bluuebunny/crossref_metadata_2025 huggingface dataset:
AUGUSTUS at EGASP: using EST, protein and genomic alignments for improved gene prediction in the human genome
Authors: Stanke Mario , Tzvetkova Ana , Morgenstern Burkhard
Published: August 2006

Background

A large number of gene prediction programs for the human genome exist. These annotation tools use a variety of methods and data sources. In the recent ENCODE genome annotation assessment project (EGASP), some of the most commonly used and recently developed gene-prediction programs were systematically evaluated and compared on test data from the human genome. AUGUSTUS was among the tools that were tested in this project.

Results

AUGUSTUS can be used as an ab initio program, that is, as a program that uses only one single genomic sequence as input information. In addition, it is able to combine information from the genomic sequence under study with external hints from various sources of information. For EGASP, we used genomic sequence alignments as well as alignments to expressed sequence tags (ESTs) and protein sequences as additional sources of information. Within the category of ab initio programs AUGUSTUS predicted significantly more genes correctly than any other ab initio program. At the same time it predicted the smallest number of false positive genes and the smallest number of false positive exons among all ab initio programs. The accuracy of AUGUSTUS could be further improved when additional extrinsic data, such as alignments to EST, protein and/or genomic sequences, was taken into account.

Conclusion

AUGUSTUS turned out to be the most accurate ab initio gene finder among the tested tools. Moreover it is very flexible because it can take information from several sources simultaneously into consideration.
A computational approach for identifying pseudogenes in the ENCODE regions
Authors: Zheng Deyou , Gerstein Mark B
Published: August 2006

Background

Pseudogenes are inheritable genetic elements showing sequence similarity to functional genes but with deleterious mutations. We describe a computational pipeline for identifying them, which in contrast to previous work explicitly uses intron-exon structure in parent genes to classify pseudogenes. We require alignments between duplicated pseudogenes and their parents to span intron-exon junctions, and this can be used to distinguish between true duplicated and processed pseudogenes (with insertions).

Results

Applying our approach to the ENCODE regions, we identify about 160 pseudogenes, 10% of which have clear 'intron-exon' structure and are thus likely generated from recent duplications.

Conclusion

Detailed examination of our results and comparison of our annotation with the GENCODE reference annotation demonstrate that our computation pipeline provides a good balance between identifying all pseudogenes and delineating the precise structure of duplicated genes.
Exogean: a framework for annotating protein-coding genes in eukaryotic genomic DNA
Authors: Djebali Sarah , Delaplace Franck , Crollius Hugues Roest
Published: August 2006

Background

Accurate and automatic gene identification in eukaryotic genomic DNA is more than ever of crucial importance to efficiently exploit the large volume of assembled genome sequences available to the community. Automatic methods have always been considered less reliable than human expertise. This is illustrated in the EGASP project, where reference annotations against which all automatic methods are measured are generated by human annotators and experimentally verified. We hypothesized that replicating the accuracy of human annotators in an automatic method could be achieved by formalizing the rules and decisions that they use, in a mathematical formalism.

Results

We have developed Exogean, a flexible framework based on directed acyclic colored multigraphs (DACMs) that can represent biological objects (for example, mRNA, ESTs, protein alignments, exons) and relationships between them. Graphs are analyzed to process the information according to rules that replicate those used by human annotators. Simple individual starting objects given as input to Exogean are thus combined and synthesized into complex objects such as protein coding transcripts.

Conclusion

We show here, in the context of the EGASP project, that Exogean is currently the method that best reproduces protein coding gene annotations from human experts, in terms of identifying at least one exact coding sequence per gene. We discuss current limitations of the method and several avenues for improvement.
Comparison of the Medical Priority Dispatch System to an Out‐of‐hospital Patient Acuity Score
Authors: Feldman Michael J. , Verbeek P. Richard , Lyons David G. , Chad Sandra J. , Craig Alan M. , Schwartz Brian
Published: August 2006

Background:
Although the Medical Priority Dispatch System (MPDS) is widely used by emergency medical services (EMS) dispatchers to determine dispatch priority, there is little evidence that it reflects patient acuity. The Canadian Triage and Acuity Scale (CTAS) is a standard patient acuity scale widely used by Canadian emergency departments and EMS systems to prioritize patient care requirements.

Objectives:
To determine the relationship between MPDS dispatch priority and out‐of‐hospital CTAS.

Methods:
All emergency calls on a large urban EMS communications database for a one‐year period were obtained. Duplicate calls, nonemergency transfers, and canceled calls were excluded. Sensitivity and specificity to detect high‐acuity illness, as well as positive predictive value (PPV) and negative predictive value (NPV), were calculated for all protocols.

Results:
Of 197,882 calls, 102,582 met inclusion criteria. The overall sensitivity of MPDS was 68.2% (95% confidence interval [CI] = 67.8% to 68.5%), with a specificity of 66.2% (95% CI = 65.7% to 66.7%). The most sensitive protocol for detecting high acuity of illness was the breathing‐problem protocol, with a sensitivity of 100.0% (95% CI = 99.9% to 100.0%), whereas the most specific protocol was the one for psychiatric problems, with a specificity of 98.1% (95% CI = 97.5% to 98.7%). The cardiac‐arrest protocol had the highest PPV (92.6%, 95% CI = 90.3% to 94.3%), whereas the convulsions protocol had the highest NPV (85.9%, 95% CI = 84.5% to 87.2%). The best‐performing protocol overall was the cardiac‐arrest protocol, and the protocol with the overall poorest performance was the one for unknown problems. Sixteen of the 32 protocols performed no better than chance alone at identifying high‐acuity patients.

