bluuebunny/crossref_metadata_2025 huggingface dataset:
Fatores associados ao letramento em saúde relacionado ao hábito etilista entre pacientes com diabetes assistidos pela atenção primária à saúde
Authors: Brito Ana Monique Gomes , Sousa Árlen Almeida Duarte de , Vicente João Victor de Jesus , Alves Stéfany Allaide Fasolak , Martins Andréa Maria Eleutério de Barros Lima
Published: March 2024

Introdução: O hábito etilista é um importante fator de desequilíbrio metabólico entre pessoas com diabetes. Contudo, indivíduos com maiores níveis de letramento em saúde podem exercer comportamentos que criam barreiras para o surgimento de complicações dessa doença. Verificar o letramento em saúde relacionado ao hábito etilista pode ser útil para reduzir as consequências do diabetes. Objetivo: Identificar os fatores associados ao letramento em saúde relacionado ao hábito etilista entre pessoas com diabetes assistidas pela atenção primária à saúde. Métodos: Estudo transversal realizado em unidades de saúde do município de Montes Claros, MG, Brasil. Foram analisadas condições demográficas e econômicas, além dos níveis de letramento em saúde avaliado a partir do instrumento Literacia em Saúde quanto ao Hábito Etilista. Resultados: Participaram do estudo 215 pessoas com diabetes com média de idade de 60,73 anos, sendo a maioria mulheres (63,7%; n=137). Menores níveis de letramento em saúde relacionado ao hábito etilista estiveram presentes em 31,2% (n=67) dos participantes. Foram associadas a menores níveis de letramento em saúde: sexo (homens, OR=0,46; IC95%=0,23-0,94; p=0,032), baixa escolaridade (educação infantil, OR=7,00; IC95%=2,55-19,20; p<0,001; analfabeto, OR=28,06; IC95%=4,40-178,83; p<0,001) e gastos com medicamentos relacionados ao diabetes (OR=2,27; IC95%=1,14-4,50; p=0,019). Conclusão: Observou-se melhores níveis de letramento em saúde relacionado ao hábito etilista entre os homens, com maior escolaridade e que não possuíam gastos com medicamentos relacionados ao diabetes.
A binding site for phosphoinositides described by multiscale simulations explains their modulation of voltage-gated sodium channels
Authors: Lin Yiechang , Tao Elaine , Champion James P , Corry Ben
Published: March 2024

Voltage-gated sodium channels (Naᵥ) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Naᵥ channels transition from the resting, closed state to an open, conductive state, before rapidly inactivating. Dysregulation of this functional cycle due to mutations causes diseases including epilepsy, pain conditions, and cardiac disorders, making Naᵥ channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P2 decreases Naᵥ1.4 activity by increasing the difficulty of channel opening, accelerating fast inactivation and slowing recovery from fast inactivation. Using multiscale molecular dynamics simulations, we show that PI(4,5)P2 binds stably to inactivated Naᵥ at a conserved site within the DIV S4–S5 linker, which couples the voltage-sensing domain (VSD) to the pore. As the Naᵥ C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesize that PI(4,5)P2 prolongs inactivation by competitively binding to this site. In atomistic simulations, PI(4,5)P2 reduces the mobility of both the DIV S4–S5 linker and the DIII–IV linker, responsible for fast inactivation, slowing the conformational changes required for the channel to recover to the resting state. We further show that in a resting state Naᵥ model, phosphoinositides bind to VSD gating charges, which may anchor them and impede VSD activation. Our results provide a mechanism by which phosphoinositides alter the voltage dependence of activation and the rate of recovery from inactivation, an important step for the development of novel therapies to treat Naᵥ-related diseases.
Adipokinetic hormone signaling mediates the fecundity of Diaphorina citri infected by ‘Candidatus Liberibacter asiaticus’
Authors: Li Jiayun , Holford Paul , Beattie George Andrew Charles , Wu Shujie , He Jielan , Tan Shijian , Wang Desen , He Yurong , Cen Yijing , Nian Xiaoge
Published: March 2024

Diaphorina citri
is the primary vector of the bacterium, ‘
Candidatus
Liberibacter asiaticus’ (
C
Las) associated with the severe Asian form of huanglongbing.
C
Las-positive
D. citri
are more fecund than their
C
Las-negative counterparts and require extra energy expenditure. Therefore, understanding the molecular mechanisms linking metabolism and reproduction is of particular importance. In this present study, we found adipokinetic hormone (
DcAKH
) and its receptor (
DcAKHR
) were essential for increased lipid metabolism and fecundity in response to
C
Las infection in
D. citri.
Knockdown of
DcAKH
and
DcAKHR
not only resulted in triacylglycerol accumulation and a decline of glycogen, but also significantly decreased fecundity and
C
Las titer in ovaries. Combined
in vivo
and
in vitro
experiments showed that miR-34 suppresses
DcAKHR
expression by binding to its 3’ untranslated region, whilst overexpression of miR-34 resulted in a decline of
DcAKHR
expression and
C
Las titer in ovaries and caused defects that mimicked
DcAKHR
knockdown phenotypes. In addition, knockdown of
DcAKH
and
DcAKHR
significantly reduced juvenile hormone (JH) titer and JH signaling pathway genes in fat bodies and ovaries, including the JH receptor,
methoprene-tolerant
(
DcMet
), and the transcription factor,
Krüppel homolog 1 (DcKr-h1)
, that acts downstream of it, as well as the egg development related genes
vitellogenin 1-like
(
DcVg-1-like
),
vitellogenin A1-like
(
DcVg-A1-like
) and the vitellogenin receptor (
DcVgR
). As a result,
C
Las hijacks AKH/AKHR-miR-34-JH signaling to improve
D. citri
lipid metabolism and fecundity, while simultaneously increasing the replication of
C
Las, suggesting a mutualistic interaction in
D. citri
ovaries with
C
Las.
Decoupling of the onset of anharmonicity between a protein and its surface water around 200 K
Authors: Zheng Lirong , Zhou Bingxin , Wu Banghao , Tan Yang , Huang Juan , Tyagi Madhusudan , García Sakai Victoria , Yamada Takeshi , O'Neill Hugh , Zhang Qiu , Hong Liang
Published: March 2024

