bluuebunny/crossref_metadata_2025 huggingface dataset:
Prenatal diagnosis of Down syndrome: Ten year experience in the Israeli population
Authors: Shohat Mordechai , Frimer Helena , Shohat‐Levy Vered , Esmailzadeh Hormoz , Appelman Zvi , Ben‐Neriah Ziva , Dar Hanna , Orr‐Urtreger Avi , Amiel Aliza , Gershoni Ruth , Manor Esther , Barkai Gad , Shalev Stavit , Gelman‐Kohen Zully , Reish Orit , Lev Dorit , Davidov Bella , Goldman Boleslaw
Published: September 2003

Second trimester maternal serum biochemical markers, introduced between 1990 and 1995, were supplemented with new ultrasound methods at 14–16 weeks and first trimester biochemical markers between 1995 and 2000. This study evaluated the effectiveness of a Down syndrome (DS) prevention program among the Israeli Jewish population between 1990 and 2000. We collected data on the total number of prenatal tests performed on Israeli Jewish women, DS cases detected prenatally and DS livebirths in Israel during these years. We also studied the use of the newer screening tests in 1990, 1992, and 2000. Between 1990 and 1995, use of chromosomal studies for DS in this population increased from 11.3% to 21.6% and the percentage of cases detected prenatally from 53% to 70%. However, between 1996 and 2000, even with the new screening methods, the utilization rate remained similar (20.7% and 19.8%, respectively) and the percentage detected prenatally decreased to 61% in 2000. The total cost per case detected increased from $47,971 in 1990 to $75,229 in 1992, and to $190,171 in 2000. Between 1990 and 1995, improvement in the percentage of cases detected prenatally was associated with a significant increase in the amniocentesis rate—both are attributed to the introduction of second trimester maternal serum biochemical marker tests. Unexpectedly, the introduction between 1995 and 2000 of new genetic methods to assess the DS risk did not improve the percentage detected or reduce the amniocentesis rate, and was accompanied by an increased cost per case detected. © 2003 Wiley‐Liss, Inc.
Left‐sided CHILD syndrome caused by a nonsense mutation in the NSDHL gene
Authors: Hummel Marybeth , Cunningham David , Mullett Charles J. , Kelley Richard I. , Herman Gail E.
Published: September 2003

Congenital hemidysplasia with ichthyosiform nevus and limb defects (CHILD) syndrome is a rare X‐linked dominant malformation syndrome characterized by unilaterally distributed ichthyosiform nevi, often sharply delimited at the midline, and ipsilateral limb defects. At least two‐thirds of cases demonstrate involvement of the right side. Mutations in an essential enzyme of cholesterol biosynthesis, NAD(P)H steroid dehydrogenase‐like [NSDHL], have been reported in five unrelated patients with right‐sided CHILD syndrome and in a sixth patient with bilaterally, symmetric nevi and mild skeletal anomalies, but not with CHILD syndrome as originally defined. Although all of the molecularly diagnosed cases with the CHILD phenotype to date have had right‐sided disease, we report here a novel nonsense mutation (E151X) of NSDHL in an infant with left‐sided CHILD syndrome. This result demonstrates that both right‐ and left‐sided CHILD syndrome can be caused by mutations in the same gene. © 2003 Wiley‐Liss, Inc.
Neonatal diabetes mellitus, congenital hypothyroidism, hepatic fibrosis, polycystic kidneys, and congenital glaucoma: A new autosomal recessive syndrome?
Authors: Taha Doris , Barbar Maha , Kanaan Hassan , Williamson Balfe John
Published: September 2003

We report on two sibs (of 4) with a syndrome of minor facial anomalies, proportionate IUGR, neonatal non‐autoimmune diabetes mellitus (NDM), severe congenital hypothyroidism (CH), cholestasis, congenital glaucoma, and polycystic kidneys. Liver disease progressed to hepatic fibrosis. The renal disease was characterized by large kidneys and multiple small cysts with deficient corticomedullary junction differentiation and normal kidney function. The phenotype observed in the two sibs was identical. Although a combination of liver, kidney, and pancreatic involvement has been described in Ivemark syndrome (hepato‐renal‐pancreatic syndrome), the coexistence of NDM, CH, and glaucoma in both sibs suggests the possibility that this combination of manifestations describes a new autosomal recessive syndrome. Mutation analysis for several candidate genes is warranted. © 2003 Wiley‐Liss, Inc.
Thyroid dysfunction in a patient with aglossia
Authors: Kantaputra Piranit , Tanpaiboon Pranoot
Published: September 2003

We report a Thai girl who had aglossia, micrognathia, microsomia, collapse of mandibular arch, persistence of buccopharyngeal membrane, microcephaly, and mild developmental delay. Thyroid function tests indicated that she had subclinical hypothyroidism. Thyroid scan revealed normal uptake of the whole thyroid gland. Tongue morphogenesis is integrally linked to the normal development of thyroid gland, and abnormal tongue morphogenesis could potentially result in a functional thyroid disorder. We propose that micrognathia, microsomia, congenital absence of mandibular incisors, and collapse of the mandibular arch are the result of abnormal tongue development. © 2003 Wiley‐Liss, Inc.
Narrowing the candidate region of Albright hereditary osteodystrophy‐like syndrome by deletion mapping in a patient with an unbalanced cryptic translocation t(2;6)(q37.3;q26)
Authors: Giardino Daniela , Finelli Palma , Gottardi Giulietta , De Canal Gabriella , Monica Matteo Della , Lonardo Fortunato , Scarano Gioacchino , Larizza Lidia
Published: September 2003

