bluuebunny/crossref_metadata_2025 huggingface dataset:
Residents’ attitudes and perceptions towards cruise tourism development: A case study of Cartagena de Indias (Colombia)
Authors: Brida Juan Gabriel , Riaño Eugenia , Aguirre Sandra Zapata
Published: December 2011

Cruise tourism is growing faster than any other sector of the tourism industry, producing different impacts on destinations. From the social and economic viewpoint, the interactions between the different actors of the exchange process – cruise passengers, crew, residents, and producers of the tourism products – can bring both positive and negative consequences. The aim of this paper is to analyse how the local population perceives the impacts of cruise tourism and which factors do affect the relationship between impacts and perceptions’ formation. The research involves primary data collection in Cartagena de Indias during the peak of the cruise season in the last trimester of 2009. The number and quality of the 1,004 questionnaires collected allowed us to perform a quantitative analysis of the hosts’ perceptions and attitudes. A cluster analysis demonstrated the existence of four different groups, within which members have common features and similar perceptions and attitudes. In general, it has revealed a positive recognition of the economic impacts of tourism. Also, social and cultural impacts are recognised to be positive, but at a lower degree. In terms of future tourism polices, the different groups identified in the cluster analysis exert different positions.
‘Estimating tomorrow’s tourist arrivals’: forecasting the demand for China’s tourism using the general-to-specific approach
Authors: Ayeh Julian K , Lin Shanshan
Published: December 2011

Accurate forecast of inbound tourism demand is vital for the tourism industry as well as government economic policy and decision making. This article sought to identify the factors which influence the demand for China’s tourism with the aid of econometric models and to generate forecasts of international tourist arrivals to China from five major long-haul source markets. Using the general-to-specific modelling approach, the demand for tourism in China by the residents of Australia, Canada, Germany, the United Kingdom and the United States of America is modelled and forecasted. The empirical results indicate that the ‘word of mouth effect’, income levels in the origin country, the costs of tourism in both China and competing destinations are the crucial factors that determine the demand for China’s tourism by residents of the five origin countries. The forecasts show sluggish growth in tourist arrivals for most of the Western source markets. Findings hold implications for policy formulations.
Dependence of premixed low-temperature diesel combustion on fuel ignitability and volatility
Authors: Li T , Moriwaki R , Ogawa H , Kakizaki R , Murase M
Published: December 2011

A comprehensive study of fuel property effects in internal combustion engines is required to enable fuel diversification as well as the development of applications to advanced engines for operation with a variety of combustion modes. The objective of this paper is to investigate the effects of fuel ignitability and volatility over a wide range of premixed low-temperature combustion (LTC) modes in diesel engines. A total of 23 fuels were prepared from commercial gasoline, kerosene, and diesel as baseline fuels and with the addition of additives, to generate a cetane number (CN) range from 11 to 75. Experiments with a single-cylinder diesel engine operated in moderately advanced-injection LTC modes were conducted to evaluate these fuels. The combustion phasing is demonstrated to be a good indicator to estimate the in-cylinder peak pressure, exhaust gas emissions, and thermal efficiency in the LTC mode. Fuel ignitability affects the combustion phasing by changing the ignition delay. The predicted cetane number (PCN) based on fuel molecular structure analysis can be fitted to the ignition delays with a higher coefficient of determination than CN, suggesting good potential as a fuel ignitability measure over a wide range. The stable operating load range in the smokeless LTC mode depends more on the actual ignition delay or PCN rather than CN. With fixed injection timing and intake oxygen concentration, O2in, only when PCN < 40, the load range can be expanded significantly to higher loads. By holding the combustion phasing at top dead centre and varying intake oxygen concentration, the nitrogen oxides and smoke emissions become limitations of the load expansion for some fuels. The effects of fuel volatility on the characteristics of LTC are small compared to ignitability. Finally, the operational injection timing range and robustness of the LTC to fuel ignitability are examined, showing that the advantageous ignitability range becomes narrower, with fuel ignitability decreasing.
Ethnography goes online: towards a user-centred methodology to research interpersonal communication on the internet
Authors: Beneito-Montagut Roser
Published: December 2011

Ethnographic research is increasingly concerned with how the internet operates within our everyday life. This article attempts to offer a methodological contribution of online communication and an exploration of initial empirical data generated with this methodology. The article calls for a specification of how ethnography can be applied appropriately to the study of relationships online. It departs from the real versus virtual dichotomy, offering a user-centred methodology to study interpersonal communications on the internet. It suggests the use of three main strategies to pay tribute to the characteristics of uses online: multi-situated, online and offline, and flexible and multimedia data collection methods. This approach facilitates a holistic analysis of the way in which social information and communication technologies operate within society in everyday life. It deals with the problem of defining the setting of research online and proposes an expanded ethnography. The article specifies details of this methodology for research into interpersonal communications and emotions online. It does so by drawing on empirical data generated in a study on everyday life and emotions on the internet. Epistemic questions related to this methodological approach will also be discussed. Overall, the exemplification suggests that the methodological approach proposed here is able to capture the uses and understandings of the internet.
Intimate reflections: private diaries in qualitative research
Authors: Harvey Laura
Published: December 2011