The protein dynamical transition at ~200 K, where the biomolecule transforms from a harmonic, non-functional form to an anharmonic, functional state, has been thought to be slaved to the thermal activation of dynamics in its surface hydration water. Here, by selectively probing the dynamics of protein and hydration water using elastic neutron scattering and isotopic labeling, we found that the onset of anharmonicity in the two components around 200 K is decoupled. The one in protein is an intrinsic transition, whose characteristic temperature is independent of the instrumental resolution time, but varies with the biomolecular structure and the amount of hydration, while the one of water is merely a resolution effect.
ASAR lncRNAs control DNA replication timing through interactions with multiple hnRNP/RNA binding proteins
Authors: Thayer Mathew , Heskett Michael B , Smith Leslie G , Spellman Paul T , Yates Phillip A
Published: March 2024

ASARs are a family of very-long noncoding RNAs that control replication timing on individual human autosomes, and are essential for chromosome stability. The eight known ASAR lncRNAs remain closely associated with their parent chromosomes. Analysis of RNA-protein interaction data (from ENCODE) revealed numerous RBPs with significant interactions with multiple ASAR lncRNAs, with several hnRNPs as abundant interactors. An ~7 kb domain within the ASAR6-141 lncRNA shows a striking density of RBP interaction sites. Genetic deletion and ectopic integration assays indicate that this ~7 kb RNA binding protein domain contains functional sequences for controlling replication timing of entire chromosomes in cis. shRNA-mediated depletion of 10 different RNA binding proteins, including HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, or HNRNPUL1, results in dissociation of ASAR lncRNAs from their chromosome territories, and disrupts the synchronous replication that occurs on all autosome pairs, recapitulating the effect of individual ASAR knockouts on a genome-wide scale. Our results further demonstrate the role that ASARs play during the temporal order of genome-wide replication, and we propose that ASARs function as essential RNA scaffolds for the assembly of hnRNP complexes that help maintain the structural integrity of each mammalian chromosome.
Mechanosensory neurons under pressure
Authors: Cram Erin
Published: March 2024

A build-up of eggs in the uterus of the nematode C. elegans triggers the release of large extracellular vesicles, called exophers, from neurons that are sensitive to mechanical forces.
Role Played by Biochemical and Hematological Parameters in the Prediction of Cardiovascular Risk
Authors: Nuwanthika W. K. T. , Welivitigoda D. I. K. , Senadeera S. P. N. N. , Kottahachchi D. U. , Ranaweera C. B. , Wijesighe N.
Published: March 2024

Introduction: The term “cardiovascular diseases” (CVDs) refers to any disease affecting the heart or blood vessels. CVDs are the most common cause of death worldwide and the most responsible reason for 10% of deaths in the early 20th century. These CVDs can emerge as myocardial infarction or ischemic heart disease, stroke, and congestive heart failure. For diagnosing CVDs, physical, radiological investigations, and laboratory investigations for cardiac enzymes and lipid profile are used. In clinical practices, cardiovascular risk prediction models are very important in the identification, prevention, and staging of the severity of CVDs. Framingham Risk Score-coronary heart disease, Framingham Risk Score-cardiovascular disease, QRESEARCH-cardiovascular risk algorithm, Joint British Society risk-calculator-3, WHO/ISH CVD risk prediction charts, and Atherosclerotic Cardiovascular Disease risk-estimator are some of the available CVD risk estimators. However, many of these estimators can be used only to evaluate individuals more than 40 years and to assess the risk for 10-years.  Therefore, new risk estimators are needed to overcome the deficiencies of the available risk estimators. Research have been done in novel directions; on ratios of routinely performing hematological and biochemical laboratory parameters such as Neutrophil to Lymphocyte Ratio (NLR), Platelet to Lymphocyte Ratio (PLR), and Aspartate aminotransferase to Alanine aminotransferase ratio (AST/ALT). The outcomes indicate a relationship between the above-said ratios and the risk for CVDs. 