We here describe a submicroscopic translocation affecting the subtelomeric regions of chromosomes 2q and 6q identified in a patient referred to us because of mental retardation, obesity, brachydactyly, and short stature. FISH analysis using subtelomeric probes showed a 46,XY,der(2)t(2;6)(q37.3;q26) in the propositus, and a balanced t(2;6) in his father and sister. FISH with region‐specific genomic clones made it possible to map the 2q37.3 breakpoint precisely to the region covered by BAC 585E12, and the 6q26 breakpoint to between the regions encompassed by 414A5 and 480A20. The 2q subtelomeric deletion has often been found in patients with Albright hereditary osteodystrophy (AHO)‐like syndrome but, to the best of our knowledge, the 2q37.3‐qter monosomy ascertained in our patient is the smallest so far described within the syndrome's critical interval, and may thus enhance the search for the responsible genes. © 2003 Wiley‐Liss, Inc.
Association between genetic variation in the Y chromosome and hypertension in myocardial infarction patients
Authors: García Eliecer Coto , González Pelayo , Castro Mónica G. , Alvarez Ruth , Reguero Julián R. , Batalla Alberto , Cortina Arturo , Alvarez Victoria
Published: September 2003

A single nucleotide polymorphism (SNP) in chromosome Y has been associated with blood pressure. In men, the risk of suffering from cardiovascular diseases, including coronary artery disease, could be influenced by one or more loci on chromosome Y. We genotyped 208 men who had suffered an early episode of myocardial infarction (MI) (≤55 years) and 178 healthy control men for two Y‐polymorphisms (a HindIII polymorphism in an alphoid satellite in the centromeric non‐recombining region and the −2627 T/C in the SRY gene). Frequencies were compared through a χ2‐test. Frequencies for the two polymorphisms did not differ between patients and controls. The alphoid‐HindIII polymorphism was not related to blood pressures in our population (HindIII+: diastolic, 80 ± 2; systolic, 129 ± 5. HindIII−: diastolic, 80 ± 2; systolic, 128 ± 3). Seventy‐six patients (37%) were hypertensives and had a significantly higher frequency of the HindIII+ allele compared to the normotensive patients (46 and 26%, respectively; P = 0.028). According to our data, the alphoid‐HindIII polymorphism in chromosome Y was not associated with differences in blood pressure in men from Asturias (Northern Spain). However, the HindIII+ allele increased the risk of suffering an early episode of MI among hypertensives. © 2003 Wiley‐Liss, Inc.
Effect of diet on dental development in four species of catarrhine primates
Authors: Dirks Wendy
Published: September 2003

In this study, dental development is described in two pairs of closely related catarrhine primate species that differ in their degree of folivory: 1) Hylobates lar and Symphalangus syndactylus, and 2) Papio hamadryas hamadryas and Semnopithecus entellus. Growth increments in histological thin sections are used to reconstruct the chronology of dental development to determine how dental development is accelerated in the more folivorous species of each pair. Although anterior tooth formation appears to be unrelated to diet, both S. syndactylus and S. entellus initiate the slowest‐forming molar earlier than the related less‐folivorous species, which supports the hypothesis that dental acceleration is related to food processing. S. syndactylus initiates M2 crown formation at an earlier age than H. lar, and S. entellus initiates and completes M3 at an earlier age than P. h. hamadryas. Similar stages of M3 eruption occur earlier in the more folivorous species; however, the sex of the individual may also play a role in creating such differences. Although the age at M3 emergence is close to that reported for the end of body mass growth in lar gibbons, hamadryas baboons, and Hanuman langurs, M3 emergence may not be coupled to body mass growth in siamangs. Am. J. Primatol. 61:29–40, 2003. © 2003 Wiley‐Liss, Inc.
Quantitative 3D analysis of the canal network in cortical bone by micro‐computed tomography
Authors: Cooper D.M.L. , Turinsky A.L. , Sensen C.W. , Hallgrímsson B.
Published: September 2003

Cortical bone is perforated by an interconnected network of porous canals that facilitate the distribution of neurovascular structures throughout the cortex. This network is an integral component of cortical microstructure and, therefore, undergoes continual change throughout life as the cortex is remodeled. To date, the investigation of cortical microstructure, including the canal network, has largely been limited to the two‐dimensional (2D) realm due to methodological hurdles. Thanks to continuing improvements in scan resolution, micro‐computed tomography (μCT) is the first nondestructive imaging technology capable of resolving cortical canals. Like its application to trabecular bone, μCT provides an efficient means of quantifying aspects of 3D architecture of the canal network. Our aim here is to introduce the use of μCT for this application by providing examples, discussing some of the parameters that can be acquired, and relating these to research applications. Although several parameters developed for the analysis of trabecular microstructure are suitable for the analysis of cortical porosity, the algorithm used to estimate connectivity is not. We adapt existing algorithms based on skeletonization for this task. We believe that 3D analysis of the dimensions and architecture of the canal network will provide novel information relevant to many aspects of bone biology. For example, parameters related to the size, spacing, and volume of the canals may be particularly useful for investigation of the mechanical properties of bone. Alternatively, parameters describing the 3D architecture of the canal network, such as connectivity between the canals, may provide a means of evaluating cumulative remodeling related change. Anat Rec (Part B: New Anat) 274B:169–179, 2003. © 2003 Wiley‐Liss, Inc.
High occurrence of a basicranial feature in Homo erectus: Anatomical description of the preglenoid tubercle
Authors: Zeitoun Valery
Published: September 2003

