson coefficient (r) (P < 0.05 for r > 0.468) and Jaccard indices (JI) were used to compare the volumes.ResultsManual delineation was reproducible with r = 0.97 and IJ = 0.65 for intra-operator, and r = 0.76 and IJ = 0.45 for inter-operator reproducibility. The preselection regions for a given lesion were different and the segmentation varied with the preselection region: r = 0.55 JI = 0.58; r = 0.85 JI = 0.83; r = 0.70 JI = 0.39 respectively for the threshold of 40%, 70% and the random walk. The segmentation differed form de manual delineation with r = 0.37 and JI = 0.16; r = 0.54 and JI = 0.42; r = 0.43 and JI = 0.37 respectively for the threshold of 40%, 70% and the random walk.ConclusionThe reproducibility of the segmentation methods depends extensively on the preselection region. The intra-operator reproducibility of cerebral lesion delineation on FET PET is satisfactory. The inter-operator reproducibility could be improved.     

Triterpenic acids-enriched fraction from Cyclocarya paliurus attenuates insulin resistance and hepatic steatosis via PI3K/Akt/GSK3β pathway

BackgroundNon-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver diseases. Cyclocarya paliurus (C. paliurus), an edible and medicinal plant in Chinese folk, has been demonstrated to ameliorate diabetes, obesity and lipid metabolism disorders. However, its effects on NAFLD and its potential molecular mechanism have not been clearly expounded.PurposeThe present study was designed to explore the therapeutic potential of triterpenic acids-enriched fraction from C. paliurus (CPT), as well as its underlying mechanism in vivo and in vitro models of NAFLD.MethodsThe metabolic effects and possible molecular mechanism of CPT were examined using HepG2 cells and primary hepatocytes (isolated from C57BL/6 J mice) models of fatty liver induced by palmitic acid (PA) and a high fat diet mouse model.ResultsIn high fat diet-induced C57BL/6 J mice, CPT significantly reduced liver weight index, serum alanine transaminase (ALT), aspartate transaminase (AST), triacylglycerol (TG), total cholesterol (TC) and hepatic TG, TC levels. Moreover, CPT dramatically decreased the contents of blood glucose, insulin, and insulin resistance (HOMA-IR) index. Meanwhile, CPT significantly increased the tyrosine phosphorylation level of IRS and the uptake of 2-deoxyglucose (2DG) in PA-induced HepG2 cells and primary hepatocytes fatty liver models. Furthermore, in PA-induced HepG2 cells and primary hepatocytes, CPT significantly decreased the number of lipid droplets and intracellular TG content. In addition, mechanism investigation showed that CPT increased the phosphorylation of phosphoinositide 3-kinase (PI3K), protein kinase B (Akt) and glycogen synthase-3β (GSK3β) in vivo and in vitro models, which were abrogated by PI3K inhibitor LY294002 in vitro models.ConclusionThese findings indicate that CPT may exert the therapeutic effects on NAFLD via regulating PI3K/Akt/GSK3β pathway.     

Atherosclerotic Plaque Component Segmentation in Combined Carotid MRI and CTA Data Incorporating Class Label Uncertainty

Atherosclerotic plaque composition can indicate plaque vulnerability. We segment atherosclerotic plaque components from the carotid artery on a combination of in vivo MRI and CT-angiography (CTA) data using supervised voxelwise classification. In contrast to previous studies the ground truth for training is directly obtained from 3D registration with histology for fibrous and lipid-rich necrotic tissue, and with CT for calcification. This registration does, however, not provide accurate voxelwise correspondence. We therefore evaluate three approaches that incorporate uncertainty in the ground truth used for training: I) soft labels are created by Gaussian blurring of the original binary histology segmentations to reduce weights at the boundaries between components, and are weighted by the estimated registration accuracy of the histology and in vivo imaging data (measured by overlap), II) samples are weighted by the local contour distance of the lumen and outer wall between histology and in vivo data, and III) 10% of each class is rejected by Gaussian outlier rejection. Classification was evaluated on the relative volumes (% of tissue type in the vessel wall) for calcified, fibrous and lipid-rich necrotic tissue, using linear discriminant (LDC) and support vector machine (SVM) classification. In addition, the combination of MRI and CTA data was compared to using only one imaging modality. Best results were obtained by LDC and outlier rejection: the volume error per vessel was 0.91.0% for calcification, 12.77.6% for fibrous and 12.18.1% for necrotic tissue, with Spearman rank correlation coefficients of 0.91 (calcification), 0.80 (fibrous) and 0.81 (necrotic). While segmentation using only MRI features yielded low accuracy for calcification, and segmentation using only CTA features yielded low accuracy for necrotic tissue, the combination of features from MRI and CTA gave good results for all studied components.     