Conclusion: On such grounds, this review describes the importance of developing a CVD risk estimator by amalgamating some of the biochemical and hematological parameters, including NLR, PLR, and AST/ALT ratios, aiming to overcome the existing shortcomings.
Digital Collaborative Tools, Strategic Communication, and Social Capital: Unveiling the Impact of Digital Transformation on Organizational Dynamics
Authors: Olaniyi Oluwaseun Oladeji , Ugonnia Jennifer Chinelo , Olaniyi Folashade Gloria , Arigbabu Aisha Temitope , Adigwe Chinasa Susan
Published: March 2024

The rapid advancement of digitalization has significantly impacted how organizations operate, with collaborative tools like Asana, Trello, and Slack becoming integral to fostering internal communication, collaboration, and social capital. Despite their widespread adoption, the comprehensive impact of these tools on organizational culture, employee engagement, and social capital remains underexplored. This study aims to bridge this gap by examining the potential of digital collaborative tools to enhance organizational social capital and exploring the role of strategic communication and digital inclusivity in this process. Utilizing a quantitative research approach, data were collected from 557 professionals through a survey distributed via LinkedIn. The questionnaire, designed with Likert scale closed-ended questions, focused on the utilization of collaborative tools and their perceived impact on communication, collaboration, and social capital within organizations. Multiple regression analysis was employed to test four hypotheses related to the effects of digital tool usage on organizational dynamics. The findings reveal that digital tools significantly enhance communication and collaboration within organizations, with the frequency and purpose of tool usage being key factors. Moreover, digital tool usage for networking and the quality of interactions facilitated by these tools were found to positively impact the development of social capital. However, challenges in integrating digital tools were shown to negatively affect this development, though technical support and training could mitigate these effects. Interestingly, the introduction of a 'social score' feature within digital tools was perceived positively, indicating its potential to further engage users and enrich organizational social capital. This study underscores the importance of adopting a strategic approach to implementing digital tools, emphasizing their role beyond operational efficiency to include the enhancement of social interactions and organizational culture. Recommendations include fostering digital tool usage for networking, providing adequate technical support and training, and exploring the integration of 'social score' features to incentivize engagement and collaboration.
Front-line Demonstration: An Effective Communication Approach for Dissemination of Sustainable Rice Production Technology
Authors: Bisen Uttam Kumar , Solanki Ramkrishna Singh , Gaur Vikram Singh
Published: March 2024

During four consecutive Kharif seasons in the years 2016, 2017, 2018 and 2019, front-line demonstrations were carried out in farmer's fields by the College of Agriculture, Waraseoni (Balaghat), Madhya Pradesh in the agroclimatic zone of the Chhattisgarh plains to assess the performance of rice Hybrids / varieties JRH-5, JRH-19, JR-81 and JRB-1 (developed by Jawaharlal Nehru Agriculture University, Jabalpur) under irrigated ecosystem. Front-line demonstrations, or FLDs, were carried out using a scientific package of rice technology practices. The yield and economic data of the plots that were on display were examined, evaluated, and examined with farmer practices (MTU-1010). With only an average of Rs. 2625/ha as an additional input cost in demonstrations using enhanced production technologies in FLDs, the mean grain yield increased by 23.80% over current farmer practices.Extension gap (10.48 q/ha), Technology gap (10.26 q/h) and the technology index is 15.79%. It is concluded that wide gap existed in potential and demonstration yield in high yielding rice varieties due to technology and extension gap in Balaghat District of Madhya Pradesh.By conductingfront line demonstrations of proven technologies, yield potential of rice can be increased to a greatextent. This will substantially increase the income as well as the livelihood of the farming community.
NMR Analysis of Thiodiglycol Oxidation by Mammalian Alcohol Dehydrogenases
Authors: Novak Mark J. , Brimfield Alan A.
Published: September 2006

Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for elucidating the metabolism of xenobiotics, as it allows for the least ambiguous assignment of chemical structure when compared to other forms of spectroscopy. In addition, it is a sensitive technique that can reveal the presence of transient species that otherwise would not be detected by utilizing either large‐scale batch processes or other forms of spectroscopic analyses. The primary focus of this unit describes the use of NMR to identify metabolites arising from the oxidation of thiodiglycol by equine and human variants of alcohol dehydrogenase (ADH). Given that it is often risky to base metabolism studies on a single form of spectroscopy, a spectrophotometric method is also presented. In addition, incorporation of independent organic syntheses in conjunction with the spectroscopic studies to further solidify structural identification of the ADH metabolites is presented.
Measurement of the Vitamin E Metabolites, Carboxyethyl Hydroxychromans (CEHCs), in Biological Samples
Authors: Leonard Scott W. , Traber Maret G.
Published: September 2006

Metabolites of α‐ and γ‐tocopherol, 2,5,7,8‐tetramethyl‐2‐(2′‐carboxyethyl)‐6‐hydroxychroman (α‐CEHC) and 2,7,8‐trimethyl‐2‐(β‐carboxyethyl)‐6‐hydroxychroman (γ‐CEHC), respectively, are produced in the liver and have been measured in biological fluids and tissue. Compared to α‐tocopherol concentrations, metabolite concentrations are as much as a factor of a thousand lower, requiring extremely sensitive methodology to attain accurate measurements. This unit presents a protocol for CEHC extraction from biological samples, and describes very specific and sensitive HPLC/MS analysis.
Overview of Biacore Systems and Their Applications
Authors: Jason‐Moller Laure , Murphy Michael , Bruno JoAnne
Published: September 2006