Conclusions:
The Medical Priority Dispatch System exhibits at least moderate sensitivity and specificity for detecting high acuity of illness or injury. This performance analysis may be used to identify target protocols for future improvements.
Low abundance of telomerase in yeast: Implications for telomerase haploinsufficiency
Authors: Mozdy Amy D. , Cech Thomas R.
Published: August 2006

Telomerase is an RNA-dependent reverse transcriptase that maintains telomeric DNA at a species-specific equilibrium length. To determine an upper limit for the number of telomerase molecules in a Saccharomyces cerevisiae cell, we have established real-time RT-PCR assays to quantify the noncoding telomerase RNA, TLC1. We find that the number of TLC1 molecules in a haploid yeast cell is ∼29, less than the number of chromosome ends (64) in late S-phase. Wild-type diploid cells contain ∼37 telomerase RNAs, while diploids heterozygous for a null tlc1 allele have half the wild-type amount, ∼19 TLC1 molecules. For comparison, there are ∼480 molecules of the U2 snRNA per haploid cell. We show that a biological consequence of this low level of telomerase is haploinsufficiency: A TLC1/tlc1Δ heterozygote maintains shorter telomeres. A dominant-negative telomerase RNA, with a deletion of the template for telomeric DNA synthesis, further demonstrates that yeast telomere length is sensitive to telomerase dosage. Sixfold overexpression of tlc1Δtemplate establishes a new telomere length set point, ∼160 bp shorter than wild type. Removing telomerase protein-interaction sites from the tlc1Δtemplate RNA mitigates the dominant-negative effect, suggesting that the tlc1Δtemplate RNA competes with wild-type TLC1 for a limited supply of telomerase proteins or for telomeres. Because yeast telomerase is tethered at chromosome ends, the finding that it may be outnumbered by its telomeric DNA substrates provides a new perspective for interpreting the results of telomere maintenance studies.
Hybrid E. coli—Mitochondrial ribonuclease P RNAs are catalytically active
Authors: Seif Elias , Cadieux Alexandre , Lang B. Franz
Published: August 2006

RNase P is a ribonucleoprotein that cleaves tRNA precursors at their 5′-end. Mitochondrion-encoded RNA subunits of mitochondrial RNase P (mtP-RNA) have been identified in jakobid flagellates such as Reclinomonas americana, in the prasinophyte alga Nephroselmis olivacea, and in several ascomycete and zygomycete fungi. While the structures of ascomycete mtP-RNAs are highly reduced, those of jakobids, prasinophytes, and zygomycetes retain most conserved features of their bacterial counterparts. Therefore, these mtP-RNAs might be active in vitro in the absence of a protein subunit, as are bacterial P-RNAs. Here we present a comparative structural analysis including seven newly characterized jakobid mtP-RNAs. We investigate ribozyme activities of mtP-RNAs and find that even the most bacteria-like molecules of jakobids are inactive in vitro. However, when certain domains of jakobid and N. olivacea mtP-RNAs are replaced with those from Escherichia coli, these hybrid RNAs show catalytic activity. In vitro mutagenesis of these hybrid mtP-RNAs shows that various structural elements play a critical role in ribozyme catalysis and provide further support for the presence of these elements in mtP-RNAs. These include GNRA tetraloops in helix P14 and P18 of Jakoba libera, and a remnant P3 pairing in Seculamonas ecuadoriensis. Finally, we will discuss reasons for the failure of mtP-RNAs to show catalytic activity in the absence of P-proteins based on our mutagenesis analysis.
FIR Spectra of [Ni(H2O)6](ClO4)2 and [Ni(D2O)6](ClO4)2 at Various Temperatures
Authors: Rachwalska M. , Ściesińska E. , Ściesiński J.
Published: August 2006

Low frequency vibrations in [Ni(H2O)6](ClO4)2 and [Ni(D2O)6](ClO4)2 were measured by the FIR Fourier Transform Spectroscopy method. They were compared with those obtained by the IINS method. No evidence of proton glass discovered by the IINS was observed via the FIR method. By applying the band contour method, the barrier to rotation U
act for H2O groups in [Ni(H2O)6](ClO4)2 crystals was determined. A III–II phase transition mechanism was proposed.
Kinetics and Dynamics of Multi-Channel Unimolecular Reactions of Alkoxyl Radicals Over an Extended Range of Temperature and Pressure. A Combined Quantum Chemical/RRKM Dynamical Study
Authors: Somnitz Holger , Zellner Reinhard
Published: August 2006

Rate coefficients and branching ratios for unimolecular reactions of alkoxyl radicals in the temperature range 200 to 1500 K and in the pressure range 10−2 to 104 bar are presented. Special emphasis is given to the change of the relative importance of different reaction channels. This is evident for longer chain alkoxyl radicals which at lower temperatures undergo isomerisation via an energetically favourable six-member ring transition state. At temperatures above >600 K, however, the rates of the alternative decomposition pathways become faster than that of isomerisation.

The theoretical approach to derive unimolecular rate coefficients makes use of extensive (G2/G3) ab initio calculations. These are used as input data for RRKM calculations to which a tunnelling probability of the generalised Eckart type has been included on the microcanonical level. A subsequent multi-channel master equation (ME) treatment yields thermal rate coefficients and complete fall-off curves for arbitrary pressure and temperature conditions.