Kirenol inhibits RANKL-induced osteoclastogenesis and prevents ovariectomized-induced osteoporosis via suppressing the Ca2+-NFATc1 and Cav-1 signaling pathways

BackgroundOsteoporosis is a threat to aged people who have excessive osteoclast activation and bone resorption, subsequently causing fracture and even disability. Inhibiting osteoclast differentiation and absorptive functions has become an efficient approach to treat osteoporosis, but osteoclast-targeting inhibitors available clinically remain rare. Kirenol (Kir), a bioactive diterpenoid derived from an antirheumatic Chinese herbal medicine Herba Siegesbeckiae, can treat collagen-induced arthritis in vivo and promote osteoblast differentiation in vitro, while the effects of Kir on osteoclasts are still unclear.PurposeWe explore the role of Kir on RANKL-induced osteoclastogenesis in vitro and bone loss in vivo.MethodsThe in vitro effects of Kir on osteoclast differentiation, bone resorption and the underlying mechanisms were evaluated with bone marrow-derived macrophages (BMMs). In vivo experiments were performed using an ovariectomy (OVX)-induced osteoporosis model.ResultsWe found that Kir remarkably inhibited osteoclast generation and bone resorption in vitro. Mechanistically, Kir significantly inhibited F-actinring formation and repressed RANKL-induced NF-κB p65 activation and p-p38, p-ERK and c-Fos expression. Moreover, Kir inhibited both the expression and nuclear translocation of NFATc1. Ca²⁺ oscillation and caveolin-1 (Cav-1) were also reduced by Kir during osteoclastogenesis in vitro. Consistent with these findings, 2–10 mg/kg Kir attenuated OVX-induced osteoporosis in vivo as evidenced by decreased osteoclast numbers and downregulated Cav-1 and NFATc1 expression.ConclusionsKir suppresses osteoclastogenesis and the Cav-1/NFATc1 signaling pathway both in vitro and in vivo and protects against OVX-induced osteoporosis. Our findings reveal Kir as a potential safe oral treatment for osteoporosis.     

Pre-culturing islets with mesenchymal stromal cells using a direct contact configuration is beneficial for transplantation outcome in diabetic mice

Background aimsWe recently showed that co-transplantation of mesenchymal stromal cells (MSCs) improves islet function and revascularization in vivo. Pre-transplant islet culture is associated with the loss of islet cells. MSCs may enhance islet cell survival or function by direct cell contact mechanisms and soluble mediators. We investigated the capacity of MSCs to improve islet cell survival or β-cell function in vitro using direct and indirect contact islet-MSC configurations. We also investigated whether pre-culturing islets with MSCs improves islet transplantation outcome.MethodsThe effect of pre-culturing islets with MSCs on islet function in vitro was investigated by measuring glucose-stimulated insulin secretion. The endothelial cell density of fresh islets and islets cultured with or without MSCs was determined by immunohistochemistry. The efficacy of transplanted islets was tested in vivo using a syngeneic streptozotocin-diabetic minimal islet mass model. Graft function was investigated by monitoring blood glucose concentrations.ResultsIndirect islet-MSC co-culture configurations did not improve islet function in vitro. Pre-culturing islets using a direct contact MSC monolayer configuration improved glucose-stimulated insulin secretion in vitro, which correlated with superior islet graft function in vivo. MSC pre-culture had no effect on islet endothelial cell number in vitro or in vivo.ConclusionsPre-culturing islets with MSCs using a direct contact configuration maintains functional β-cell mass in vitro and the capacity of cultured islets to reverse hyperglycemia in diabetic mice.     

Participation of hemocyte proteinase in dissociation of the fat body on pupation of Sarcophaga peregrina (flesh fly)

In holometabolous insects, larval tissues are degraded on pupation. Previously, we established an in vitro system in which Sarcophaga fat body dissociates in the presence of pupal hemocytes, mimicking the situation in vivo (Kutata et al., J. Insect Physiol.35, 559–565, 1989). In this paper, we showed that chymostatin and phenylmethylsulfonyl fluoride prevented dissociation of fat body in this system. Moreover, of the various proteinases tested, only chymotrypsin degraded the fat body in vitro. These results suggested that a chymotrypsin-like proteinase of hemocytes participates in dissociation of the fat body at the early pupal stage.     

Using DNA mechanics to predict in vitro nucleosome positions and formation energies

In eukaryotic genomes, nucleosomes function to compact DNA and to regulate access to it both by simple physical occlusion and by providing the substrate for numerous covalent epigenetic tags. While competition with other DNA-binding factors and action of chromatin remodeling enzymes significantly affect nucleosome formation in vivo, nucleosome positions in vitro are determined by steric exclusion and sequence alone. We have developed a biophysical model, DNABEND, for the sequence dependence of DNA bending energies, and validated it against a collection of in vitro free energies of nucleosome formation and a set of in vitro nucleosome positions mapped at high resolution. We have also made a first ab initio prediction of nucleosomal DNA geometries, and checked its accuracy against the nucleosome crystal structure. We have used DNABEND to design both strong and weak histone- binding sequences, and measured the corresponding free energies of nucleosome formation. We find that DNABEND can successfully predict in vitro nucleosome positions and free energies, providing a physical explanation for the intrinsic sequence dependence of histone–DNA interactions.