Surface plasmon resonance (SPR) allows for the investigation of the functional nature of binding interactions and provides detailed kinetic information across a wide range of molecular weights, including small molecules, all without the use of labels. Here the various Biacore instrument platforms and their primary uses, ranging from semi‐automated systems designed for simple, flexible basic research to fully automated, high‐throughput systems, and systems designed to function in regulated environments, are all highlighted. The available sensor chip surfaces and immobilization techniques are also discussed. Biacore SPR biosensors can be used for a wide variety of assays, including specificity, active concentration measurement, kinetics, and affinity and thermodynamic parameters. Biacore SPR biosensors, which measure real‐time analysis of biospecific interactions without the use of labeled molecules, can be used for a wide variety of protein interaction assays. In this unit, examples and recommendations for studying protein interactions with a variety of molecules are provided. This unit also shows how the technology can be used to determine binding specificity, active concentration measurements, and the determination of kinetic and thermodynamic parameters.
Determination of Compound Binding to Plasma Proteins
Authors: Dow Natasha
Published: September 2006

The pharmacokinetic and pharmacodynamic properties of a compound are profoundly affected by the extent of its binding to plasma proteins. Consequently, the determination of the plasma protein binding properties of a compound is essential during drug development and is increasingly required during lead prioritization. The protocols described in this unit detail the techniques of ultrafiltration and equilibrium dialysis for determination of plasma protein binding. Equilibrium dialysis is the more robust of the two techniques, whereas ultrafiltration is the more rapid. Two versions of the equilibrium dialysis technique are provided: the traditional approach and a higher‐throughput 96‐well plate version.
In Vitro Species Comparisons and Metabolite Identification
Authors: Dow Paul
Published: September 2006

The metabolism and elimination of a drug has a direct bearing on its potential to cause toxicity in humans as well as to influence its clinical activity. The confidence with which data from preclinical safety studies, particularly toxicology studies, can be extrapolated to humans depends upon knowing whether humans are exposed to the same chemical entities (i.e., a parent drug and its metabolites) as the laboratory animals used to study toxicity and pharmacological responses. To address this issue, the FDA Guidance for Industry (1997) recommends the use of in vitro interspecies metabolic comparisons at an early stage in the drug development process. Knowledge about the metabolism of a compound is also important from a drug discovery perspective. In vitro studies provide a convenient and cost‐effective way to obtain this information. This unit presents protocols for comparing metabolite profiles across several species, as well as methods for identifying metabolites.
Using Biacore to Measure the Binding Kinetics of an Antibody‐Antigen Interaction
Authors: Murphy Michael , Jason‐Moller Laure , Bruno JoAnne
Published: September 2006

The optical phenomenon of surface plasmon resonance (SPR) used by Biacore systems enables the detection and measurement of protein‐protein interactions in real time, without the use of labels. In this unit, the application of Biacore technology to measure a protein‐protein interaction is described using an antibody and its antigen as an example. The affinity of the antibody for its antigen is determined by measuring the binding kinetics of the interaction. The protocols are divided into three major steps that are required for measuring binding kinetics using Biacore: (1) surface preparation, (2) assay development, and (3) kinetic analysis.
Generation, Isolation, and Maintenance of Rodent Mast Cells and Mast Cell Lines
Authors: Jensen Bettina M. , Swindle Emily J. , Iwaki Shoko , Gilfillan Alasdair M.
Published: September 2006

Antigen‐mediated mast cell activation, with subsequent mediator release, is a major initiator of the inflammatory allergic response associated with such conditions as asthma. A comprehensive understanding of the principles involved in this process therefore is key to the development of novel therapies for the treatment of these disease states. In vitro models of mast cell function have allowed significant progress to be made in the recognition of the fundamental principles of mast cell activation via the high‐affinity IgE receptor (FcɛRI) and, more recently, other receptors expressed on mast cells. In addition to human mast cells, the major cell culture systems employed to investigate these responses are rat and mouse peritoneal mast cells, mouse bone‐marrow‐derived mast cells, the rat basophilic leukemia cell line RBL‐2H3, and the mouse MC/9 mast cell line. In this unit, we describe the protocols used for the isolation and/or culture of these cells and discuss the relative merits of their use.
Safe Use of Radioisotopes
Authors: Meisenhelder Jill , Semba Kentaro
Published: September 2006

The use of radioisotopes to label specific molecules in a defined way has greatly furthered the discovery and dissection of biochemical pathways. The development of methods to synthesize such tagged biological compounds inexpensively on an industrial scale has enabled them to be used routinely in laboratory protocols, including many detailed in this manual. Although most of these protocols involve the use of only microcurie amounts of radioactivity, some (particularly those describing the metabolic labeling of proteins or nucleic acids within cells) require amounts on the order of millicuries. In all cases where radioisotopes are used, depending on the quantity and nature of the isotope, certain precautions must be taken to ensure the safety of the scientist. It is essential to use good safety practices and proper protection to handle radioactive substances. This unit discusses handling, storage, and disposal of the isotopes most frequently used in biological research.
Measuring Retrograde Transport to the Trans‐Golgi Network
Authors: Amessou Mohamed , Popoff Vincent , Yelamos Belèn , Saint‐Pol Agnès , Johannes Ludger
Published: September 2006