This article explores the use of private diaries in qualitative research about intimate everyday experiences. The article first reflects on existing diary-based research, then examines data from a small-scale UK study about the negotiation of condom use in heterosex to pose questions about the kinds of data made available when participants use private diaries as a prompt in qualitative interviews. The article discusses the use of private diaries as a way to explore ambivalent, everyday experiences and interrogates the role of diaries as a form of confessional or measurement of private life. It explores how combined researcher intimate diaries and field note journals can be used in reflexive qualitative sexualities research. The article finally examines the potential for diaries as a form of research intervention in participants’ intimate lives.
Mundane reason, membership categorization practices and the everyday ontology of space and place in interview talk
Authors: Housley William , Smith Robin James
Published: December 2011

In this article we aim to utilise and apply ethnomethodological and interactionist principles to the analysis of members’ situated accounts of regenerated urban space. With reference to previous empirical studies we apply membership categorization analysis and the concept of mundane reason to data gathered from situated street level interviews carried out as part of a programme of ethnographic research into the regenerated setting of Cardiff Bay. The article demonstrates that these data yield sociological insight into social actors’ interpretive and interactional reasoning in relation to the negotiation, navigation and comprehension of space and place. Through this work the patterned signatures of the urban interactional order can be identified. Furthermore, we illustrate the forms of emic rationality associated with the everyday and ubiquitous constitution of urban space as a meaningful, and thence cultural, milieu. It is our claim that an appreciation of these urban forms of reasoning is important in the ethnographic, sociological and geographical analysis of space and place.
The co-construction of couplehood in dementia
Authors: Molyneaux Victoria J. , Butchard Sarah , Simpson Jane , Murray Craig
Published: December 2011

Much research has investigated the impact of dementia on spousal relationships. Most often this is from the point of view of the spouse, but occasionally people with dementia are interviewed independently or in parallel. The current study was novel in its aim to understand ‘couplehood’ as it is co-constructed by the couple when one partner has dementia and employed an innovative design that involved interviewing couples together about their relationship. Using grounded theory methodology, the study identified the changing identities within couplehood and the strategies couples employ to maintain their relationship, including normalizing, externalizing, sharing the experience and reframing the experience in light of the past. These findings are discussed in relation to other relevant research and suggest that co-constructed accounts of couplehood are valuable not only when researching the experiences of couples with dementia but also in their clinical assessment and treatment.
The state of the digital humanities
Authors: Liu Alan
Published: December 2011

The scholarly field of the digital humanities has recently expanded and integrated its fundamental concepts, historical coverage, relationship to social experience, scale of projects, and range of interpretive approaches. All this brings the overall field (including the related area of new media studies) to a tipping point where it has the potential not just to facilitate the work of the humanities but to represent the state of the humanities at large in its changing relation to higher education in the postindustrial state. Are the digital humanities up to this larger task?
Temp Cyborgs
Authors: Johnson Robin S.
Published: December 2011

This article analyzes three primetime television series: Chuck and Bionic Woman on NBC and Dollhouse on Fox. The narratives and representations in the three programs raise questions about the role of immaterial labor, gender, and technology in contemporary life. Each series articulates and reifies the unequal sexual division of labor around prized technological work. The texts also express the instability of humanity as it becomes ever more intimate with new technology. As cyborgs, the main protagonists are temporary subjects because technology is driven by capital’s need for newer products and newer iterations of existing technology. The potential for liberation and collective activity that comes with the newfound power, skill, and interconnection of cyborgs is closed off. The contemporary cyborg in Chuck and Bionic Woman is alienated and never far from being obsolete, while Dollhouse shows the possibility for working-class resistance.
A Conversation with danah boyd, Microsoft Research
Authors: Turner Fred
Published: December 2011

On July 7, 2011, new media historian Fred Turner interviewed Microsoft Research Senior Researcher danah boyd about her research on teens and new media, queer identities, and the role of the media researcher as a public intellectual in the United States.
Cerebellar Ataxia Rehabilitation Trial in Degenerative Cerebellar Diseases
Authors: Miyai Ichiro , Ito Mizuki , Hattori Noriaki , Mihara Masahito , Hatakenaka Megumi , Yagura Hajime , Sobue Gen , Nishizawa Masatoyo
Published: December 2011

