A hyperparameter-tuning approach to automated inverse planning.

BACKGROUND: In current practice, radiotherapy inverse planning often requires treatment planners to modify multiple parameters in the treatment planning system's objective function to produce clinically acceptable plans. Due to the manual steps in this process, plan quality can vary depending on the planning time available and the planner's skills. PURPOSE: This study investigates the feasibility of two hyperparameter-tuning methods for automated inverse planning. Because this framework does not train a model on previously optimized plans, it can be readily adapted to practice pattern changes, and the resulting plan quality is not limited by that of a training cohort. METHOD: We retrospectively selected 10 patients who received lung stereotactic body radiation therapy using manually generated clinical plans. We implemented random sampling and Bayesian optimization to automatically tune objective function parameters using linear-quadratic utility functions based on 11 clinical goals. Normalizing all plans to deliver a minimum dose of 48 Gy to 95% of the planning target volume, we compared plan quality for the automatically generated plans to the manually generated plans. We also investigated the impact of iteration count on the automatically generated plans, comparing planning time and plan utility for randomized and Bayesian plans with and without stopping criteria. RESULTS: Without stopping criteria, the median planning time was 1.9 and 2.3 h for randomized and Bayesian plans, respectively. The organ-at-risk (OAR) doses in the randomized and Bayesian plans had a median percent difference (MPD) of 48.7% and 60.4% below clinical dose limits and an MPD of 2.8% and 3.3% below clinical plan doses. With stopping criteria, the utility decreased by an MPD of 5.3% and 3.9% for randomized and Bayesian plans, but the median planning time was reduced to 0.5 and 0.7 h, and the OAR doses still had an MPD of 42.9% and 49.7% below clinical dose limits and an MPD of 0.3% and 1.8% below clinical plan doses. CONCLUSIONS: This study demonstrates that hyperparameter-tuning approaches to automated inverse planning can reduce the treatment planner's active planning time with plan quality that is similar to or better than manually generated plans.

Genomic profiling of Acute lymphoblastic leukemia in ataxia telangiectasia patients reveals tight link between ATM mutations and chromothripsis.

Recent developments in sequencing technologies led to the discovery of a novel form of genomic instability, termed chromothripsis. This catastrophic genomic event, involved in tumorigenesis, is characterized by tens to hundreds of simultaneously acquired locally clustered rearrangements on one chromosome. We hypothesized that leukemias developing in individuals with Ataxia Telangiectasia, who are born with two mutated copies of the ATM gene, an essential guardian of genome stability, would show a higher prevalence of chromothripsis due to the associated defect in DNA double-strand break repair. Using whole-genome sequencing, fluorescence in situ hybridization and RNA sequencing, we characterized the genomic landscape of Acute Lymphoblastic Leukemia (ALL) arising in patients with Ataxia Telangiectasia. We detected a high frequency of chromothriptic events in these tumors, specifically on acrocentric chromosomes, as compared with tumors from individuals with other types of DNA repair syndromes (27 cases total, 10 with Ataxia Telangiectasia). Our data suggest that the genomic landscape of Ataxia Telangiectasia ALL is clearly distinct from that of sporadic ALL. Mechanistically, short telomeres and compromised DNA damage response in cells of Ataxia Telangiectasia patients may be linked with frequent chromothripsis. Furthermore, we show that ATM loss is associated with increased chromothripsis prevalence in additional tumor entities.

Hybrid responsive hydrogel carriers for oral delivery of low molecular weight therapeutic agents.

Hydrogels have been influential in the development of controlled release systems for a wide variety of therapeutic agents. These materials are attractive as carriers for transmucosal and intracellular drug delivery because of their inherent biocompatibility, tunable physicochemical properties, basic synthesis, and ability to be physiologically responsive. Due to their hydrophilic nature, hydrogel-based carrier systems are not always the best systems for delivery of small molecular weight, hydrophobic therapeutic agents. In this work, versatile hydrogel-based carriers composed of copolymers of methyl methacrylate (MMA) and acrylic acid (AA) were designed and synthesized to create formulations for oral delivery of small molecular weight therapeutic agents. Through practical material selection and careful design of copolymer composition and molecular architecture, we engineered systems capable of responding to physiological changes, with tunable physicochemical properties that are optimized to load, protect, and deliver their payloads to their intended site of action. The synthesized carriers' ability to respond to changes in pH, to load and release small molecular weight drugs, and biocompatibility were investigated. Our results suggest these hydrophilic networks have great potential for controlled delivery of small-molecular weight, hydrophobic and hydrophilic agents.

Mirion--a software package for automatic processing of mass spectrometric images.