The recently described retrograde transport route is a highly selective pathway that allows some internalized molecules to reach the trans‐Golgi network from early/recycling endosomes, bypassing the recycling route to the plasma membrane and the late endocytic pathway. The non‐toxic receptor‐binding B‐subunit of bacterial Shiga toxin has played an important role in the discovery and molecular dissection of membrane trafficking at the early/recycling endosomes–TGN interface. This unit describes several recent methods for quantitative biochemical and morphological analysis of retrograde transport. The sulfation assay permits the detection and quantification of cargo protein transport from endosomes to the TGN, describing how sulfation‐site peptides can be chemically coupled to cargo proteins. Furthermore, a variant of the sulfation assay on permeabilized cells is presented. The chemical crosslinking theme is extended to horseradish peroxidase for the ultrastructural study of the Shiga toxin–containing early/recycling endosomes by whole mount analysis. Finally, an endocytosis assay describes concomitant analysis of cellular uptake of Shiga toxin and transferrin.
Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
Authors: Snapp Erik L. , Hegde Ramanujan S.
Published: September 2006

Fluorescence resonance energy transfer (FRET) refers to the nonradiative transfer of energy from one fluorescent molecule (the donor) to another fluorescent molecule (the acceptor). Measurement of FRET between two fluorophore‐labeled proteins can be used to infer the subnanometer spatial and temporal characteristics of protein interactions in their native cellular environment. Multiple experimental methods exist for measuring FRET. The method that can be most widely and simply implemented, quantified, and interpreted is the acceptor‐photobleaching FRET technique. In this method, the presence of FRET between a donor and acceptor is revealed upon destruction (by photobleaching) of the acceptor. Acceptor photobleaching can be exploited to detect changes in the composition and organization of subunit proteins within a multiprotein complex and to even gain insight into relative stoichiometries of proteins within the complex. In this unit, strategies, tools, and background for designing and interpreting acceptor‐photobleaching FRET experiments in cells are described.
Comparative Protein Structure Modeling Using Modeller
Authors: Eswar Narayanan , Webb Ben , Marti‐Renom Marc A. , Madhusudhan M.S. , Eramian David , Shen Min‐yi , Pieper Ursula , Sali Andrej
Published: September 2006

Functional characterization of a protein sequence is one of the most frequent problems in biology. This task is usually facilitated by accurate three‐dimensional (3‐D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3‐D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3‐D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target‐template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from
Trichomonas vaginalis
(TvLDH) is described as an example. The download and installation of the MODELLER software is also described.
Detection, Isolation, and Identification of Vibrio cholerae from the Environment
Authors: Huq Anwar , Grim Christopher , Colwell Rita R. , Nair G. Balakrish
Published: September 2006

Microbiological techniques for sampling the aquatic realm have become increasingly sophisticated, especially with advances in molecular biology. These techniques have been used to detect microorganisms that cannot be cultured by conventional bacteriological methods. This has resulted in a deeper and a clearer understanding of the ecology and epidemiology of microorganisms. Important advances have been made in isolation, detection, and identification of Vibrio cholerae over the past decade. The understanding that V. cholerae, like several other pathogenic bacteria, can enter into a state known as “viable but nonculturable” (VBNC) provided important clues on the epidemiology of the pathogen and its ability to cause sudden explosive epidemics at multiple places almost simultaneously. The advances in techniques have also allowed investigators to discern the intricate aspects of the ecology of this pathogen in the aquatic world. In this unit, we present the most accepted methods for the isolation and detection of V. cholerae.
Hamster Model of Leptospirosis
Authors: Haake David A.
Published: September 2006

This unit describes the techniques involved in the hamster model of leptospirosis. Hamsters are exquisitely susceptible to infection with pathogenic Leptospira species. The LD50 of virulent Leptospiral strains by intraperitoneal inoculation of 3‐ to 4‐week‐old hamsters is as low as a single organism. The pathogenesis of severe leptospirosis in the hamster is a model of accidental infections observed in nature in many mammals, including humans. Animal husbandry issues and the reproducibility of the hamster model make it the animal model of choice for Leptospiral vaccine and antibiotic treatment studies.
Human Immunodeficiency Viruses: Propagation, Quantification, and Storage
Authors: Richards Kathryn H. , Clapham Paul R.
Published: September 2006

Described in this unit are basic protocols frequently used in the research of human immunodeficiency viruses (HIVs). Provided are methods for propagating and quantifying HIV, as well as recommendations for long‐term storage. Background information about these methods is also provided and includes their advantages, disadvantages, and troubleshooting.
Diffusion and reaction in a porous catalyst slab: Perturbation solutions
Authors: Cardoso Silvana S. S. , Rodrigues Alírio E.
Published: September 2006

The problem of diffusion and reaction in a porous catalyst slab is revisited. A combination of perturbation and integral mathematical techniques are used to derive approximate analytical solutions for the concentration and temperature profiles, as well as the effectiveness factor, for a first order, non‐isothermal reaction. The development of thermal and concentration boundary layers near the surface of the particle is characterized. The internal particle resistances to mass and heat transfer are also calculated. Our analytical solutions are validated by comparison with previous numerical results. © 2006 American Institute of Chemical Engineers AIChE J, 2006
Role of neurofilament aggregation in motor neuron disease
Authors: Lin Hong , Schlaepfer William W.
Published: September 2006