Objective. To investigate short- and long-term effects of intensive rehabilitation on ataxia, gait, and activities of daily living (ADLs) in patients with degenerative cerebellar disease. Methods. A total of 42 patients with pure cerebellar degeneration were randomly assigned to the immediate group or the delayed-entry control group. The immediate group received 2 hours of inpatient physical and occupational therapy, focusing on coordination, balance, and ADLs, on weekdays and 1 hour on weekends for 4 weeks. The control group received the same intervention after a 4-week delay. Short-term outcome was compared between the immediate and control groups. Long-term evaluation was done in both groups at 4, 12, and 24 weeks after the intervention. Outcome measures included the assessment and rating of ataxia, Functional Independence Measure, gait speed, cadence, functional ambulation category, and number of falls. Results. The immediate group showed significantly greater functional gains in ataxia, gait speed, and ADLs than the control group. Improvement of truncal ataxia was more prominent than limb ataxia. The gains in ataxia and gait were sustained at 12 weeks and 24 weeks, respectively. At least 1 measure was better than at baseline at 24 weeks in 22 patients. Conclusions. Short-term benefit of intensive rehabilitation was evident in patients with degenerative cerebellar diseases. Although functional status tended to decline to the baseline level within 24 weeks, gains were maintained in more than half of the participants.
Quadriceps Muscle Weakness, Activation Deficits, and Fatigue With Parkinson Disease
Authors: Stevens-Lapsley Jennifer , Kluger Benzi M. , Schenkman Margaret
Published: December 2011

Background. People with Parkinson disease (PD) typically have complaints of weakness. The mechanisms underlying this deficit have not been well established, although many factors may contribute. Objective. This investigation aimed to characterize quadriceps muscle weakness and activation failure in people with PD and explore whether these deficits were related to disease severity. The authors further sought to examine quadriceps muscle fatigability. Methods. This was a cross-sectional comparison of 17 people with mild-severe PD and 17 healthy adults matched by age, sex, and body mass index (BMI). The Unified Parkinson’s Disease Rating Scale motor score (UPDRS motor) ranged from 9.5 to 61.0. Participants were divided into those with low-PD motor signs (UPDRS motor < 31.7) and high-PD motor signs (UPDRS motor ≥ 31.7). Measures of quadriceps performance included isometric torque, central activation using doublet interpolation, and an isokinetic fatigue test. Results. Participants with high-PD motor signs had significantly more quadriceps weakness and central activation deficits than those with low-PD motor signs or healthy controls. Strength and activation deficits correlated strongly with UPDRS motor score. Quadriceps muscle fatigue was present in healthy controls and in those with low-PD motor signs but not in those with high-PD motor signs. Conclusions. These findings provide additional evidence for lower-extremity strength loss with PD; central activation deficits may account for some of the strength deficits, especially with increased PD motor signs. Also, muscle fatigue did not occur in individuals with a greater degree of PD motor signs, most likely because of insufficient central activation to allow for muscle overload to induce metabolic fatigue.
A Randomized Controlled Trial Comparing 2 Interventions for Visual Field Loss With Standard Occupational Therapy During Inpatient Stroke Rehabilitation
Authors: Mödden Claudia , Behrens Marion , Damke Iris , Eilers Norbert , Kastrup Andreas , Hildebrandt Helmut
Published: December 2011

Background and Purpose. Compensatory and restorative treatments have been developed to improve visual field defects after stroke. However, no controlled trials have compared these interventions with standard occupational therapy (OT). Methods. A total of 45 stroke participants with visual field defect admitted for inpatient rehabilitation were randomized to restorative computerized training (RT) using computer-based stimulation of border areas of their visual field defects or to a computer-based compensatory therapy (CT) teaching a visual search strategy. OT, in which different compensation strategies were used to train for activities of daily living, served as standard treatment for the active control group. Each treatment group received 15 single sessions of 30 minutes distributed over 3 weeks. The primary outcome measures were visual field expansion for RT, visual search performance for CT, and reading performance for both treatments. Visual conjunction search, alertness, and the Barthel Index were secondary outcomes. Results. Compared with OT, CT resulted in a better visual search performance, and RT did not result in a larger expansion of the visual field. Intragroup pre–post comparisons demonstrated that CT improved all defined outcome parameters and RT several, whereas OT only improved one. Conclusions. CT improved functional deficits after visual field loss compared with standard OT and may be the intervention of choice during inpatient rehabilitation. A larger trial that includes lesion location in the analysis is recommended.
Contralesional Motor Cortex Activation Depends on Ipsilesional Corticospinal Tract Integrity in Well-Recovered Subcortical Stroke Patients
Authors: Lotze Martin , Beutling Willy , Loibl Moritz , Domin Martin , Platz Thomas , Schminke Ulf , Byblow Winston D.
Published: December 2011

Background. The relationship between structural and functional integrity of descending motor pathways can predict the potential for motor recovery after stroke. The authors examine the relationship between brain imaging biomarkers within contralesional and ipsilesional hemispheres and hand function in well-recovered patients after subcortical stroke at the level of the internal capsule. Objective. Measures of functional activation and integrity of the ipsilesional corticospinal tract might predict paretic hand function. Methods. A total of 14 patients in the chronic stable phase of motor recovery after subcortical stroke and 24 healthy age-matched individuals participated in the study. Functional MRI was used to examine BOLD contrast during passive wrist flexion–extension and paced or maximum-velocity active fist clenching. Functional integrity of the corticospinal pathway was assessed by transcranial magnetic stimulation to obtain motor-evoked potentials (MEPs) in the first dorsal interosseus muscle of the paretic and nonparetic hands. Fractional anisotropy and the proportion of traces between hemispheres in the posterior limb of both internal capsules were quantified using diffusion-weighted MRI. Results. Patients with smaller MEPs had a weaker paretic hand and more primary motor cortex activation in their affected hemisphere. Asymmetry between white matter tracts of either hemisphere was associated with reduced precision grip strength and increased BOLD activation within the contralesional dorsal premotor cortex for demanding hand tasks. Conclusion. There may be beneficial reorganization in contralesional secondary motor areas with increasing damage to the corticospinal tract after subcortical stroke. Associations between clinical, functional, and structural integrity measures in chronic stroke may lead to a better understanding of motor recovery processes.
Controversies on clitoroplasty
Authors: Şenaylı Atilla
Published: December 2011