The kinetics of two linear chain alkoxyl radicals, 1-butoxyl and 2-pentoxyl, have been analysed to show the expected complex temperature and pressure behaviour resulting from the competition of multiple accessible reaction channels, each with its specific p,T-dependence. Additional results are reported for the minimum temperatures where channel switching can occur, the trajectory of these characteristic points on the p,T-surface, and the temperature dependence of the fractional ratios of the unimolecular reaction channels and the bimolecular reaction with oxygen at ambient atmospheric pressure.
Churg-Strauss Syndrome in Children: A Clinical and Pathologic Review
Authors: Boyer Debra , Vargas Sara O. , Slattery Dubhfeasa , Rivera-Sanchez Yadira M. , Colin Andrew A.
Published: August 2006

Churg-Strauss syndrome is a vasculitis accompanied by asthma and eosinophilia. It is generally considered a disease of adults; occurrence in children has been reported infrequently. Here we report 2 pediatric patients with Churg-Strauss syndrome manifesting with prominent pulmonary involvement. One, a 16-year-old with a previous history of asthma, presented with pleuritic chest pain and a peripheral pulmonary nodule complicated by an eosinophilic pleural effusion. The other patient presented at age 6 with cough, weight loss, and radiographic infiltrates. Lung biopsies revealed elements characteristic of Churg-Strauss syndrome, including eosinophilic microabscesses and vasculitis. Three- and 5-year follow-up showed continued symptoms in both patients despite medical therapy. Both patients illustrate many of the typical features of Churg-Strauss syndrome. We report these cases to expand the scant knowledge about Churg-Strauss syndrome in pediatric patients and to heighten awareness that this serious disease may affect the pediatric population. The relevant literature on Churg-Strauss syndrome, with specific reference to childhood cases, is reviewed.
Cognitive Function and Behavior of Children With Adenotonsillar Hypertrophy Suspected of Having Obstructive Sleep-Disordered Breathing
Authors: Suratt Paul M. , Peruggia Mario , D'Andrea Lynn , Diamond Robert , Barth Jeffrey T. , Nikova Margarita , Perriello Vito A. , Johnson Michael L.
Published: August 2006

OBJECTIVE. The purpose of this study was to determine whether risks of impaired cognitive function could be predicted for children or groups of children with adenotonsillar hypertrophy who were suspected of having obstructive sleep-disordered breathing, from historical and polysomnographic variables used separately or in combination.

METHODS. We studied 114 consecutive 6- to 12-year-old children with adenotonsillar hypertrophy, who were referred because of suspected obstructive sleep-disordered breathing, with questionnaires, assessment of tonsil size, general and memory cognitive tests, and attended polysomnography with the use of nasal pressure recording to detect flow.

RESULTS. There were important significant relationships between snore group (snored every night versus less often), sleep efficiency, and race and 2 of 3 general cognitive tests (vocabulary and similarities). Significant but weaker relationships were observed between sleep latency and 2 memory indices (verbal memory and general memory) and between sleep efficiency and 2 behavior indices (attention-deficit/hyperactivity disorder summary and hyperactive-impulsive summary). The number of episodes of apnea and hypopnea per 1 hour of sleep predicted the vocabulary score as well as did the snore group, but it did not predict other tests as well as other variables. Tonsil size did not predict any cognitive or behavior score. Confidence intervals for group means were small, whereas prediction intervals for individual children were large.

CONCLUSIONS. Risk of impaired cognitive function and behavior can be predicted from snoring history, sleep efficiency, sleep latency, and race but not tonsil size. The combination of snoring history and polysomnographic variables predicted impaired cognitive scores better than did either alone. The snoring history predicted more test scores than the number of episodes of apnea and hypopnea per 1 hour of sleep.
Weakly periodic-like rings and commutativity
Authors: Yaqub Adil
Published: August 2006

A ring R is called periodic if, for every x in R, there exist distinct positive integers m and n such that xm=xn. An element x is called potent if xk=x for some integer k≯1. A ring R is called weakly periodic if every x in R can bewritten in the form x=a+b for some nilpotent element a and some potent element b. A ring R is called weakly periodic-like if every x in R which is not in the center of R can be written in the form x=a+b, where a is nilpotent and b is potent. Our objective is to study the structure of weakly periodic-like rings, with particular emphasis on conditions which yield such rings commutative, or conditions which render the nilpotents N as an ideal of R and R/N as commutative.
A Korovkin type approximation theorem in statistical sense
Authors: Erkuş Esra , Duman Oktay
Published: August 2006

In this study, using the concept of A-statistical convergence we investigate a Korovkin type approximation result for a sequence of positive linear operators defined on the space of all continuous real valued functions on any compact subset of the real m-dimensional space.
Distributed attitude alignment in spacecraft formation flying
Authors: Ren Wei
Published: August 2006

In this paper, we consider a distributed attitude alignment problem for a team of deep space formation flying spacecraft through local information exchange. We propose control laws for three different cases. In the first case, multiple spacecraft converge to their (possibly time‐varying) desired attitudes while maintaining the same attitude or given relative attitudes during formation maneuvers under an undirected communication graph. In the second case, multiple spacecraft converge to the same rotation rate while aligning their attitudes during formation maneuvers under an undirected communication graph. In the third case, attitude alignment under a directed information exchange graph is addressed. Simulation results for attitude alignment among six spacecraft demonstrate the effectiveness of our approach. Copyright © 2006 John Wiley & Sons, Ltd.
Synthesis of Nearly Monodisperse Iron Oxide and Oxyhydroxide Nanocrystals
Authors: Liang X. , Wang X. , Zhuang J. , Chen Y. , Wang D. , Li Y.
Published: August 2006