The degree of differentiation between Western and Eastern Early Middle Pleistocene hominids (Homo erectus) is still under debate. On the one hand, the two populations are believed by some to belong to two separate taxonomic categories possessing their own exclusive features. On the other hand, they are considered by others to belong to a single but ubiquitous global population with regional specificities that are mainly due to variations in the frequency of certain features. In this context, an additional trait, the preglenoid tubercle (tuberculum anterius fossae mandibularis), may well shed light on the matter. The tubercle is often present on the anterior part of the fossa mandibularis at the anterior extent of the joint capsule and articular tubercle (tuber articulare) of fossils usually assigned to Homo erectus. This study describes the anatomy of the preglenoid tubercle, to indicate its occurrence and to try to improve its use in taxonomy and phylogeny, although its origin and functional significance have not yet been clarified. Anat Rec (Part B: New Anat) 274B:148–156, 2003. © 2003 Wiley‐Liss, Inc.
Circularly polarized light standards for investigations of collagen fiber orientation in bone
Authors: Bromage Timothy G. , Goldman Haviva M. , McFarlin Shannon C. , Warshaw Johanna , Boyde Alan , Riggs Christopher M.
Published: September 2003

Bone exhibits positive form birefringence dominated by and dependent upon the orientation of its collagen. The biomechanical efficacy of bone as a tissue is largely determined by collagen fibers of preferred orientation and distribution (and corresponding orientation of mineral crystallites), and evidence is accumulating to demonstrate that this efficacy extends to function at the organ level. This study has three aims. The first is to provide a Background to the study of circularly polarized light (CPL) investigations of collagen fiber orientation in bone. The significance of preferred collagen fiber orientation in bone, linearly polarized light and CPL imaging principles, and a short history of CPL studies of mammalian functional histology are reviewed. The second is to describe, in some detail, methodological considerations relating to specimen preparation and imaging appropriate for the quantitative analysis of preferentially oriented collagen. These include section transparency, section thickness, the uniformity of the illuminating system, and CPL paraphernalia. Finally, we describe a grey‐level standard useful for quantitative CPL, based upon mineralized turkey tendon, which shall be provided to investigators upon request. When due consideration is paid to specimen preparation and imaging conditions, quantitative assessment of collagen fiber orientation provides insight into the effects of mechanical loading on the skeleton. Anat Rec (Part B: New Anat) 274B:157–168, 2003. © 2003 Wiley‐Liss, Inc.
Joint Modelling of Repeated Measures and Survival Time Data
Authors: Ha Il Do , Park Taesung , Lee Youngjo
Published: September 2003

In many clinical trials both repeated measures data and event history data are simultaneously observed from the same subject. These two types of responses are usually correlated, because they are from the same subject. In this article, we propose a joint model for the combined analysis of repeated measures data and event history data in the framework of hierarchical generalized linear models. The correlation between repeated measures and event time is modelled by introducing a shared random effect. The model parameters are estimated using the hierarchical‐likelihood approach. The proposed model is illustrated using a real data set for the renal transplant patients.
Utilities of the P‐value Distribution Associated with Effect Size in Clinical Trials
Authors: Hung H.M. James , O'Neill Robert T.
Published: September 2003

The P‐value, which is widely used for assessing statistical evidence in randomized comparative clinical trials, is a function of the observed effect size of the experimental treatment relative to the control treatment. The relationship of the P‐value with the observed effect size at study completion and the effect size anticipated at the design stage has potential usefulness in providing guidance for planning and interpretation of a clinical trial. The post‐trial power associated with a statistically significant P‐value from a completed study is also a random variable and its use may assist in planning a follow‐up trial to confirm the statistically significant findings in an initial study. A measure of robustness is explored to quantify the degree of sensitivity of the observed P‐value to potential bias that may be contained in the observed effect size.
Non‐Parametric Ecological Regression and Spatial Variation
Authors: Natário Isabel , Knorr‐Held Leonhard
Published: September 2003

Ecological studies aim to analyse the variation of disease risk in relation to exposure variables that are measured at an area unit level. In practice it is rarely possible to use the exposure variables themselves, either because the corresponding data are not available or because the causes of the disease are not fully understood. It is therefore quite common to use crude proxies of the real exposure to the disease in question. These proxies are rarely able to explain the disease variation and hence additional area level random effects are introduced to account for the residual variation. In this paper we investigate the possibility to model the effect of ecological covariates non‐parametrically, with and without additional random effects for the residual spatial variation. We illustrate the issues arising through analyses of simulated and real data on larynx cancer mortality in Germany, during the years of 1986 to 1990, where we use the corresponding lung cancer rates as a proxy for smoking consumption.
Poisson Regression with Change‐Point Prior in the Modelling of Disease Risk around a Point Source
Authors: Kokki Esa , Penttinen Antti
Published: September 2003