A review of the literature regarding the current state of knowledge of the anatomic and physiologic features of the female clitoris was conducted. Based on this evaluation, operations on the clitoris were reviewed. The anatomic and physiologic reconstruction problems of surgical techniques for female pseudohermaphroditism that have previously been reported were reviewed. The author suggests that clitoroplasty is essential for patients with ambiguous genitalia, but the decision regarding the correct procedure, taking into account anatomic and physiologic success, can be controversial. This may be because of unclear anatomic and physiologic definitions, even for healthy people. As a temporary solution, more conservative procedures for maximally effective treatment are suggested.
Nanocomposites of moisture-sensitive polymers and biopolymers with enhanced performance for flexible packaging applications
Authors: Lagaron Jose M , Núñez Eugenia
Published: December 2011

Ethylene–vinyl alcohol copolymers, polyamides, polyvinyl alcohol and, since more recently, some biopolymers are materials with broad applications in high-barrier packaging due to transparency and superior oxygen barrier. However, these materials suffer from strong plasticization in properties due to sorption of moisture, which handicaps their application under high-moisture conditions such as those applied in many packaging cases. This article shows the development of novel nanocomposites based on an optimized kaolinite grade, commercially marketed as O2Block®, which exhibit enhanced ultraviolet and decreased oxygen permeability, by up to 70%, as well as reduced both water permeability, by up to 50%, and sensitivity.
Distribution of temperature during fibre laser radiation and effects thereon phase transformation of ZrO2 engineering ceramic
Authors: Shukla P P , Lawrence J
Published: December 2011

Distribution of surface and the bulk temperature was recorded during fibre laser surface treatment of ZrO2 engineering ceramic. The experimental readings were then compared with a finite element model which showed the flow and distribution of the laser induced heat as a result of the fibre laser surface treatment. Moreover, thermogravimetry–differential scanning calorimetry was used to collect data with respect to physical changes during heating and cooling of the ZrO2 engineering ceramic. The thermogravimetry–differential scanning calorimetry data and the finite element model predictions were then used to map the phase transitions within the ZrO2 engineering ceramic resulting from fibre laser surface treatment. The mapping revealed that the fibre laser surface treatment had generally resulted in a phase transformation of the ZrO2 engineering ceramic from the monoclinic (M) state to a mixture of tetragonal and cubic (T+C) followed by partially formed liquid (L) phase during fibre laser surface treatment and from L to T+C then T, followed by the M state during solidification.
Ti: Sapphire femtosecond laser interaction with human dental dentine
Authors: Ji L F , Li L , Devlin H , Liu Z , Whitehead D , Wang Z B , Wang W , Jiao J
Published: December 2011

This paper reports an investigation of the characteristics of human dentine ablated by a femtosecond laser of 800 nm wavelength for potential pain free dental restoration applications. The work has shown that precise femtosecond laser ablation with clear boundaries and without cracks and debris can be obtained. The transmittance of the dentine tissue was ∼2·7%, and the absorption coefficient was ∼3·19 cm−1 at 800 nm. The laser processed dentine morphology was investigated over a wide range of laser fluences and pulse numbers. The fluence threshold of dentine was determined as 0·45 J cm−2 according to the experimental dependence of the ablated depth versus laser fluence. The thermal energy conduction effects of dentine tubules were analysed. The SEM–EDX and Raman mapping analysis of phosphate indicate that the chemical components of the dentine remained intact before and after ablation.
Preparation and electrocatalytic activity of gold nanoparticle embedded in highly ordered TiO2 nanotube array electrode for electro-oxidation of galactose
Authors: Hosseini M G , Momeni M M , Faraji M
Published: December 2011

Gold nanoparticle–TiO2 nanotube electrodes with high surface area have been successfully prepared using a two step process consisting of galvanic deposition of gold nanoparticles on TiO2 nanotubes/Ti electrodes as support. TiO2 nanotubes/Ti electrodes are fabricated by anodising titanium foil in a dimethyl sulphoxide fluoride containing electrolyte. Scanning electron microscopy and energy dispersive X-ray spectroscopy results indicate that the TiO2 nanotube layers consist of individual tubes of about 60–90 nm diameters, and gold nanoparticles with a size range of 30–40 nm are well dispersed on the surface of the TiO2 nanotube support. The electrocatalytic properties of gold nanoparticle–TiO2 nanotube electrodes towards the electro-oxidation of galactose are investigated by cyclic voltammetry. Compared with pure gold electrodes, gold nanoparticles–TiO2 nanotube electrodes show much higher electrochemical activity. This novel electrode can be used repeatedly and exhibits stable electrocatalytic activity for galactose oxidation.
Analysis of critical doping level of sprayed SnO2:F films
Authors: Chinnappa L , Ravichandran K , Saravanakumar K , Jabenabegum N , Sakthivel B
Published: December 2011