Uniform magnetite, hematite, and goethite nanocrystals were prepared through an attractive method based on an oleic acid/alcohol/water system. By adjusting the synthetic parameters (base concentration, alcohol content, categories of alcohols, etc.), the controlled synthesis of uniform magnetite, hematite, and goethite nanocrystals can be easily achieved. Detailed investigations on the effect of the experimental parameters on the morphology of the final products and the phase transitions among the magnetite, hematite, and goethite phases were carried out. Finally, a method of doping other metal ions into magnetite was developed and the magnetic properties of magnetite doped with different metal elements were studied.
A pH‐Responsive Gating Membrane System with Pumping Effects for Improved Controlled Release
Authors: Qu J.‐B. , Chu L.‐Y. , Yang M. , Xie R. , Hu L. , Chen W.‐M.
Published: August 2006

In this study, we report on a novel composite membrane system for pH‐responsive controlled release, which is composed of a porous membrane with linear grafted, positively pH‐responsive polymeric gates acting as functional valves, and a crosslinked, negatively pH‐responsive hydrogel inside the reservoir working as a functional pumping element. The proposed system features a large responsive release rate that goes effectively beyond the limit of concentration‐driven diffusion due to the pumping effects of the negatively pH‐responsive hydrogel inside the reservoir. The pH‐responsive gating membranes were prepared by grafting poly(methacrylic acid) (PMAA) linear chains onto porous polyvinylidene fluoride (PVDF) membrane substrates using a plasma‐graft pore‐filling polymerization, and the crosslinked poly(N,N‐dimethylaminoethyl methacrylate) (PDM) hydrogels were synthesized by free radical polymerization. The volume phase‐transition characteristics of PMAA and PDM were opposite. The proposed system opens new doors for pH‐responsive “smart” or “intelligent” controlled‐release systems, which are highly attractive for drug‐delivery systems, chemical carriers, sensors, and so on.
On the use of detailed models in the MPC algorithm: The pressure‐swing distillation case
Authors: Fissore Davide , Pin Marco , Barresi Antonello A.
Published: August 2006

A novel approach to the design of a model predictive control (MPC) algorithm for a complex plant (with energy integration and mass recycles) is given. Sensitivity analysis and steady‐state optimization are used to determine the manipulated variables that have the strongest influence on the objective function of the operation. This allows a reduction of the number of variables that are optimized on‐line, as well as the use of detailed, first‐principle–based models in the MPC algorithm, thus resulting in more reliable predictions. Moreover, the same algorithm can be used to control plants of different size, without the need of a new calibration of the parameters of the model. The application of this procedure to a pressure‐swing distillation unit is given as an example. © 2006 American Institute of Chemical Engineers AIChE J, 2006
Intraskeletal variability in bone mass
Authors: Peck Joshua J. , Stout Sam D.
Published: August 2006

For methodological or other reasons, a variety of skeletal elements are analyzed and subsequently used as a basis for describing general bone loss and mass. However, bone loss and mass may not be uniform within and among skeletal elements of the same individual because of biomechanical factors. We test the hypothesis that a homogeneity in bone mass exists among skeletal elements of the same individual. Measures indicative of bone mass were calculated from the midshafts of six skeletal elements from the same individuals (N = 41). The extent of intraskeletal variability in bone mass (relative cortical area) was then examined for the entire sample, according to age, sex, and pathological status. The results of the analysis showed that all measures reflect a heterogeneity in bone mass (P ≤ 0.001). Specifically, differences were observed between the bones of the upper limb and those of the lower limb. Both sexes showed intraskeletal variability in bone mass, but the difference between the sexes is not significant (P = 0.509). When the sample is subdivided according to age, all groups show intraskeletal variability in bone mass, but the difference did not differ significantly among the groups (P = 0.217). However, significant differences in intraskeletal variability are observed between individuals below and above the age of 50. Pathological individuals show intraskeletal variability in bone mass, but the difference between the pathological and non‐pathological groups is not significant (P = 0.095). These results indicate that the bone mass of any particular skeletal element is intricately tethered to its specific mechanical loading environment. Am J Phys Anthropol, 2006. © 2006 Wiley‐Liss, Inc.
Ruthenium‐Catalyzed Oxidation of Alkenes, Alkynes, and Alcohols to Organic Acids with Aqueous Hydrogen Peroxide
Authors: Che Chi‐Ming , Yip Wing‐Ping , Yu Wing‐Yiu
Published: August 2006

A protocol that adopts aqueous hydrogen peroxide as a terminal oxidant and [(Me3tacn)(CF3CO2)2RuIII(OH2)]CF3CO2 (1; Me3tacn=1,4,7‐trimethyl‐1,4,7‐triazacyclononane) as a catalyst for oxidation of alkenes, alkynes, and alcohols to organic acids in over 80 % yield is presented. For the oxidation of cyclohexene to adipic acid, the loading of 1 can be lowered to 0.1 mol %. On the one‐mole scale, the oxidation of cyclohexene, cyclooctene, and 1‐octanol with 1 mol % of 1 produced adipic acid (124 g, 85 % yield), suberic acid (158 g, 91 % yield), and 1‐octanoic acid (129 g, 90 % yield), respectively. The oxidative CC bond‐cleavage reaction proceeded through the formation of cis‐ and trans‐diol intermediates, which were further oxidized to carboxylic acids via CC bond cleavage.
Highly Enantiomerically Enriched Planar Chiral Naphthalene Tricarbonylchromium Complexes
Authors: Cumming Graham R. , Bernardinelli Gérald , Kündig E. Peter
Published: August 2006