A major question in the pathogenesis of motor neuron disease is why motor neurons are selectively susceptible to mutations in widely expressed gene products. Reexamination of motor neuron degeneration due to alterations of neurofilament (NF) expression suggests that disruption of assembly with aggregation of the light neurofilament (NFL) protein may be an upstream event and contributing factor leading to the preferential degeneration of motor neurons. The implications of these findings are that aggregation of NFL is not only a triggering mechanism to account for the hallmark aggregates of NF protein in sporadic and familial forms of amyotrophic lateral sclerosis, but that aggregates of NFL may also promote aggregation of wildly expressed proteins that are destabilized by missense mutations, such as by mutations in superoxide dismutase‐1 protein. This review examines the potential role of NFs in determining and promoting the preferential degeneration of motor neurons in motor neuron disease. The underlying premise is that motor neurons are selectively susceptible to alterations in NF expression, that alterations in NF expression lead to NF aggregates in motor neurons, and that elevated levels of NF aggregates provide a favorable microenvironment for the formation of neurotoxic aggregation and degeneration of motor neurons. Ann Neurol 2006
Cleavage of cystatin C is not associated with multiple sclerosis
Authors: Del Boccio Piero , Pieragostino Damiana , Lugaresi Alessandra , Di Ioia Maria , Pavone Barbara , Travaglini Daniela , D'Aguanno Simona , Bernardini Sergio , Sacchetta Paolo , Federici Giorgio , Di Ilio Carmine , Gambi Domenico , Urbani Andrea
Published: September 2006

Recently, Irani and colleagues proposed a C‐terminal cleaved isoform cystatin C (12.5kDa) in cerebrospinal fluid as a marker of multiple sclerosis. In this study, we demonstrate that the 12.5kDa product of cystatin C is formed by degradation of the first eight N‐terminal residues. Moreover, such a degradation is not specific in the cerebrospinal fluid of multiple sclerosis, but rather is given by an inappropriate sample storage at −20°C. We conclude that the use of the 12.5kDa product of cystatin C in cerebrospinal fluid might lead to a fallacious diagnosis of multiple sclerosis. Preanalytical validation procedure is mandatory for proteomics investigations. Ann Neurol 2007
Nanocrystalline Ag from Supramolecular Stabilization of Metals in 4‐tert‐Butylcalix[4]arene Lattices
Authors: Brown Philip O. , Enright Gary D. , Ripmeester John A.
Published: September 2006

Competition between intermolecular forces is a prime method for directing the formation of self‐assembled structures without resorting to the use of covalently modified host molecules. In this study, we demonstrate how the competition between hydrogen bonding, coordinate bonding, and van der Waals interactions guides the formation of a complex crystalline clathrate based on a Ag–ethylenediamine coordination complex and 4‐tert‐butylcalix[4]arene. Owing to the coordinative flexibility of Ag and the stabilizing interactions with the calixarene framework, a four‐coordinate Ag complex is included in the structure. Subsequent removal of the amine by heating the crystalline inclusion compound results in reduction of the Ag+ ion such that metallic Ag nanocrystals are formed, thus indicating that this self‐assembly approach is a viable method for obtaining simple calixarene frameworks loaded with silver. In the light of the experimental evidence, we comment on how this process relates to the reduction of silver in other systems.
Endoplasmic reticulum‐retention C‐terminal sequence enhances production of an 11S seed globulin fromAmaranthus hypochondriacusinPichia pastoris
Authors: Medina‐Godoy Sergio , Valdez‐Ortiz Angel , Valverde María Elena , Paredes‐López Octavio
Published: September 2006

The methylotrophic yeastPichia pastoriswas used to express an 11S seed globulin fromAmaranthus hypochondriacus.Three different plasmids were tested for expression of amarantin. One of them, which included the untranslated regions (UTR) of the full cDNA, failed to express the amarantin under tested conditions, whereas the other plasmids, one without UTR and the other similar but including the endoplasmic reticulum‐retention signal KDEL, were able to express the proamarantin inP. pastoris. After 48 h of induction, KDEL‐proamarantin had accumulated quite significantly compared to unmodified proamarantin. Different solubilization patterns were also obtained from both proamarantin versions; only soluble protein was obtained from the system that included the KDEL retrieval signal. Protein fractionation was carried out by differential precipitation with ammonium sulfate, and proamarantin purification was performed using an HPLC ion exchange column. The endoplasmic reticulum‐retention C‐terminal sequence (KDEL retrieval signal), not commonly employed in this heterologous expression system, can therefore be used to enhance accumulation of recalcitrant protein inP. pastoris.The results obtained here also suggest that this expression system is suitable for expression and evaluation of engineered seed globulin proteins.
Plant growth promoting potential of free‐living diazotrophs and other rhizobacteria isolated from Northern Indian soil
Authors: Ahmad Farah , Ahmad Iqbal , Aqil Farrukh , Ahmed Wani Aijaz , Sousche Yogesh S.
Published: September 2006