Bayesian estimation of the risk of a disease around a known point source of exposure is considered. The minimal requirements for data are that cases and populations at risk are known for a fixed set of concentric annuli around the point source, and each annulus has a uniquely defined distance from the source. The conventional Poisson likelihood is assumed for the counts of disease cases in each annular zone with zone‐specific relative risk and parameters and, conditional on the risks, the counts are considered to be independent. The prior for the relative risk parameters is assumed to be piecewise constant at the distance having a known number of components. This prior is the well‐known change‐point model. Monte Carlo sampling from the posterior results in zone‐specific posterior summaries, which can be applied for the calculation of a smooth curve describing the variation in disease risk as a function of the distance from the putative source. In addition, the posterior can be used in the calculation of posterior probabilities for interesting hypothesis. The suggested model is suitable for use in geographical information systems (GIS) aimed for monitoring disease risks. As an application, a case study on the incidence of lung cancer around a former asbestos mine in eastern Finland is presented. Further extensions of the model are discussed.
Parameter Redundancy in Multistate Capture‐Recapture Models
Authors: Gimenez Olivier , Choquet Rémi , Lebreton Jean‐Dominique
Published: September 2003

Multistate capture‐recapture models are a powerful tool to address a variety of biological questions concerning dispersal and/or individual variability in wild animal populations. However, biologically meaningful models are often over‐parameterized and consequently some parameters cannot be estimated separately. Identifying which quantities are separately estimable is crucial for proper model selection based upon likelihood tests or information criteria and for the interpretation of the estimates obtained. We show how to investigate parameter redundancy in multistate capture‐recapture models, based on formal methods initially proposed by Catchpole and his associates for exponential family distributions (Catchpole, Freeman and Morgan, 1996. Journal of the Royal Statistical Society Series B 58, 763–774). We apply their approach to three models of increasing complexity.
Improvement Over Transformed Auxiliary Variable in Estimating the Finite Population Mean
Authors: Shabbir J. , Yaab M.Z.
Published: September 2003

For estimating the finite population mean $ \bar Y $ of the study variable y, we propose a ratio‐type estimator which gives an improvement over estimators given by Upadhyaya and Singh (1999), Sisodia and Dwivedi (1981), and Singh and Kakran (1993). These estimators are compared by observing the bias and mean square error (MSE). In this empirical study, the suggested estimator under the optimal condition is found to be more efficient than the estimators mentioned above.
Describing Ordinal Odds Ratios for Stratifiedr×cTables
Authors: Liu Ivy
Published: September 2003

For anr×ctable with ordinal responses, odds ratios are commonly used to describe the relationship between the row and column variables. This article shows two types of ordinal odds ratios where local‐global odds ratios are used to compare several groups on ac‐category ordinal response and a global odds ratio is used to measure the global association between a pair of ordinal responses. When there is a stratification factor, we consider Mantel‐Haenszel (MH) type estimators of these odds ratios to summarize the association from several strata. Like the ordinary MH estimator of the common odds ratio for several 2 × 2 contingency tables, the estimators are used when the association is not expected to vary drastically among the strata. Also, the estimators are consistent under the ordinary asymptotic framework in which the number of strata is fixed and also under sparse asymptotics in which the number of strata grows with the sample size. Compared to the maximum likelihood estimators, simulations find that the MH type estimators perform better especially when each stratum has few observations. This article provides variances and covariances formulae for the local‐global odds ratios estimators and applies the bootstrap method to obtain a standard error for the global odds ratio estimator. At the end, we discuss possible ways of testing the homogeneity assumption.
A note on the Design of Unreplicated Trials
Authors: Williams E.R. , John J.A.
Published: September 2003

One approach to the design of early generation trials is to have a number of replicated check varieties and a larger number of unreplicated or new varieties. For such trials we define the class of α–α‐designs as an extension to the augmented lattice square designs of Federer (2002). Efficient latinized α–α‐designs can be conveniently constructed using the design generation package CycDesigN.
A Stochastic Model Relating the Number of Cells at X‐Inactivation to the Allelic Ratio in Normal Heterozygous Adult Females
Authors: Chen George S. , Chen Shande
Published: September 2003

Early in development, one X‐chromosome in each cell of the female embryo is inactivated. Knowing the number of certain human tissue cells at the time of X‐inactivation can improve our understanding of certain diseases such as cancer or genetic disorders as well as cellular development. However, the moment of X‐inactivation in humans is difficult to observe directly. In this study, we developed a mathematical model using branching processes and asymptotic normal approximation that will more accurately determine a relationship between the number of cells at X‐inactivation with the proportion of one allele found in normal heterozygous adult females. We then conducted computer simulations to show the adequacy of this model. Finally, this model was used to more accurately estimate the number of hemopoietic stem cells at X‐inactivation using a real life data set.
Multiple Comparisons in Nonlinear Repeated Measurements
Authors: Hyakutake Hiroto
Published: September 2003