Lithiation/electrophile trapping reactions were carried out with the highly enantiomerically enriched complex [Cr(5‐bromonaphthalene)(CO)3]. Electrophile quenching with ClPPh2, PhCHO, and (Me3SiO)2 afforded the enantiomerically enriched (>97 % ee) planar chiral 5‐substituted naphthalene complexes with PPh2, CH(Ph)OH, and OH substituents, respectively. Very mild Pd‐catalyzed Suzuki–Miyaura cross‐coupling reactions were developed and applied to the highly labile [Cr(5‐bromonaphthalene)(CO)3] to give nine new planar chiral aryl‐, heteroaryl‐, alkynyl‐, and alkenylnaphthalene chromium complexes with high enantiomeric purity. The efficient ambient‐temperature coupling reactions with borinates prepared in situ were also applied to a number of chlorobenzene complexes and to aryl and vinyl halides.
Confirmatory Seamless Phase II/III Clinical Trials with Hypotheses Selection at Interim: Applications and Practical Considerations
Authors: Schmidli Heinz , Bretz Frank , Racine Amy , Maurer Willi
Published: August 2006

Adaptive seamless phase II/III designs combine a phase II and a phase III study into one single confirmatory clinical trial. Several examples of such designs are presented, where the primary endpoint is binary, time‐to‐event or continuous. The interim adaptations considered include the selection of treatments and the selection of hypotheses related to a pre‐specified subgroup of patients. Practical aspects concerning the planning and implementation of adaptive seamless confirmatory studies are also discussed. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
An Overview of Statistical Approaches for Adaptive Designs and Design Modifications
Authors: Schäfer Helmut , Timmesfeld Nina , Müller Hans‐Helge
Published: August 2006

With adaptive design methods for clinical trials, design elements such as sample size or further interim sample sizes may be changed during the course of the trial depending on all previously collected data. The focus of the overview is on group sequential approaches where the types of adaptations need not be specified in advance. An overview of the different statistical approaches for adaptive design methods proposed in the literature is given, relations between these methods are described and some perspectives of application and for future research are discussed. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Opening the Adaptive Toolbox
Authors: Wittes Janet , Lachenbruch Peter A.
Published: August 2006

This is a discussion of the following three papers appearing in this special issue on adaptive designs: ‘A regulatory view on adaptive/flexible clinical trial design’ by H. M. James Hung, Robert T. O'Neill, Sue‐Jane Wang and John Lawrence; ‘Confirmatory clinical trials with an adaptive design’ by Armin Koch; and ‘FDA's critical path initiative: A perspective on contributions of biostatistics’ by Robert T. O'Neill. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Some Aspects of the Application of Internal Pilot Studies
Authors: Lachenbruch Peter A. , Wittes Janet
Published: August 2006

This is a discussion of the paper' Sample size recalculation in internal pilot study designs' by Tim Friede and Meinhard Kieser, appearing in this special issue on adaptive designs. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Methodological Developments vs. Regulatory Requirements
Authors: Bauer Peter
Published: August 2006

This is a discussion of the following three papers appearing in this special issue on adaptive designs: ‘FDA’s critical path initiative: A perspective on contributions of biostatistics' by Robert T. O'Neill, ‘A regulatory view on adaptive/flexible clinical trial design’ by H. M. James Hung, Robert T. O'Neill, Sue‐Jane Wang and John Lawrence; and ‘Confirmatory clinical trials with an adaptive design’ by Armin Koch. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Adaptive Designs from the Viewpoint of an Academic Biostatistician
Authors: Schäfer Helmut
Published: August 2006

This is a discussion of the following three papers appearing in this special issue on adaptive designs: ‘FDA's critical path initiative: A perspective on contributions of biostatistics’ by Robert T. O'Neill, ‘A regulatory view on adaptive/flexible clinical trial design’ by H. M. James Hung, Robert T. O'Neill, Sue‐Jane Wang and John Lawrence; and ‘Confirmatory clinical trials with an adaptive design’ by Armin Koch. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
A Consultant's Perspective on the Regulatory Hurdles to Adaptive Trials
Authors: Mehta Cyrus R. , Jemiai Yannis
Published: August 2006

This is a discussion of the following two papers appearing in this special issue on adaptive designs: ‘A regulatory view on adaptive/flexible clinical trial design’ by H. M. James Hung, Robert T. O'Neill, Sue‐Jane Wang and John Lawrence and ‘Confirmatory clinical trials with an adaptive design’ by Armin Koch. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Challenges in Implementing Adaptive Designs: Comments on the Viewpoints Expressed by Regulatory Statisticians
Authors: Gallo Paul , Maurer Willi
Published: August 2006