The viable count of free‐living diazotrophic bacteria in different crop rhizospheres varied from 1.11 × 104 to 8.5 × 105 CFU/g of soil. The majority of the diazotrophs phenotypically belong to either Azotobacter chroococcum, non‐A. chroococcum type and to a heterogenous group tentatively named putative nitrogen‐fixing (PNF) bacteria. In this study, 25 isolates of the PNF group were screened for their multiple plant growth‐promoting (PGP) traits and grouped into 5 PGP types. An isolate, PNF11 showed promising PGP potential in vitro and was characterized as a species of Achromobacter by 16S rRNA analysis. The isolate PNF11 along with three other previously isolated PGP bacteria, Azotobacter sp. (AZS3), fluorescent pseudomonas (Ps5), Bacillus sp. (Bc1) were selected for crop inoculation response in green house experiment on Vigna radiata var.T44. Plants from inoculated and control pots were sampled and analyzed at 30, 45 and 60 days after sowing for various vegetative, nodule‐related data and yield parameters. The findings indicated that selected isolate of PNF bacteria, and other PGP isolates with multiple activities significantly improve the plant growth parameters, yield parameters of Vigna radiata T44 over control and also show good compatibility with Bradyrhizobium inoculation.
Genetic analysis and epigenetic silencing ofAt4CL1andAt4CL2expression in transgenicArabidopsis
Authors: Soltani Bahram M. , Ehlting Jürgen , Douglas Carl J.
Published: September 2006

4‐coumarate::CoA ligase (4CL) gene family members are involved in channeling carbon flow into branch pathways of phenylpropanoid metabolism. TransgenicArabidopsisplants containing theAt4CL1orAt4CL2promoter fused to the beta‐glucuronidase (GUS) reporter gene show developmentally regulated GUS expression in the xylem tissues of the root and shoot. To identify regulatory genes involved in the developmental regulation ofAt4CLand other phenylpropanoid‐specific genes, we generated ethyl methyl sulfate mutagenized populations ofAt4CL1::GUSandAt4CL2::GUStransgenic lines and screened ∼16 000 progeny for reduced or altered GUS expression. Several lines with reproducible patterns of reduced GUS expression were identified. However, the GUS‐expression phenotype segregated in a non‐Mendelian manner in all of the identified lines. Also, GUS expression was restored by 5‐azacytidine (aza) treatment, suggesting inhibitory DNA methylation of the transgene. Southern analysis confirmed DNA methylation of the proximal promoter sequences of the transgene only in the mutant lines. In addition, retransformation ofAt4CL::GUSlines with furtherAt4CLpromoter constructs enhanced the GUS‐silencing phenotype. Taken together, these results suggest that the isolated mutants are epimutants. Apparently, two different modes of silencing were engaged in theAt4CL1::GUSandAt4CL2::GUSsilenced lines. While silencing in the seedlings of theAt4CL1::GUSlines was root specific in seedlings, it affected all organs in theAt4CL2::GUSlines. Also,At4CL1::GUStransgene silencing was confined to the transgene butAt4CL2::GUSsilencing extended to the endogenousAt4CL2gene. Organ‐specific silencing of theAt4CL1::GUStransgene cannot be explained by current models in the literature.
Two new disposable bioreactors for plant cell culture: The wave and undertow bioreactor and the slug bubble bioreactor
Authors: Terrier Bénédicte , Courtois Didier , Hénault Nicolas , Cuvier Arnaud , Bastin Maryse , Aknin Aziz , Dubreuil Julien , Pétiard Vincent
Published: September 2006

The present article describes two novel flexible plastic‐based disposable bioreactors. The first one, the WU bioreactor, is based on the principle of a wave and undertow mechanism that provides agitation while offering convenient mixing and aeration to the plant cell culture contained within the bioreactor. The second one is a high aspect ratio bubble column bioreactor, where agitation and aeration are achieved through the intermittent generation of large diameter bubbles, “Taylor‐like” or “slug bubbles” (SB bioreactor). It allows an easy volume increase from a few liters to larger volumes up to several hundred liters with the use of multiple units. The cultivation of tobacco and soya cells producing isoflavones is described up to 70 and 100 L working volume for the SB bioreactor and WU bioreactor, respectively. The bioreactors being disposable and pre‐sterilized before use, cleaning, sterilization, and maintenance operations are strongly reduced or eliminated. Both bioreactors represent efficient and low cost cell culture systems, applicable to various cell cultures at small and medium scale, complementary to traditional stainless‐steel bioreactors. Biotechnol. Bioeng. 2007;96:914–923. © 2006 Wiley Periodicals, Inc.
Expansion of mouse embryonic stem cells on microcarriers
Authors: Abranches Elsa , Bekman Evguenia , Henrique Domingos , Cabral Joaquim M.S.
Published: September 2006