It is interest to compare functions of parameters in nonlinear models for repeated measurements. In a pharmacokinetic model, the maximum value of the model would be a nonlinear function of some unknown parameters. In this paper, simultaneous confidence intervals of functions of parameters in a nonlinear model for repeated mesurement data are considered to compare the populations.
Analysis of Escherichia coli anaplerotic metabolism and its regulation mechanisms from the metabolic responses to altered dilution rates and phosphoenolpyruvate carboxykinase knockout
Authors: Yang Chen , Hua Qiang , Baba Tomoya , Mori Hirotada , Shimizu Kazuyuki
Published: September 2003

The gluconeogenic phosphoenolpyruvate (PEP) carboxykinase is active in Escherichia coli during its growth on glucose. The present study investigated the influence of growth rates and PEP carboxykinase knockout on the anaplerotic fluxes in E. coli. The intracellular fluxes were determined using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U‐13C6]glucose labeling experiments and 2D nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids and glycerol. Significant activity of PEP carboxykinase was identified in wild‐type E. coli, and the ATP dissipation for the futile cycling via this reaction accounted for up to 8.2% of the total energy flux. Flux analysis of pck deletion mutant revealed that abolishment of PEP carboxykinase activity resulted in a remarkably reduced flux through the anaplerotic PEP carboxylase and the activation of the glyoxylate shunt, with 23% of isocitrate found being channeled in the glyoxylate shunt. The changes in intracellular metabolite concentrations and specific enzyme activities associated with different growth rates and pck deletion, were also determined. Combining the measurement data of in vivo fluxes, metabolite concentrations and enzyme activities, the in vivo regulations of PEP carboxykinase flux, PEP carboxylation, and glyoxylate shunt in E. coli are discussed. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 129–144, 2003.
Stability of recombinant plasmids on the continuous culture of Bifidobacterium animalis ATCC 27536
Authors: González Vara Antonio , Rossi Maddalena , Altomare Luigi , Eikmanns Bernhard , Matteuzzi Diego
Published: September 2003

Bifidobacterium animalis ATCC 27536 represents among bifidobacteria a host‐model for cloning experiments. The segregational and structural stabilities of a family of cloning vectors with different molecular weights but sharing a common core were studied in continuous fermentation of the hosting B. animalis without selective pressure. The rate of plasmid loss (R) and the specific growth rate difference (δμ) between plasmid‐free and plasmid‐carrying cells were calculated for each plasmid and their relationship with plasmid size was studied. It was observed that both R and the numerical value of δμ increased exponentially with plasmid size. The exponential functions correlating the specific growth rate difference and the rate of plasmid loss with the plasmid molecular weight were determined. Furthermore, the smallest of the plasmids studied, pLAV (4.3‐kb) was thoroughly characterized by means of its complete nucleotide sequence. It was found that it contained an extra DNA fragment, the first bifidobacterial insertion sequence characterised, named IS 1999. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 145–150, 2003.
Binding and reversibility of Thermobifida fusca Cel5A, Cel6B, and Cel48A and their respective catalytic domains to bacterial microcrystalline cellulose
Authors: Jung Hyungil , Wilson David B. , Walker Larry P.
Published: September 2003

The binding and reversibility of Thermobifida fusca intact Cel5A, Cel5B, and Cel48A and their corresponding catalytic domains (CDs) to bacterial microcrystalline cellulose (BMCC) were studied at 5°C. The binding of the intact cellulases and of corresponding CDs to BMCC was irreversible in all regions: Langmuir binding (region I), interstice penetration (region II), and interstice saturation (region III). The three cellulose binding domains (CBMs) bind reversibly in “region I” although their respective CDs do not. The irreversible binding of these enzymes in the Langmuir region does not satisfy the Langmuir assumption; however, the overall fit of the Interstice Saturation model, which includes binding in MBCC interstices as well as on the freely accessible surface (Jung et al., 2002a) is good. The main limitation of the model is that it does not explicitly address a mechanism for forming the enzyme‐substrate complex within the active site of the CDs. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 151–159, 2003.
Simultaneous nitrification, denitrification, and phosphorus removal in a lab‐scale sequencing batch reactor
Authors: Zeng Raymond J. , Lemaire Romain , Yuan Zhiguo , Keller Jürg
Published: September 2003