Fluorine doped tin oxide films were deposited using a simplified spray technique. The concentration of the host precursor (SnCl2.2H2O) in the starting solution is varied between 0·2 and 1·0M. The critical doping level of fluorine for which the sheet resistance Rsh acquires a minimum value is observed for each host precursor concentration (HPC). It is found that for a 0·2M solution, Rsh is minimum (42·59 Ω sq−1) when the fluorine doping level is 40 at-%, and the critical doping level gradually increases as the HPC increases. This variation in the critical doping level is correlated with the increase in the number of oxygen sites in the SnO2 lattice caused by the increase in the HPC. The transmittance in the visible range is 80% when the HPC is 0·2M, and the transmittance decreases as the HPC increases.
Phagocytosis by macrophages and endothelial cells inhibits procoagulant and fibrinolytic activity of acute promyelocytic leukemia cells
Authors: Xie Rui , Gao Chunyan , Li Wen , Zhu Jiuxin , Novakovic Valerie , Wang Jing , Ma Ruishuang , Zhou Jin , Gilbert Gary E. , Shi Jialan
Published: December 2011

The coagulopathy of acute promyelocytic leukemia (APL) is mainly related to procoagulant substances and fibrinolytic activators of APL blasts, but the fate of these leukemic cells is unknown. The aim of this study was to investigate the removal of APL blasts by macrophages and endothelial cells in vitro and consequent procoagulant and fibrinolytic activity of APL cells. We found that human umbilical vein endothelial cells as well as THP-1 and monocyte-derived macrophages bound, engulfed, and subsequently degraded immortalized APL cell line NB4 and primary APL cells. Lactadherin promoted phagocytosis of APL cells in a time-dependent fashion. Furthermore, factor Xa and prothrombinase activity of phosphatidylserine-exposed target APL cells was time-dependently decreased after incubation with phagocytes (THP-1–derived macrophages or HUVECs). Thrombin production on target APL cells was reduced by 40%-45% after 2 hours of coincubation with phagocytes and 80% by a combination of lactadherin and phagocytes. Moreover, plasmin generation of target APL cells was inhibited 30% by 2 hours of phagocytosis and ∼ 50% by lactadherin-mediated engulfment. These results suggest that engulfment by macrophages and endothelial cells reduce procoagulant and fibrinolytic activity of APL blasts. Lactadherin and phagocytosis could cooperatively ameliorate the clotting disorders in APL.
Functional characterization of CD4+ T cells in aplastic anemia
Authors: Kordasti Shahram , Marsh Judith , Al-Khan Sufyan , Jiang Jie , Smith Alexander , Mohamedali Azim , Abellan Pilar Perez , Veen Caroline , Costantini Benedetta , Kulasekararaj Austin G. , Benson-Quarm Nana , Seidl Thomas , Mian Syed A. , Farzaneh Farzin , Mufti Ghulam J.
Published: December 2011

The role of CD4+ T cells in the pathogenesis of aplastic anemia (AA) is not well characterized. We investigate CD4+ T-cell subsets in AA. Sixty-three patients with acquired AA were studied. Th1 and Th2 cells were significantly higher in AA patients than in healthy donors (HDs; P = .03 and P = .006). Tregs were significantly lower in patients with severe AA than in HDs (P < .001) and patients with non-severe AA (P = .01). Th17 cells were increased in severe AA (P = .02) but normal in non-severe AA. Activated and resting Tregs were reduced in AA (P = .004; P = .01), whereas cytokine-secreting non-Tregs were increased (P = .003). Tregs from AA patients were unable to suppress normal effector T cells. In contrast, AA effector T cells were suppressible by Tregs from HDs. Th1 clonality in AA, investigated by high-throughput sequencing, was greater than in HDs (P = .03). Our results confirm that Th1 and Th2 cells are expanded and Tregs are functionally abnormal in AA. The clonally restricted expansion of Th1 cells is most likely to be antigen-driven, and induces an inflammatory environment, that exacerbate the functional impairment of Tregs, which are reduced in number.
AAV-mediated gene transfer in the perinatal period results in expression of FVII at levels that protect against fatal spontaneous hemorrhage
Authors: Binny Christopher , McIntosh Jenny , Della Peruta Marco , Kymalainen Hanna , Tuddenham Edward G. D. , Buckley Suzanne M. K. , Waddington Simon N. , McVey John H. , Spence Yunyu , Morton Christopher L. , Thrasher Adrian J. , Gray John T. , Castellino Francis J. , Tarantal Alice F. , Davidoff Andrew M. , Nathwani Amit C.
Published: December 2011