Embryonic stem (ES) cells have been shown to differentiate in vitro into a wide variety of cell types having significant potential for tissue regeneration. Therefore, the operational conditions for the ex vivo expansion and differentiation should be optimized for large‐scale cultures. The expansion of mouse ES cells has been evaluated in static culture. However, in this system, culture parameters are difficult to monitor and scaling‐up becomes time consuming. The use of stirred bioreactors facilitates the expansion of cells under controlled conditions but, for anchorage‐dependent cells, a proper support is necessary. Cytodex‐3, a microporous microcarrier made up of a dextran matrix with a collagen layer at the surface, was tested for its ability to support the expansion of the mouse S25 ES cell line in spinner flasks. The effect of inocula and microcarrier concentration on cell growth and metabolism were analyzed. Typically, after seeding, the cells exhibited a growth curve consisting of a short death or lag phase followed by an exponential phase leading to the maximum cell density of 2.5–3.9 × 106 cells/mL. Improved expansion was achieved using an inoculum of 5 × 104 cells/mL and a microcarrier concentration of 0.5 mg/mL. Medium replacement allowed the supply of the nutrients and the removal of waste products inhibiting cell growth, leading to the maintenance of the cultures in steady state for several days. These conditions favored the preservation of the S25 cells pluripotent state, as assessed by quantitative real‐time PCR and immunostaining analysis. Biotechnol. Bioeng. 2007;96:1211–1221. © 2006 Wiley Periodicals, Inc.
High and compact formation of baculoviral polyhedrin‐induced inclusion body by co‐expression of baculoviral FP25 in Escherichia coli
Authors: Li Lin , Kim Young Soo , Hwang Dong Soo , Seo Jeong Hyun , Jung Hee Jung , Du Juan , Cha Hyung Joon
Published: September 2006

Previously, we found that baculoviral polyhedrin (Polh) can successfully be used in Escherichia coli as a fusion partner for the expression of special foreign proteins as inclusion bodies, and the resulting, easily isolatable Polh‐induced fusion inclusion bodies had almost the same characteristics as the native Polh. Here, we investigated the effects of co‐expression of baculoviral FP25 protein on Polh‐induced inclusion‐body production in an E. coli expression system, as FP25 is known to be involved specifically in polyhedra formation. Using several analytical tools, including SDS–PAGE, pronase proteolysis, solubilization under alkaline conditions, and electron microscopy, we found that co‐expressed FP25 was associated with Polh‐induced inclusion bodies and that its co‐expression led to formation of compact inclusion bodies as well as high production levels. We confirmed that FP25 co‐expression induced higher production levels of other heterologous protein, antimicrobial peptide Hal18, fused with aggregation‐prone Polh. Therefore, co‐expression of baculoviral FP25 can be promisingly used to increase the levels of baculoviral Polh‐fused foreign proteins, especially harmful proteins, expressed as inclusion bodies in an E. coli expression system. Biotechnol. Bioeng. 2007;96:1183–1190. © 2006 Wiley Periodicals, Inc.
Sulfidogenic fluidized‐bed treatment of metal‐containing wastewater at low and high temperatures
Authors: Sahinkaya Erkan , Özkaya Bestamin , Kaksonen Anna H. , Puhakka Jaakko A.
Published: September 2006

The applicability of a fluidized‐bed reactor (FBR)‐based sulfate reducing bioprocess was investigated for the treatment of iron‐containing (40–90 mg/L) acidic wastewater at low (8°C) and high (65°C) temperatures. The FBRs operated at low and high temperatures were inoculated with cultures of sulfate‐reducing bacteria (SRB) originally enriched from arctic and hot mining environments, respectively. Ethanol was supplemented as carbon and electron source for SRB. At 8°C, ethanol oxidation and sulfate reduction rates increased steadily and reached 320 and 265 mg/L·day, respectively, after 1 month of operation. After this point, the rates did not change significantly during 130 days of operation. Despite the complete ethanol oxidation and iron precipitation, the average sulfate reduction efficiency was 35 ± 4% between days 30 and 130 due to the accumulation of acetate. At 65°C, a rapid startup was observed as 99.9, 46, and 29% ethanol, sulfate, acetate removals, in respective order, were observed after 6 days. The feed pH was decreased gradually from its initial value of 6 to around 3.7 during 100 days of operation. The wastewater pH of 4.3–4.4 was neutralized by the alkalinity produced in acetate oxidation and the average effluent pH was 7.8 ± 0.8. As in the low temperature FBR, acetate accumulated. Hence, the oxidation of acetate is the rate‐limiting step in the sulfidogenic ethanol oxidation by thermophilic and psychrotrophic SRB. The sulfate reduction rate is three times and acetate oxidation rate is four times higher at 65°C than at 8°C. Biotechnol. Bioeng. 2007;96:1064–1072. © 2006 Wiley Periodicals, Inc.
Modeling and rendering of realistic waterfall scenes with dynamic texture sprites
Authors: Guan Yu , Chen Wei , Zou Lincan , Zhang Long , Peng Qunsheng
Published: September 2006

We propose an efficient approach for authoring dynamic and realistic waterfall scenes based on an acquired video sequence. Traditional video based techniques generate new images by synthesizing 2D samples, i.e., texture sprites chosen from a video sequence. However, they are limited to one fixed viewpoint and cannot provide arbitrary walkthrough into 3D scenes. Our approach extends this scheme by synthesizing dynamic 2D texture sprites and projecting them into 3D space. We first generate a set of basis texture sprites, which capture the representative appearance and motions of waterfall scenes contained in the video sequence. To model the shape and motion of a new waterfall scene, we interactively construct a set of flow lines taking account of physical principles. Along each flow line, the basis texture sprites are manipulated and animated dynamically, yielding a sequence of dynamic texture sprites in 3D space. These texture sprites are displayed using the point splatting technique, which can be accelerated efficiently by graphics hardware. By choosing varied basis texture sprites, waterfall scenes with different appearance and shapes can be conveniently simulated. The experimental results demonstrate that our approach achieves realistic effects and real‐time frame rates on consumer PC platforms. Copyright © 2006 John Wiley & Sons, Ltd.