Mass spectrometric imaging (MSI) techniques are of growing interest for the Life Sciences. In recent years, the development of new instruments employing ion sources that are tailored for spatial scanning allowed the acquisition of large data sets. A subsequent data processing, however, is still a bottleneck in the analytical process, as a manual data interpretation is impossible within a reasonable time frame. The transformation of mass spectrometric data into spatial distribution images of detected compounds turned out to be the most appropriate method to visualize the results of such scans, as humans are able to interpret images faster and easier than plain numbers. Image generation, thus, is a time-consuming and complex yet very efficient task. The free software package "Mirion," presented in this paper, allows the handling and analysis of data sets acquired by mass spectrometry imaging. Mirion can be used for image processing of MSI data obtained from many different sources, as it uses the HUPO-PSI-based standard data format imzML, which is implemented in the proprietary software of most of the mass spectrometer companies. Different graphical representations of the recorded data are available. Furthermore, automatic calculation and overlay of mass spectrometric images promotes direct comparison of different analytes for data evaluation. The program also includes tools for image processing and image analysis.

[Value of fall-risk tests for patients with rheumatoid arthritis]. / Wertigkeit von Sturzrisikotests bei Patienten mit rheumatoider Arthritis.

The aim of the study was to quickly and efficiently determine the risk of falling in patients with rheumatoid arthritis over the age of 46 with established methods, to discover parameters which influence the risk of falling and fractures. The study group consisted of 67 patients (median age 69±7.4 years, duration of disease <10 years 71%).With the help of the present data on fractures the performance of the chair-rising (CR) test, the timed up-and-go (TUG) test and the tandem stand (TS) test plus determination of the average daily and cumulative glucocorticoid (GC) dosage, it was possible to detect parameters which influence the risk of falling and fractures.Higher age (>60 years), overweight, deficits in muscle strength in the lower extremities and very low GC dosage (≤5 mg) were found to be associated with an increased risk of falling, which is accompanied by an increased risk of fractures.

Coherence during scattering of fast H atoms from a LiF(001) surface.

The coherence for diffraction effects during grazing scattering of fast hydrogen and helium atoms from a LiF(001) surface with energies up to some keV is investigated via the coincident detection of two-dimensional angular distributions for scattered projectiles with their energy loss. For keV H atoms, we identify electronic excitations of the target surface as the dominant mechanism for decoherence, whereas for He atoms this contribution is small. The suppression of electronic excitations owing to the band gap of insulators plays an essential role for preserving quantum coherence and thus for the application of fast atom diffraction as a surface analytical tool.

GlycoCT-a unifying sequence format for carbohydrates.

As part of the EUROCarbDB project (www.eurocarbdb.org) we have carefully analyzed the encoding capabilities of all existing carbohydrate sequence formats and the content of publically available structure databases. We have found that none of the existing structural encoding schemata are capable of coping with the full complexity to be expected for experimentally derived structural carbohydrate sequence data across all taxonomic sources. This gap motivated us to define an encoding scheme for complex carbohydrates, named GlycoCT, to overcome the current limitations. This new format is based on a connection table approach, instead of a linear encoding scheme, to describe the carbohydrate sequences, with a controlled vocabulary to name monosaccharides, adopting IUPAC rules to generate a consistent, machine-readable nomenclature. The format uses a block concept to describe frequently occurring special features of carbohydrate sequences like repeating units. It exists in two variants, a condensed form and a more verbose XML syntax. Sorting rules assure the uniqueness of the condensed form, thus making it suitable as a direct primary key for database applications, which rely on unique identifiers. GlycoCT encompasses the capabilities of the heterogeneous landscape of digital encoding schemata in glycomics and is thus a step forward on the way to a unified and broadly accepted sequence format in glycobioinformatics.

Retargeting of retroviral integration sites for the predictable expression of transgenes and the analysis of cis-acting sequences.

The transcriptional activity of transgenes in eukaryotic host cells critically depends on the sites of their integration where it is modulated by interactions between the promoter and surrounding chromatin structures. Retroviruses integrate their genome into chromosomal loci favoring expression from one long-terminal repeat (LTR). We have therefore developed a strategy in which retroviral vectors are provided with "tags", that is, targets for a site-specific recombinase (Flp). Presence of two 48 bp Flp recognition target (FRT) sequences permits the excision of a selection marker whereby the reading frame of a reporter gene (lacZ) is restored and beta-galactosidase activity can be monitored to characterize the integration site regarding the level and stability of expression. The location of the remaining FRT tag permits the subsequent Flp-mediated insertion of a truncated selection marker which is then expressed from the LTR. This step represents an authentic site-specific recombination event which can be demonstrated by a number of criteria, among these its reversibility in the presence of Flp activity. Thereby the "expression trap" principle permits the efficient recovery of stable insertion events, and if a gene of interest is linked to the truncated marker, the established properties of a given genomic site can be utilized for transcription studies or for the generation of highly expressing clones, even for biotechnological purposes.

A thymocyte factor SATB1 suppresses transcription of stably integrated matrix-attachment region-linked reporter genes.