Simultaneous nitrification and denitrification (SND) via the nitrite pathway and anaerobic–anoxic‐enhanced biological phosphorus removal (EBPR) are two processes that can significantly reduce the energy and COD demand for nitrogen and phosphorus removal. The combination of these two processes has the potential of achieving simultaneous nitrogen and phosphorus removal with a minimal requirement for COD. A lab‐scale sequencing batch reactor (SBR) was operated in alternating anaerobic–aerobic mode with a low dissolved oxygen (DO) concentration (0.5 mg/L) during the aerobic period, and was demonstrated to accomplish nitrification, denitrification, and phosphorus removal. Under anaerobic conditions, COD was taken up and converted to polyhydroxyalkanoates (PHAs), accompanied by phosphorus release. In the subsequent aerobic stage, PHA was oxidized and phosphorus was taken up to <0.5 mg/L by the end of the cycle. Ammonia was also oxidized during the aerobic period, but without accumulation of nitrite or nitrate in the system, indicating the occurrence of simultaneous nitrification and denitrification. However, off‐gas analysis showed that the final denitrification product was mainly nitrous oxide (N2O), not N2. Further experimental results demonstrated that nitrogen removal was via nitrite, not nitrate. These experiments also showed that denitrifying glycogen‐accumulating organisms (DGAOs), rather than denitrifying polyphosphate‐accumulating organisms (DPAOs), were responsible for the denitrification activity. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 170–178, 2003.
Generic/matrix evaluation of SV40 clearance by anion exchange chromatography in flow‐through mode
Authors: Curtis Sherrie , Lee Kitty , Blank Gregory S. , Brorson Kurt , Xu Yuan
Published: September 2003

The potential of viral contamination is a regulatory concern for continuous cell line‐derived pharmaceutical proteins. Complementary and redundant safety steps, including an evaluation of the viral clearance capacity of unit operations in the purification process, are performed prior to registration and marketing of biotechnology pharmaceuticals. Because process refinement is frequently beneficial, CBER/FDA has published guidance facilitating process improvement by delineating specific instances where the bracketing and generic approaches are appropriate for virus removal validation. In this study, a generic/matrix study was performed using Q‐Sepharose Fast Flow (QSFF) chromatography to determine if bracketing and generic validation can be applied to anion exchange chromatography. Key operational parameters were varied to upper and lower extreme values and the impact on viral clearance was assessed using simian virus 40 (SV40) as the model virus. Operational ranges for key chromatography parameters were identified where an SV40 log10 reduction value (LRV) of ≥4.7 log10 is consistently achieved. On the basis of the apparent robustness of SV40 removal by Q‐anion exchange chromatography, we propose that the concept of “bracketed generic” validation can be applied to this and potentially other chromatography unit operations. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 179–186, 2003.
Kinetics of thermophilic, anaerobic oxidation of straight and branched chain butyrate and valerate
Authors: Batstone D. J. , Pind P. F. , Angelidaki I.
Published: September 2003

The degradation kinetics of normal and branched chain butyrate and valerate are important in protein‐fed anaerobic systems, as a number of amino acids degrade to these organic acids. Including activated and primary wastewater sludge digesters, the majority of full‐scale systems digest feeds with a significant or major fraction of COD as protein. This study assesses the validity of using a common kinetic parameter set and biological catalyst to represent butyrate, n‐valerate, and i‐valerate degradation in dynamic models. The i‐valerate degradation stoichiometry in a continuous, mixed population system is also addressed, extending previous pure‐culture and batch studies. A previously published mathematical model was modified to allow competitive uptake of i‐valerate, and used to model a thermophilic manure digester operated over 180 days. The digester was periodically pulsed with straight and branched chain butyrate and valerate. Parameters were separately optimized to describe butyrate, i‐valerate, and n‐valerate degradation, as well as a lumped set optimized for all three substrates, and nonlinear, correlated parameter spaces estimated using an F distribution in the objective function (J). Each parameter set occupied mutually exclusive parameter spaces, indicating that all were statistically different from each other. However, qualitatively, the influence on model outputs was similar, and the lumped set would be reasonable for mixed acid digestion. The main characteristic not represented by Monod kinetics was a delay in i‐valerate uptake, and was compensated for by a decreased maximum uptake rate (km). Therefore, the kinetics need modification if fed predominantly i‐valerate. Butyrate (i‐ and n‐) and n‐valerate could be modeled using stoichiometry consistent with β‐oxidation degradation pathways. However, i‐valerate produced acetate only, supporting the stoichiometry of a reaction determined by other researchers in pure culture. Therefore, lumping i‐valerate stoichiometry with that of n‐valerate will not allow good system representation, especially when the feed consists of proteins high in leucine (which produces i‐valerate), and the modified model structure and stoichiometry as proposed here should be used. This requires no additional kinetic parameters and one additional dynamic concentration state variable (i‐valerate) in addition to the variables in the base model. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 195–204, 2003.
Oscillating perfusion of cell suspensions through three‐dimensional scaffolds enhances cell seeding efficiency and uniformity
Authors: Wendt D. , Marsano A. , Jakob M. , Heberer M. , Martin I.
Published: September 2003