We explored adeno-associated viral vector (AAV)–mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease associated with early postnatal life-threatening hemorrhage. In young adult mice with plasma FVII < 1% of normal, a single tail vein administration of AAV (1 × 1013 vector genomes [vg]/kg) resulted in expression of murine FVII at 266% ± 34% of normal for ≥ 67 days, which mediated protection against fatal hemorrhage and significantly improved survival. Codon optimization of human FVII (hFVIIcoop) improved AAV transgene expression by 37-fold compared with the wild-type hFVII cDNA. In adult macaques, a single peripheral vein injection of 2 × 1011 vg/kg of the hFVIIcoop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7% ± 3.1%) and females (12.3% ± 0.8%). In utero delivery of this vector in the third trimester to fetal monkeys conferred expression of hFVII at birth of 20.4% ± 3.7%, with a gradual decline to > 1% by 7 weeks. Re-administration of an alternative serotype at 12 months postnatal age increased hFVII levels to 165% ± 6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus, perinatal AAV-mediated gene transfer shows promise for disorders with onset of pathology early after birth.
Comparison of lists of genes based on functional profiles
Authors: Salicrú Miquel , Ocaña Jordi , Sánchez-Pla Alex
Published: December 2011

Background

How to compare studies on the basis of their biological significance is a problem of central importance in high-throughput genomics. Many methods for performing such comparisons are based on the information in databases of functional annotation, such as those that form the Gene Ontology (GO). Typically, they consist of analyzing gene annotation frequencies in some pre-specified GO classes, in a class-by-class way, followed by p-value adjustment for multiple testing. Enrichment analysis, where a list of genes is compared against a wider universe of genes, is the most common example.

Results

A new global testing procedure and a method incorporating it are presented. Instead of testing separately for each GO class, a single global test for all classes under consideration is performed. The test is based on the distance between the functional profiles, defined as the joint frequencies of annotation in a given set of GO classes. These classes may be chosen at one or more GO levels. The new global test is more powerful and accurate with respect to type I errors than the usual class-by-class approach. When applied to some real datasets, the results suggest that the method may also provide useful information that complements the tests performed using a class-by-class approach if gene counts are sparse in some classes. An R library, goProfiles, implements these methods and is available from Bioconductor, http://bioconductor.org/packages/release/bioc/html/goProfiles.html.

Conclusions

The method provides an inferential basis for deciding whether two lists are functionally different. For global comparisons it is preferable to the global chi-square test of homogeneity. Furthermore, it may provide additional information if used in conjunction with class-by-class methods.
Desert hedgehogis a mammal-specific gene expressed during testicular and ovarian development in a marsupial
Authors: O'Hara William A , Azar Walid J , Behringer Richard R , Renfree Marilyn B , Pask Andrew J
Published: December 2011

Background

Desert hedgehog (DHH) belongs to the hedgehog gene family that act as secreted intercellular signal transducers. DHH is an essential morphogen for normal testicular development and function in both mice and humans but is not present in the avian lineage. Like other hedgehog proteins, DHH signals through the patched (PTCH) receptors 1 and 2. Here we examine the expression and protein distribution of DHH, PTCH1 and PTCH2 in the developing testes of a marsupial mammal (the tammar wallaby) to determine whether DHH signalling is a conserved factor in gonadal development in all therian mammals.

Results

DHH, PTCH1 and PTCH2 were present in the marsupial genome and highly conserved with their eutherian orthologues. Phylogenetic analyses indicate that DHH has recently evolved and is a mammal-specific hedgehog orthologue. The marsupial PTCH2 receptor had an additional exon (exon 21a) not annotated in eutherian PTCH2 proteins. Interestingly we found evidence of this exon in humans and show that its translation would result in a truncated protein with functions similar to PTCH1. We also show that DHH expression was not restricted to the testes during gonadal development (as in mice), but was also expressed in the developing ovary. Expression of DHH, PTCH1 and PTCH2 in the adult tammar testis and ovary was consistent with findings in the adult mouse.

Conclusions

These data suggest that there is a highly conserved role for DHH signalling in the differentiation and function of the mammalian testis and that DHH may be necessary for marsupial ovarian development. The receptors PTCH1 and PTCH2 are highly conserved mediators of hedgehog signalling in both the developing and adult marsupial gonads. Together these findings indicate DHH is an essential therian mammal-specific morphogen in gonadal development and gametogenesis.
AluScan: a method for genome-wide scanning of sequence and structure variations in the human genome
Authors: Mei Lingling , Ding Xiaofan , Tsang Shui-Ying , Pun Frank W , Ng Siu-Kin , Yang Jianfeng , Zhao Cunyou , Li Dezhi , Wan Weiqing , Yu Chi-Hung , Tan Tze-Ching , Poon Wai-Sang , Leung Gilberto Ka-Kit , Ng Ho-Keung , Zhang Liwei , Xue Hong
Published: December 2011

Background

To complement next-generation sequencing technologies, there is a pressing need for efficient pre-sequencing capture methods with reduced costs and DNA requirement. The Alu family of short interspersed nucleotide elements is the most abundant type of transposable elements in the human genome and a recognized source of genome instability. With over one million Alu elements distributed throughout the genome, they are well positioned to facilitate genome-wide sequence amplification and capture of regions likely to harbor genetic variation hotspots of biological relevance.