This is a discussion of the following three papers appearing in this special issue on adaptive designs: ‘FDA's critical path initiative: A perspective on contributions of biostatistics’ by Robert T. O'Neill; ‘A regulatory view on adaptive/flexible clinical trial design’ by H. M. James Hung, Robert T. O'Neill, Sue‐Jane Wang and John Lawrence; and ‘Confirmatory clinical trials with an adaptive design’ by Armin Koch. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Mapping and partial characterization of proteases expressed by a CHO production cell line
Authors: Sandberg H. , Lütkemeyer D. , Kuprin S. , Wrangel M. , Almstedt A. , Persson P. , Ek V. , Mikaelsson M.
Published: August 2006

The proteolytic activities expressed by a Chinese hamster ovary (CHO) cell line in the cultivation supernatant during the production of recombinant factor VIII were mapped with a broad spectrum protease assay and a series of different types of protease inhibitors. The destabilizing effect on the product emanated from a metalloproteinase, which could be effectively blocked by chelating agents to lead to product stabilization. Amino acid sequences of the isolated metalloproteinase were found to have sequence homology with matrix metalloproteinases (MMPs) MMP3, MMP10, and MMP12. Several species with metalloproteinase activity were characterized and found to be related to each other. The results indicate that an MMP pro‐enzyme of ≥200 kDa was released from the CHO cells during the production phase. The enzyme expressed collagenase/gelatinase activity when activated. Due to autoproteolysis, a number of smaller, less specific MMPs were formed with the smallest form, a 19.4 kDa protein, being the most active. These results may be of particular relevance for other production processes using CHO cells for the expression of recombinant proteins. © 2006 Wiley Periodicals, Inc.
Derivation and characterization of cholesterol‐independent non‐GS NS0 cell lines for production of recombinant antibodies
Authors: Hartman Taymar E. , Sar Nalin , Genereux Kimberly , Barritt Diana S. , He Yimin , Burky John E. , Wesson Mark C. , Tso J. Yun , Tsurushita Naoya , Zhou Weichang , Sauer Paul W.
Published: August 2006

Presented is an antibody production platform based on the fed‐batch culture of recombinant NS0‐derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein‐free, cholesterol‐free medium. The resulting host cell line was designated NS0‐PFCF (protein‐free, cholesterol‐free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0‐PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine‐guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19–64% increase in antibody productivity when four mother–daughter cell pairs were cultured in a fed‐batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0‐derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed‐batch bioreactor process, consistently achieving an average specific productivity of 20–60 pg/cell‐day, and a volumetric productivity exceeding 120 mg/L‐day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS‐NS0), these NS0 cells are cholesterol‐independent, grow well in a protein‐free medium, use a non‐proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies. Biotechnol. Bioeng. 2007;96: 294–306. © 2006 Wiley Periodicals, Inc.
Thermally induced gelable polymer networks for living cell encapsulation
Authors: Lu Hong‐Fang , Targonsky Elisha D. , Wheeler Michael B. , Cheng Yu‐Ling
Published: August 2006

We report the encapsulation of MIN6 cells, a pancreatic β‐cell line, using thermally induced gelable materials. This strategy uses aqueous solvent and mild temperatures during encapsulation, thereby minimizing adverse effects on cell function and viability. Using a 2:1 mixture of PNIPAAm‐PEG‐PNIPAAm tri‐block copolymer and PNIPAAm homopolymer that exhibit reversible sol‐to‐gel transition at ∼30°C, gels were formed that exhibit mechanical integrity, and are stable in H2O, PBS and complete DMEM with negligible mass loss at 37°C for 60 days. MTT assays showed undetectable cytotoxicity of the polymers towards MIN6 cells. A simple microencapsulation process was developed using vertical co‐extrusion and a 37°C capsule collection bath containing a paraffin layer above DMEM. Spherical capsules with diameters ranging from 500 to 900 µm were formed. SEM images of freeze‐dried capsules with PBS as the core solution showed homogenous gel capsule membranes. Confocal microscopy revealed that the encapsulated cells tended to form small aggregates over 5 days, and staining for live and dead cells showed high viability post‐encapsulation. A static glucose challenge with day‐5 cultured microencapsulated cells exhibited glucose‐dependent insulin secretion comparable to controls of free MIN6 cells grown in monolayers. These results demonstrate the potential use of these thermo‐responsive polymers as cell encapsulation membranes. Biotechnol. Bioeng. 2007;96: 146–155. © 2006 Wiley Periodicals, Inc.
Salt stress effects on the central and carnitine metabolisms of Escherichia coli
Authors: Cánovas M. , Bernal V. , Sevilla A. , Iborra J.L.
Published: August 2006