We developed a bioreactor for automated cell seeding of three‐dimensional scaffolds by continuous perfusion of a cell suspension through the scaffold pores in oscillating directions. Using quantitative biochemical and image analysis techniques, we then evaluated the efficiency and uniformity of perfusion seeding of Polyactive foams as compared to conventional static and spinner flask methods. Finally, we assessed the efficacy of the perfusion seeding technique for different scaffolds and cell types. Perfusion seeding of chondrocytes into Polyactive foams resulted in “viable cell seeding efficiencies,” defined as the percentages of initially loaded cells that were seeded and remained viable, that were significantly higher (75 ± 6%) than those by static (57% ± 5%) and spinner flask seeding (55% ± 8%). In addition, as compared to static and spinner flask methods, cells seeded by perfusion were respectively 2.6‐fold and 3.8‐fold more uniformly distributed and formed more homogeneously sized cell clusters. Chondrocytes seeded by perfusion into Hyaff®‐11 nonwoven meshes were 26% and 63%, respectively, more uniformly distributed than following static and spinner flask seeding. Bone marrow stromal cells seeded by perfusion into ChronOS™ porous ceramics were homogeneously distributed throughout the scaffold volume, while following the static method, cells were found only near the top surface of the ceramic. In summary, we demonstrated that our cell seeding perfusion bioreactor generated constructs with remarkably uniform cell distributions at high efficiencies, and was effective for a variety of scaffolds and different mesenchymal cell types. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 205–214, 2003.
Comparison of antibody functionality using different immobilization methods
Authors: Danczyk R. , Krieder B. , North A. , Webster T. , HogenEsch H. , Rundell A.
Published: September 2003

This study investigates the influence of antibody immobilization methods on antigen capture. Adsorption and two surface chemistries, an aminosilane chemistry and a common heterobifunctional crosslinker (N‐gamma‐maleimidobutyryloxy‐succinimide ester, GMBS), were compared and evaluated for their ability to immobilize antibodies and capture antigen. The role of protein A as an orienting protein scaffold component in each of these techniques was also evaluated. Through experimentation it was determined that the GMBS technique immobilized the highest amount of antibody and minimized nonspecific binding. For all techniques, the most functional antibodies were found to be those immobilized with protein A. Interestingly, the aminosilane technique demonstrated the highest antigen capture with antibody alone but also exhibited the highest level of nonspecific binding. © 2003 Wiley Periodicals. Biotechnol Bioeng 84: 215–223, 2003.
Tyrosinase: Polybrene noncovalent complexes in water‐ethanol mixtures
Authors: Shipovskov Stepan , Levashov Andrey
Published: September 2003

Formation of noncovalent complexes between tyrosinase from mushrooms and a cationic polyelectrolyte, polybrene (PB, poly (1,5‐dimethyl‐1,5‐diazaundecamethyelene) bromide), was shown to activate and stabilize tyrosinase in water–ethanol mixtures. In the reaction of catechol oxidation in aqueous solutions, catalytic activity (kcat) of tyrosinase‐PB complex ([PB]/[tyrosinase] molar ratio 100:1, per mole of polymer) in a wide range of pH was higher than that of free tyrosinase. In aqueous solutions and in water–ethanol mixtures at moderate concentrations of ethanol (10–40% v/v), the value of kcat of tyrosinase‐PB complex exceeded the activity of free enzyme, from 1.2–2‐fold, accompanied by the essential (up to 10‐fold) increase in the value of the specificity constant (kcat/Km). The results are of practical importance for the construction of biocatalysts working successfully in polar organic media. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84 258–263, 2003.
Natural antimicrobial agent (reuterin) produced by lactobacillus reuteri for sanitization of biological tissues inoculated with pseudomonas aeruginosa
Authors: Liang Hsiang‐Fa , Chen Chiun‐Nan , Chang Yen , Sung Hsing‐Wen
Published: September 2003

The study was done to evaluate the efficacy of using reuterin produced by Lactobacillus reuteri to sanitize biological tissues. The microorganism tested in the study was Pseudomonas aeruginosa, a common cause of nosocomial biomaterial‐related infections. The inhibitory effect of reuterin on P. aeruginosa for an inoculated tissue was investigated at different conditions of concentration, temperature, and pH. Additionally, the cellular compatibility of the reuterin‐sanitized tissue was evaluated. Glutaraldehyde was employed as a control. It was noted that the minimum inhibitory concentration (MIC, 33.0 ± 2.9 ppm) and minimum bactericidal concentration (MBC, 50.0 ± 0.0 ppm) values of reuterin for P. aeruginosa were significantly lower than their glutaraldehyde counterparts (MIC, 130.0 ± 8.2 ppm and MBC, 180.0 ± 18.3 ppm). This indicated that reuterin was more efficient than glutaraldehyde as an antimicrobial agent. The addition of reuterin on the inoculated tissue led to a reduced viability of P. aeruginosa. The reduction in the P. aeruginosa culture was more pronounced with increasing the concentration of reuterin (0–100 ppm). At increasing temperature (25–45°C), there was an increasing effect of reuterin on its sanitization activity. However, it should be pointed out that the growth of P. aeruginosa in the nutrient broth was also significantly affected by temperature. The sanitization activity of reuterin was more evident with increasing the pH level (pH 6.5–8.5). The cytotoxicity of reuterin was significantly lower than that of glutaraldehyde. Additionally, the cellular compatibility of the reuterin‐sanitized tissue was superior to its glutaraldehyde‐sanitized counterpart. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 233–239, 2003.
Development of foamed emulsion bioreactor for air pollution control
Authors: Kan Eunsung , Deshusses Marc A.
Published: September 2003