Results

Here we report on the use of inter-Alu PCR with an enhanced range of amplicons in conjunction with next-generation sequencing to generate an Alu-anchored scan, or 'AluScan', of DNA sequences between Alu transposons, where Alu consensus sequence-based 'H-type' PCR primers that elongate outward from the head of an Alu element are combined with 'T-type' primers elongating from the poly-A containing tail to achieve huge amplicon range. To illustrate the method, glioma DNA was compared with white blood cell control DNA of the same patient by means of AluScan. The over 10 Mb sequences obtained, derived from more than 8,000 genes spread over all the chromosomes, revealed a highly reproducible capture of genomic sequences enriched in genic sequences and cancer candidate gene regions. Requiring only sub-micrograms of sample DNA, the power of AluScan as a discovery tool for genetic variations was demonstrated by the identification of 357 instances of loss of heterozygosity, 341 somatic indels, 274 somatic SNVs, and seven potential somatic SNV hotspots between control and glioma DNA.

Conclusions

AluScan, implemented with just a small number of H-type and T-type inter-Alu PCR primers, provides an effective capture of a diversity of genome-wide sequences for analysis. The method, by enabling an examination of gene-enriched regions containing exons, introns, and intergenic sequences with modest capture and sequencing costs, computation workload and DNA sample requirement is particularly well suited for accelerating the discovery of somatic mutations, as well as analysis of disease-predisposing germline polymorphisms, by making possible the comparative genome-wide scanning of DNA sequences from large human cohorts.
Evaluation of the NOD/SCID xenograft model for glucocorticoid-regulated gene expression in childhood B-cell precursor acute lymphoblastic leukemia
Authors: Bhadri Vivek A , Cowley Mark J , Kaplan Warren , Trahair Toby N , Lock Richard B
Published: December 2011

Background

Glucocorticoids such as prednisolone and dexamethasone are critical drugs used in multi-agent chemotherapy protocols used to treat acute lymphoblastic leukemia (ALL), and response to glucocorticoids is highly predictive of outcome. The NOD/SCID xenograft mouse model of ALL is a clinically relevant model in which the mice develop a systemic leukemia which retains the fundamental biological characteristics of the original disease. Here we report a study evaluating the NOD/SCID xenograft mouse model to investigate glucocorticoid-induced gene expression. Cells from a glucocorticoid-sensitive xenograft derived from a child with B-cell precursor ALL were inoculated into NOD/SCID mice. When highly engrafted the mice were randomized into groups of 4 to receive dexamethasone 15 mg/kg by intraperitoneal injection or vehicle control. Leukemia cells were harvested from mice spleens at 0, 8, 24 or 48 hours thereafter, and gene expression analyzed on Illumina WG-6_V3 chips, comparing all groups to time 0 hours.

Results

The 8 hour dexamethasone-treated timepoint had the highest number of significantly differentially expressed genes, with fewer observed at the 24 and 48 hour timepoints, and with minimal changes seen across the time-matched controls. When compared to publicly available datasets of glucocorticoid-induced gene expression from an in vitro cell line study and from an in vivo study of patients with ALL, at the level of pathways, expression changes in the 8 hour xenograft samples showed a similar response to patients treated with glucocorticoids. Replicate analysis revealed that at the 8 hour timepoint, a dataset with high signal and differential expression, using data from 3 replicates instead of 4 resulted in excellent recovery scores of > 0.9. However at other timepoints with less signal very poor recovery scores were obtained with 3 replicates.

Conclusions

The NOD/SCID xenograft mouse model provides a reproducible experimental system in which to investigate clinically-relevant mechanisms of drug-induced gene regulation in ALL; the 8 hour timepoint provides the highest number of significantly differentially expressed genes; time-matched controls are redundant and excellent recovery scores can be obtained with 3 replicates.
Relative impact of key sources of systematic noise in Affymetrix and Illumina gene-expression microarray experiments
Authors: Kitchen Robert R , Sabine Vicky S , Simen Arthur A , Dixon J Michael , Bartlett John MS , Sims Andrew H
Published: December 2011

Background

Systematic processing noise, which includes batch effects, is very common in microarray experiments but is often ignored despite its potential to confound or compromise experimental results. Compromised results are most likely when re-analysing or integrating datasets from public repositories due to the different conditions under which each dataset is generated. To better understand the relative noise-contributions of various factors in experimental-design, we assessed several Illumina and Affymetrix datasets for technical variation between replicate hybridisations of Universal Human Reference (UHRR) and individual or pooled breast-tumour RNA.