The aim was to understand how interaction of the central carbon and the secondary carnitine metabolisms is affected under salt stress and its effect on the production of L‐carnitine by Escherichia coli. The biotransformation of crotonobetaine into L‐carnitine by resting cells of E. coli O44 K74 was improved by salt stress, a yield of nearly twofold that for the control being obtained with 0.5 M NaCl. Crotonobetaine and the L‐carnitine formed acted as an osmoprotectant during cell growth and biotransformation in the presence of NaCl. The enzyme activities involved in the biotransformation process (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl‐CoA/acetate (pyruvate dehydrogenase, acetyl‐CoA synthetase [ACS] and ATP/acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid cycle (isocitrate dehydrogenase [ICDH]) and glyoxylate shunt (isocitrate lyase [ICL]) were followed in batch with resting cells both in the presence and absence of NaCl and in perturbation experiments performed on growing cells in a high density cell recycle membrane reactor. Further, the levels of carnitine, crotonobetaine, γ‐butyrobetaine and ATP and the NADH/NAD+ ratio were measured in order to know how the metabolic state was modified and coenzyme pools redistributed as a result of NaCl's effect on the energy content of the cell. The results provided the first experimental evidence of the important role played by salt stress during resting and growing cell biotransformation (0.5 M NaCl increased the L‐carnitine production in nearly 85%), and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the main metabolic pathways and carbon flow operating during cell biotransformation was that controlled by the ICDH/ICL ratio, which decreased from 8.0 to 2.5, and the phosphotransferase/ACS ratio, which increased from 2.1 to 5.2, after a NaCl pulse fivefold the steady‐state level. Resting E. coli cells were seen to be made up of heterogeneous populations consisting of several types of subpopulation (intact, depolarized, and permeabilized cells) differing in viability and metabolic activity as biotransformation run‐time and the NaCl concentration increased. The results are discussed in relation with the general stress response of E. coli, which alters the NADH/NAD+ ratio, ATP content, and central carbon enzyme activities. Biotechnol. Bioeng. 2007;96:722–737. © 2006 Wiley Periodicals, Inc.
Successful transfection of hepatoma cells after encapsulation of plasmid DNA into negatively charged liposomes
Authors: Zhang Hong‐Wei , Zhang Ling , Sun Xun , Zhang Zhi‐Rong
Published: August 2006

The application of conventional cationic liposomes/DNA complexes in gene transfer was hampered due to their large size, instability, and limited transfection site in vivo. In this report, we described a dialysis‐based method and produced small, stable, and negatively charged DNA‐containing liposomes composed of low content of cationic lipid and high content of fusogenic lipid. The liposomes were relatively spherical with a condensed core inside, and exhibited small size with narrow particle size distribution. The encapsulation efficiency of the liposomes was 42.53 ± 2.29%. They were stable and showed enough protective ability to plasmid DNA from degradation after incubation with different amounts of DNase. Twenty‐fold higher transfection efficiency for the liposomes was achieved when compared with that of naked plasmid DNA and no toxicities to hepatocellular carcinoma cells were observed. Our results indicate that the negatively charged DNA‐containing liposomes can facilitate gene transfer in cultured cells, and may alleviate the drawbacks of the conventional cationic liposomes/DNA complexes for gene delivery in vivo. Biotechnol. Bioeng. 2007;96: 118–124. © 2006 Wiley Periodicals, Inc.
Morphological and molecular course of mitochondrial pathology in cultured human cells exposed long‐term to Zidovudine
Authors: Divi Rao L. , Haverkos Kathryn J. , Humsi Juliette A. , Shockley Marie E. , Thamire Chandrasekhar , Nagashima Kunio , Olivero Ofelia A. , Poirier Miriam C.
Published: August 2006

Long‐term use of antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs) as therapy for human immunodeficiency virus‐1 (HIV‐1) infection is limited by mitochondrial toxicity. Here we document mitochondrial pathology during the long‐term culture of human HeLa cells in the presence or absence of the NRTI Zidovudine® (AZT, 800 μM) for up to 77‐passages (p), with samples taken at early (p5–p11), middle (p36 and p37), and late (p70–p77) passages. Samples were analyzed for changes in mitochondrial morphology, mitochondrial (mt)DNA quantity, nuclear and mitochondrial gene expression, and mitochondrial membrane potential. Mitochondria showed abnormal proliferation at p5 and abnormal morphology ≥p36. mtDNA quantity was increased at p5 and p11, and 65% depleted at p71. Hierarchical clustering of nuclear gene expression, examined at p37 by the NCI cDNA microarray in AZT‐exposed cells, showed down‐regulation of 13 out of 16 lipid‐metabolizing genes, and up‐regulation of most oxidative phosphorylation (OXPHOS) genes. OXPHOS genes encoded by mtDNA, examined at p5, p36, and p75 using the Mitochondrial Gene Mini Array, revealed up‐regulation of genes coding for polypeptides of NADH dehydrogenase, ATP synthase, and cytochrome c oxidase. Mitochondrial membrane potential, monitored by JC1 staining, was elevated at p10 and p32, and essentially completely absent at p71. The data show that during chronic exposure of HeLa cells to AZT, a compensatory response was induced at the earlier passages (p5–p37), and by p71 there was widespread mitochondrial morphological damage, severe mtDNA depletion, and a substantial loss of mitochondrial membrane potential. Environ. Mol. Mutagen., 2006. Published 2006 Wiley‐Liss, Inc.
Imbalances of chromosome arm 1p in pediatric and adult germ cell tumors are caused by true allelic loss: A combined comparative genomic hybridization and microsatellite analysis
Authors: Zahn Susanne , Sievers Sonja , Alemazkour Katayoun , Orb Sandra , Harms Dieter , Schulz Wolfgang A. , Calaminus Gabriele , Göbel Ulrich , Schneider Dominik T.
Published: August 2006