SATB1 specifically recognizes and binds to specialized genomic regions with an ATC sequence context with high base-unpairing propensity. Such base-unpairing regions (BURs) are typically identified within nuclear scaffold- or matrix-attachment regions (S/MARs). SATB1 is a homeodomain protein and is predominantly expressed in thymocytes. We obtained BHK cell lines expressing low levels of SATB1 by stable transfection and investigated its effect on stably integrated MAR-linked SV40 enhancer/promoter-driven luciferase reporter genes. For this study, both naturally occurring and synthetic MARs, as well as an AT-rich non-MAR control, were tested. Previous studies demonstrated that MAR sequences augment transcription of the linked reporter luciferase gene. Here, we show that SATB1 dramatically reduces the high levels of MAR-linked luciferase gene transcription. Transcription was virtually abolished for a reporter gene surrounded by two MARs at the 5' and 3' ends of the gene, which otherwise confer the highest level of transcriptional augmentation. On the other hand, SATB1 did not affect expression of an AT-rich non-MAR-linked luciferase gene or of endogenous housekeeping genes. This study shows that SATB1 acts as a strong transcriptional suppressor on a reporter gene linked to MARs when it is stably integrated into chromatin.

Scaffold/matrix-attached regions act upon transcription in a context-dependent manner.

Scaffold/matrix-attached regions (S/MARs) are cis-acting elements with a function outside transcribed regions and in introns. Although they usually augment transcriptional rates, their action is highly context-dependent. We cloned an 800 bp S/MAR element from the upstream border of the human interferon-beta domain at various positions within a transcribed region of 4.3 kb. By use of retroviral gene transfer, the vector could be integrated into target cells as a single copy enabling a rigorous definition of the distance between the S/MAR and the transcriptional start site. At a distance of about 4 kb, the S/MAR supported transcriptional initiation, whereas at distances below 2.5 kb, transcription was essentially shut off. Controls proved the functionally of all constructs in the transient expression phase and ruled out any influence of S/MAR position on transcript stability. Moreover, no pausing or premature termination was observed within these elements. We suggest that the protein binding partners of S/MARs change according to the topological status, explaining these divergent S/MAR effects.

Anatomy of highly expressing chromosomal sites targeted by retroviral vectors.

The eukaryotic genome contains chromosomal loci with a high transcription-promoting potential. For their identification in cultured cells, transfer of a reporter gene has to be performed by a technique that grants the integration of individual copies. We have applied retroviral vectors in conjunction with inverse polymerase chain reaction techniques to reconstruct a number of these sites for a further characterization. Remarkably, all examples conform to the same design in that the process of retroviral infection selected a scaffold- or matrix-attached region (S/MAR) that was flanked by DNA with high bending potential. The S/MARs are of an unusual type in that they show a high incidence of certain dinucleotide repeats and the potential to act as topological sinks. The anatomy of retroviral integration sites reveals principles that can be exploited for the development of predictable transgenic systems on the basis of expression and targeting vectors.

Permeability of the blood-retinal barrier and the blood-aqueous barrier in type I diabetes without diabetic retinopathy: simultaneous evaluation with fluorophotometry.

For the evaluation of a possible malfunction of the blood-retinal barrier (BRB) and the blood-aqueous barrier (BAB) in type I diabetes without manifest angiopathy after i.v. injection of sodium fluorescein, the permeability of the BRB (P) and the permeability coefficient of the BAB [P(a)] were simultaneously determined by fluorophotometry in 34 eyes of 34 type-I diabetics [hemoglobin (Hb)A1c = 6.6% +/- 0.9%] without retinopathy whose age ranged from 19 to 38 years (mean, 30.5 +/- 5 years); the diabetes duration was between 5 and 18 years. Fluorescein angiography was performed to exclude nonperfused areas. In all, 34 eyes of 34 healthy volunteers whose age ranged between 23 and 34 years (mean, 28.5 +/- 3.3 years) served as controls; in this group, fluorophotometry was performed twice to evaluate reproducibility. The mean BAB permeability coefficient in diabetics [P(a) = 5.3 +/- 1.8 x 10(-4)/min] was significantly increased (P = 0.00003) as compared with control values [P(a) = 3.7 +/- 0.7 x 10(-4)/min]; BRB permeability in diabetics (P = 3.2 +/- 1.4 x 10(-7) cm/s) was raised, with this elevation being of lower significance (P = 0.019; controls, P = 2.6 +/- 0.7 x 10(-7) cm/s). We found a decrease in BRB permeability depending on diabetes duration (r = -0.15; P = 0.007) that was not significant in the BAB (r = -0.1; P = 0.24). No correlation was found to exist between permeability and hemoglobin (Hb)A1c values either in the BAB or in the BRB. The reproducibility in controls was 9% in BRB determinations and 12% in BAB measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

Scaffold-attached regions (SAR elements) mediate transcriptional effects due to butyrate.