Results

A varying degree of systematic noise was observed in each of the datasets, however in all cases the relative amount of variation between standard control RNA replicates was found to be greatest at earlier points in the sample-preparation workflow. For example, 40.6% of the total variation in reported expressions were attributed to replicate extractions, compared to 13.9% due to amplification/labelling and 10.8% between replicate hybridisations. Deliberate probe-wise batch-correction methods were effective in reducing the magnitude of this variation, although the level of improvement was dependent on the sources of noise included in the model. Systematic noise introduced at the chip, run, and experiment levels of a combined Illumina dataset were found to be highly dependant upon the experimental design. Both UHRR and pools of RNA, which were derived from the samples of interest, modelled technical variation well although the pools were significantly better correlated (4% average improvement) and better emulated the effects of systematic noise, over all probes, than the UHRRs. The effect of this noise was not uniform over all probes, with low GC-content probes found to be more vulnerable to batch variation than probes with a higher GC-content.

Conclusions

The magnitude of systematic processing noise in a microarray experiment is variable across probes and experiments, however it is generally the case that procedures earlier in the sample-preparation workflow are liable to introduce the most noise. Careful experimental design is important to protect against noise, detailed meta-data should always be provided, and diagnostic procedures should be routinely performed prior to downstream analyses for the detection of bias in microarray studies.
Revealing the missing expressed genes beyond the human reference genome by RNA-Seq
Authors: Chen Geng , Li Ruiyuan , Shi Leming , Qi Junyi , Hu Pengzhan , Luo Jian , Liu Mingyao , Shi Tieliu
Published: December 2011

Background

The complete and accurate human reference genome is important for functional genomics researches. Therefore, the incomplete reference genome and individual specific sequences have significant effects on various studies.

Results

we used two RNA-Seq datasets from human brain tissues and 10 mixed cell lines to investigate the completeness of human reference genome. First, we demonstrated that in previously identified ~5 Mb Asian and ~5 Mb African novel sequences that are absent from the human reference genome of NCBI build 36, ~211 kb and ~201 kb of them could be transcribed, respectively. Our results suggest that many of those transcribed regions are not specific to Asian and African, but also present in Caucasian. Then, we found that the expressions of 104 RefSeq genes that are unalignable to NCBI build 37 in brain and cell lines are higher than 0.1 RPKM. 55 of them are conserved across human, chimpanzee and macaque, suggesting that there are still a significant number of functional human genes absent from the human reference genome. Moreover, we identified hundreds of novel transcript contigs that cannot be aligned to NCBI build 37, RefSeq genes and EST sequences. Some of those novel transcript contigs are also conserved among human, chimpanzee and macaque. By positioning those contigs onto the human genome, we identified several large deletions in the reference genome. Several conserved novel transcript contigs were further validated by RT-PCR.

Conclusion

Our findings demonstrate that a significant number of genes are still absent from the incomplete human reference genome, highlighting the importance of further refining the human reference genome and curating those missing genes. Our study also shows the importance of de novo transcriptome assembly. The comparative approach between reference genome and other related human genomes based on the transcriptome provides an alternative way to refine the human reference genome.
Rapid eye movements during sleep in mice: High trait-like stability qualifies rapid eye movement density for characterization of phenotypic variation in sleep patterns of rodents
Authors: Fulda Stephany , Romanowski Christoph PN , Becker Andreas , Wetter Thomas C , Kimura Mayumi , Fenzl Thomas
Published: December 2011

Background

In humans, rapid eye movements (REM) density during REM sleep plays a prominent role in psychiatric diseases. Especially in depression, an increased REM density is a vulnerability marker for depression. In clinical practice and research measurement of REM density is highly standardized. In basic animal research, almost no tools are available to obtain and systematically evaluate eye movement data, although, this would create increased comparability between human and animal sleep studies.

Methods

We obtained standardized electroencephalographic (EEG), electromyographic (EMG) and electrooculographic (EOG) signals from freely behaving mice. EOG electrodes were bilaterally and chronically implanted with placement of the electrodes directly between the musculus rectus superior and musculus rectus lateralis. After recovery, EEG, EMG and EOG signals were obtained for four days. Subsequent to the implantation process, we developed and validated an Eye Movement scoring in Mice Algorithm (EMMA) to detect REM as singularities of the EOG signal, based on wavelet methodology.

Results

The distribution of wakefulness, non-REM (NREM) sleep and rapid eye movement (REM) sleep was typical of nocturnal rodents with small amounts of wakefulness and large amounts of NREM sleep during the light period and reversed proportions during the dark period. REM sleep was distributed correspondingly. REM density was significantly higher during REM sleep than NREM sleep. REM bursts were detected more often at the end of the dark period than the beginning of the light period. During REM sleep REM density showed an ultradian course, and during NREM sleep REM density peaked at the beginning of the dark period. Concerning individual eye movements, REM duration was longer and amplitude was lower during REM sleep than NREM sleep. The majority of single REM and REM bursts were associated with micro-arousals during NREM sleep, but not during REM sleep.

Conclusions

Sleep-stage specific distributions of REM in mice correspond to human REM density during sleep. REM density, now also assessable in animal models through our approach, is increased in humans after acute stress, during PTSD and in depression. This relationship can now be exploited to match animal models more closely to clinical situations, especially in animal models of depression.