A new type of bioreactor for air pollution control has been developed. The new process relies on an organic‐phase emulsion and actively growing pollutant‐degrading microorganisms, made into a foam with the air being treated. This new reactor is referred to as a foamed emulsion bioreactor (FEBR). As there is no packing in the reactor, the FEBR is not subject to clogging. Mathematical modeling of the process and proof of concept using a laboratory prototype revealed that the foamed emulsion bioreactor greatly surpasses the performance of existing gas‐phase bioreactors. Experimental results showed a toluene elimination capacity as high as 285 gtoluene m−3reactor h−1 with a removal efficiency of 95% at a gas residence time of 15 s and a toluene inlet concentration of 1–1.3 g m−3. Oxygen limited the reactor performance at toluene concentration above about 0.7–1.0 g m−3; consequently, performance was significantly improved when pure oxygen was added to the contaminated air. The elimination capacity increased from 204 to 408 g m−3 h−1 with >77% toluene removal at toluene inlet concentrations of 2–2.2 g m−3. Overall, the results show that the performance of the FEBR far exceeds that of currently used bioreactors for air pollution control. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 240–244, 2003.
Synergistic cellulose hydrolysis can be described in terms of fractal‐like kinetics
Authors: Väljamäe Priit , Kipper Kalle , Pettersson Göran , Johansson Gunnar
Published: September 2003

A fractal‐like kinetics model was used to describe the synergistic hydrolysis of bacterial cellulose by Trichoderma reesei cellulases. The synergistic action of intact cellobiohydrolase Cel7A and endoglucanase Cel5A at low enzyme‐to‐substrate ratios showed an apparent substrate inhibition consistent with a case where two‐dimensional (2‐D) surface diffusion of the cellobiohydrolase is rate‐limiting. The action of Cel7A core and Cel5A was instead consistent with a three‐dimensional (3‐D) diffusion‐based mode of action. The synergistic action of intact Cel7A was far superior to that of the core at a high enzyme‐to‐substrate ratio, but this effect was gradually reduced at lower enzyme‐to‐substrate ratios. The apparent fractal kinetics exponent h obtained by nonlinear fit of hydrolysis data to the fractal‐like kinetics analogue of a first‐order reaction was a useful empirical parameter for assessing the rate retardation and its dependence on the reaction conditions. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 254–257, 2003.
The Blasticidin S Biosynthesis Gene Cluster from Streptomyces griseochromogenes: Sequence Analysis, Organization, and Initial Characterization
Authors: Cone Martha C. , Yin Xihou , Grochowski Laura L. , Parker Morgan R. , Zabriskie T. Mark
Published: September 2003

Blasticidin S is a potent antifungal and cytotoxic peptidyl nucleoside antibiotic from Streptomyces griseochromogenes. The mixed biosynthesis of the compound is evident from the three distinct structural components: a cytosine base, an amino deoxyglucuronic acid, and N‐methyl β‐arginine. The blasticidin S biosynthesis gene cluster was cloned from S. griseochromogenes and the pathway heterologously expressed in S. lividans from a cosmid harboring a 36.7‐kb fragment of S. griseochromogenes DNA. The complete DNA sequence of this insert has now been determined and evidence suggests a contiguous 20‐kb section defines the blasticidin S biosynthesis cluster. The predicted functions of several open reading frames are consistent with the expected biochemistry and include an arginine 2,3‐aminomutase, a cytosylglucuronic acid synthase, and a guanidino N‐methyltransferase. Insight into other steps in the assembly of blasticidin S was evident from sequence homology with proteins of known function and heterologous expression of fragments of the cluster. Additionally, the gene that directs the production of free cytosine, blsM, was subcloned and expressed in Escherichia coli. Characterization of BlsM revealed that cytidine monophosphate serves as the precursor to cytosine.
Fluorescent Cellular Sensors of Steroid Receptor Ligands
Authors: Muddana Smita S. , Peterson Blake R.
Published: September 2003

Steroid hormone receptors comprise a major class of therapeutic drug targets that control gene expression by binding steroid hormone ligands. These small molecule–protein interactions are typically characterized in living cells by quantification of ligand‐mediated reporter gene expression. As an alternative, non‐transcriptional approach, we constructed fluorescent cellular sensors by expressing yellow fluorescent protein (YFP) fused to the ligand binding domains (LBDs) of estrogen receptor‐alpha (ERα), estrogen receptor‐beta (ERβ), androgen receptor (AR), and the glucocorticoid receptor (GR). These proteins were tethered through a short two amino acid linker and expressed in S. cerevisiae yeast. Recombinant yeast treated with cognate steroid receptor ligands exhibited dose‐dependent fluorescence enhancements that were correlated with known relative receptor binding affinity values. These effects generally paralleled ligand‐mediated receptor dimerization quantified with analogous yeast two‐hybrid transcriptional assays, suggesting that the majority of the observed fluorescence enhancements were conferred by conformational changes coupled with receptor dimerization, such as ligand‐mediated stabilization of protein folding. Remarkably, certain interactions such as the binding of cortisol, progesterone, and dexamethasone to the GR were undetectable with yeast two‐hybrid assays. However, these interactions were detected with the fluorescent cellular sensors, indicating the sensitivity of this system to subtle ligand‐induced conformational effects. These sensors provide a novel, non‐transcriptional, and high‐throughput method to identify and analyze ligands of nuclear hormone receptors.