The expression of certain genes has been reported to respond positively to sodium butyrate. This study demonstrates the same feature for two marker genes under the control of five different promoters. In all examples, the stimulatory effect is largest if one or especially two scaffold/matrix-attached regions (SAR/MAR elements) are present adjacent to the gene, and in one case, the stimulation depends entirely upon this situation. These results are observed with several SAR sequences including those obtained by oligomerizing short stretches of DNA surrounding a core motif. It is suggested that butyrate exerts important actions at the level of the chromatin structure.

Scaffold-attached regions from the human interferon beta domain can be used to enhance the stable expression of genes under the control of various promoters.

We have transfected DNA corresponding to the complete chromatin domain of human interferon beta (huIFN-beta) gene into mouse L cells. In this construct, which is flanked by scaffold-attached regions (SARs), the gene's transcription was enhanced 20-30-fold with respect to DNAs containing only the immediate regulatory elements. To elucidate the role of SAR elements in the transcriptional enhancement, their position was varied relative to several artificial promoter-gene combinations. It was found that SARs enhance general promoter functions in an orientation- and partially distance-independent manner; their effect is restricted to the integrated state of transfected templates. During the phase of transient expression, SAR elements were generally found to have an antagonizing effect.

[Comparative methodologic studies of closing volume and increase in airway resistance volumes]. / Vergleichende methodologische Untersuchungen über das Closing Volume und das Strömungswiderstandsanstiegsvolumen.

The early recognition of obstructive lung diseases plays an important role in the subsequent therapy; the closing volume, determined by means of single-breath oxygen test has been established as a suitable parameter. This technique, however, has not succeeded as a routine method due to prohibitive costs. In a clinical study the closing volume and the easier estimatable so-called flow-resistance elevation volume (Ros-volume-curve parameter obtained from oscilloresistometry/volumetry) were compared and a correlation sought. It could be concluded, that for characterization of small airways diseases the closing volume is a more suitable parameter than the flow-resistance elevation volume.

Chromatin domain surrounding the human interferon-beta gene as defined by scaffold-attached regions.

Regions attached to the nuclear scaffold have been traced after transfecting a 36-kilobase (kb) piece of DNA, surrounding the human interferon-beta gene, into mouse L-cells. An extended attached region starts 1.7 kb upstream from the gene and a moderate binding site immediately downstream. These findings could be confirmed by reconstitution experiments in vitro which predict another scaffold-attached region (SAR) starting 12 kb downstream from the gene. Since no other transcripts originate from DNA between the major SARs, these elements could be involved in interferon gene regulation.

Chromatin structure and induction-dependent conformational changes of human interferon-beta genes in a mouse host cell.

Multiple copies of a human interferon-beta gene introduced into a mouse host cell line can be activated by induction with double-stranded RNA. Several induction-dependent changes of the chromatin structure could be traced by mapping techniques using four different agents [DNase I, micrococcus nuclease, bromoacetaldehyde and methidiumpropyl-EDTA X iron(II)]. Our data show that all copies of the interferon gene have adopted a very similar conformation in the host cell and respond to the inducing stimulus in a highly synchronous fashion. Detailed induction-specific changes were observed best with the chemical reagents which disclose a specific hypersensitive site within a sequence that has been shown to be required for the induction process (around position -80) and three other regions which, in addition to the transcribed region itself, gain single-strand character by an auxiliary process which can be mimicked by the addition of butyrate to the medium and may therefore involve histone hyperacetylation. Six discrete 'phased' nucleosomes are present upstream from the gene and are modulated by induction. At least four nucleosomes are located downstream. The interferon genes are largely protected from micrococcus nuclease in the inactive state. Gene activation increases access to micrococcus nuclease and DNase I indicating gross conformational changes on a higher level of chromatin structure.

Links between effects of butyrate on histone hyperacetylation and regulation of interferon synthesis in Namalva and FS-4 cell lines.

The human Namalva lymphoma cell line being an established producer, predominantly of alpha interferon, has been reported to enhance interferon synthesis after a preincubation in butyrate containing media. We have performed the corresponding experiments with FS-4 fibroblast cells and show that the synthesis of human beta interferon is adversely affected by this treatment. Both cell types are hyperacetylated by the fatty acid to a comparable extent. However, after the withdrawal of butyrate, the persistence of highly acetylated forms of histone H4 is insufficient in the case of FS-4 to endure the interferon induction period. Concerning fibroblasts, deacetylation proceeds to a hypoacetylated state which is reversed only slowly. With lymphoid cells on the other hand, acetylated H4 specimens are much more stable and persist for more than eight hours in the absence of butyrate. Moreover, the acetylation reactions are supported by other Friend cell stimulators which by themselves are no inhibitors of deacetylase activities.