Previous studies on childhood germ cell tumors (GCTs) report highly variable frequencies of losses at chromosome arm 1p. Since deletions at 1p portend a poor prognosis in other embryonal tumors, this study aims to clarify the question of the frequency of true allelic loss at 1p and whether it constitutes a prognostic parameter. We analyzed 13 GCTs from different gonadal and extragonadal sites of children (4 teratomas, 9 malignant GCTs) and 18 GCTs of adolescents and adults (3 teratomas; 15 malignant GCTs) using automated microsatellite analysis with 23 polymorphic markers and chromosomal “high resolution” comparative genomic hybridization (HR‐CGH). With this combined approach, we detected loss of heterozygosity (LOH) at 1p in 8/9 childhood malignant GCTs with concordant data from HR‐CGH and microsatellite analyses. In contrast, LOH at 1p was not detected in childhood teratomas (0/4) and constituted a rare event in GCTs of adolescence and adulthood (3/18). The commonly deleted region was located at distal 1p36‐pter, with a proximal boundary between the markers D1S450 and D1S2870. These data unequivocally demonstrate that deletion at 1p is common in childhood GCTs and results in allelic loss. This observation argues for the presence of a classical tumor suppressor at distal 1p. Considering the high frequency of LOH at 1p and the overall favorable prognosis of childhood GCTs, a prognostic impact of LOH at 1p in childhood GCTs appears unlikely. However, since two postpubertal tumors with LOH at 1p progressed, a prognostic relevance in this age group seems possible, warranting a prospective evaluation. © 2006 Wiley‐Liss, Inc.
Genomic alterations in lobular neoplasia: A microarray comparative genomic hybridization signature for early neoplastic proliferationin the breast
Authors: Mastracci Teresa L. , Shadeo Ashleen , Colby Sarah M. , Tuck Alan B. , O'Malley Frances P. , Bull Shelley B. , Lam Wan L. , Andrulis Irene L.
Published: August 2006

The identification of genomic alterations occurring in neoplastic lesions provides insight into both lesion occurrence and disease progression. In this study, we used microarray comparative genomic hybridization (CGH) to investigate genetic changes in atypical lobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS), as the presence of these lobular neoplastic lesions is an indicator of risk in the development of invasive breast cancer. DNA was extracted from microdissected archival breast tissue containing ALH or LCIS, lacking adjacent invasive carcinoma, and subjected to whole‐genome tiling path microarray‐CGH using the submegabase resolution tiling set (SMRT)‐array platform. Twelve ALH and 13 LCIS lesions were examined. Copy number alterations were identified using statistical criteria and validated with Real‐Time PCR and fluorescence in situ hybridization. From statistical analysis, a greater number of alterations were observed in ALH compared to LCIS. Alterations common to ALH include gain at 2p11.2 and loss at 7p11–p11.1 and 22q11.1. Alterations common to LCIS include gain at 20q13.13 and loss at 19q13.2–q13.31. In both ALH and LCIS, we observed loss of 16q21–q23.1, an altered region previously identified in lobular neoplasia and invasive carcinoma. The validation of select alterations reinforces the genomic signature. This study represents the first whole‐genome investigation of lobular neoplastic breast lesions using clinical archival specimens. The identified genomic signature includes copy number alterations not previously identified for lobular neoplasia. This genomic signature, common to ALH and LCIS, suggests a role for the acquisition of novel genomic alterations in the aberrant cellular proliferation that defines lobular neoplasia. © 2006 Wiley‐Liss, Inc.
Molecular dissection of t(11;17) in acute myeloid leukemia reveals a variety of gene fusions with heterogeneous fusion transcripts and multiple splice variants
Authors: Strehl Sabine , König Margit , Meyer Claus , Schneider Björn , Harbott Jochen , Jäger Ulrich , von Bergh Anne R. M. , Loncarevic Ivan F. , Jarosova Marie , Schmidt Helmut H. , Moore Steven D. P. , Marschalek Rolf , Haas Oskar A.
Published: August 2006

The majority of translocations that involve the long arms of chromosomes 11 and 17 in acute myeloid leukemia appear identical on the cytogenetic level. Nevertheless, they are diverse on the molecular level. At present, two genes are known in 11q23 and four in 17q12‐25 that generate five distinct fusion genes: MLL‐MLLT6/AF17, MLL‐LASP1, MLL‐ACACA or MLL‐SEPT9/MSF, and ZBTB16/PLZF‐RARA. We analyzed 14 cases with a t(11;17) by fluorescence in situ hybridization and molecular genetic techniques and determined the molecular characteristics of their fusion genes. We identified six different gene fusions that comprised seven cases with a MLL‐MLLT6/AF17, three with a MLL‐SEPT9/MSF, and one each with MLL‐LASP1, MLL‐ACACA, and ZBTB16/PLZF‐RARA fusions. In the remaining case, a MLL‐SEPT6/Xq24 fusion suggested a complex rearrangement. The MLL‐MLLT6/AF17 transcripts were extremely heterogeneous and the detection of seven different in‐frame transcript and splice variants enabled us to predict the protein domains relevant for leukemogenesis. The putative MLL‐MLLT6 consensus chimeric protein consists of the AT‐hook DNA‐binding, the methyltransferase, and the CXXC zinc‐finger domains of MLL and the highly conserved octapeptide and the leucine‐zipper dimerization motifs of MLLT6. The MLL‐SEPT9 transcripts showed a similar high degree of variability. These analyses prove that the diverse types of t(11;17)‐associated fusion genes can be reliably identified and delineated with a proper combination of cytogenetic and molecular genetic techniques. The heterogeneity of transcripts encountered in cases with MLL‐MLLT6/AF17 and MLL‐SEPT9/MSF fusions clearly demonstrates that thorough attention has to be paid to the appropriate selection of primers to cover all these hitherto unrecognized fusion variants. © 2006 Wiley‐Liss, Inc.