Identification of Potential Molecular Determinants of Murine Embryonic Stem Cell Differentiation by a Transposon-Based Approach

Embryonic stem cells (ESCs) are self-renewing pluripotent cells, capable of differentiating into all somatic cell types. The molecular control of self-renewal is relatively well-characterized, whereas how ESCs exit pluripotent state to differentiate is poorly understood. Here we identify two genes are required for differentiation and dozens of intergenic regions that potentially regulate ESC differentiation. We used PiggyBac (PB) transposon-based approach to randomly mutate the genome of ESCs, and generated hundreds of clones that resisted differentiation signals. Each clone was sequenced to determine genomic regions mutated by PB insertion. Intriguingly, many mutations were localized among intergenic regions and we identified two genes are required for differentiation. This study should facilitate further exploration of novel molecular determinants of embryonic stem cell differentiation.     

The Use of a Predictive Habitat Model and a Fuzzy Logic Approach for Marine Management and Planning

Bottom trawl survey data are commonly used as a sampling technique to assess the spatial distribution of commercial species. However, this sampling technique does not always correctly detect a species even when it is present, and this can create significant limitations when fitting species distribution models. In this study, we aim to test the relevance of a mixed methodological approach that combines presence-only and presence-absence distribution models. We illustrate this approach using bottom trawl survey data to model the spatial distributions of 27 commercially targeted marine species. We use an environmentally- and geographically-weighted method to simulate pseudo-absence data. The species distributions are modelled using regression kriging, a technique that explicitly incorporates spatial dependence into predictions. Model outputs are then used to identify areas that met the conservation targets for the deployment of artificial anti-trawling reefs. To achieve this, we propose the use of a fuzzy logic framework that accounts for the uncertainty associated with different model predictions. For each species, the predictive accuracy of the model is classified as ‘high’. A better result is observed when a large number of occurrences are used to develop the model. The map resulting from the fuzzy overlay shows that three main areas have a high level of agreement with the conservation criteria. These results align with expert opinion, confirming the relevance of the proposed methodology in this study.     

基于模糊支持向量机的膜蛋白折叠类型预测

现有的基于支持向量机（support vector machine,SVM）来预测膜蛋白折叠类型的方法．利用的蛋白质序列特征并不充分．并且在处理多类蛋白质分类问题时存在不可分区域,针对这两类问题．提取蛋白质序列的氨基酸和二肽组成特征,并计算加权的多阶氨基酸残基指数相关系数特征,将3类特征融和作为分类器的输入特征矢量．并采用模糊SVM（fuzzy SVM,FSVM）算法解决对传统SVM不可分数据的分类．在无冗余的数据集上测试结果显示．改进的特征提取方法在相同分类算法下预测性能优于已有的特征提取方法：FSVM在相同特征提取方法下性能优于传统的SVM．二者相结合的分类策略在独立性数据集测试下的预测精度达到96．6％．优于现有的多种预测方法．能够作为预测膜蛋白和其它蛋白质折叠类型的有效工具．     

The Role of Personalised Choice in Decision Support: A Randomized Controlled Trial of an Online Decision Aid for Prostate Cancer Screening

ImportanceDecision support tools can assist people to apply population-based evidence on benefits and harms to individual health decisions. A key question is whether “personalising” choice within decisions aids leads to better decision quality.ObjectiveTo assess the effect of personalising the content of a decision aid for prostate cancer screening using the Prostate Specific Antigen (PSA) test.DesignRandomized controlled trial.SettingAustralia.Participants1,970 men aged 40–69 years were approached to participate in the trial.Intervention1,447 men were randomly allocated to either a standard decision aid with a fixed set of five attributes or a personalised decision aid with choice over the inclusion of up to 10 attributes.Results5% of men in the fixed attribute group scored ‘Have a PSA test’ as the opinion generated by the aid, as compared to 62% of men in the personalised choice group (χ² = 569.38, 2df, p< 0001). Those men who used the personalised decision aid had slightly higher decision quality (t = 2.157, df = 1444, p = 0.031). The men in the personalised choice group made extensive use of the additional decision attributes. There was no difference between the two groups in terms of their stated intention to undergo screening in the next 12 months.ConclusionsTogether, these findings suggest that personalised decision support systems could be an important development in shared decision-making and patient-centered care.

Trial Registration

Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12612000723886     

A Clinical Decision Support System for Integrating Tuberculosis and HIV Care in Kenya: A Human-Centered Design Approach

With the aim of integrating HIV and tuberculosis care in rural Kenya, a team of researchers, clinicians, and technologists used the human-centered design approach to facilitate design, development, and deployment processes of new patient-specific TB clinical decision support system for medical providers. In Kenya, approximately 1.6 million people are living with HIV and have a 20-times higher risk of dying of tuberculosis. Although tuberculosis prevention and treatment medication is widely available, proven to save lives, and prioritized by the World Health Organization, ensuring that it reaches the most vulnerable communities remains challenging. Human-centered design, used in the fields of industrial design and information technology for decades, is an approach to improving the effectiveness and impact of innovations that has been scarcely used in the health field. Using this approach, our team followed a 3-step process, involving mixed methods assessment to (1) understand the situation through the collection and analysis of site observation sessions and key informant interviews; (2) develop a new clinical decision support system through iterative prototyping, end-user engagement, and usability testing; and, (3) implement and evaluate the system across 24 clinics in rural West Kenya. Through the application of this approach, we found that human-centered design facilitated the process of digital innovation in a complex and resource-constrained context.     

Identification of Infected B-Cell Populations by Using a Recombinant Murine Gammaherpesvirus 68 Expressing a Fluorescent Protein

Infection of inbred mice with murine gammaherpesvirus 68 (MHV68) has proven to be a powerful tool to study gammaherpesvirus pathogenesis. However, one of the limitations of this system has been the inability to directly detect infected cells harvested from infected animals. To address this issue, we generated a transgenic virus that expresses the enhanced yellow fluorescent protein (YFP), driven by the human cytomegalovirus immediate-early promoter and enhancer, from a neutral locus within the viral genome. This virus, MHV68-YFP, replicated and established latency as efficiently as did the wild-type virus. During the early phase of viral latency, MHV68-YFP efficiently marked latently infected cells in the spleen after intranasal inoculation. Staining splenocytes for expression of various surface markers demonstrated the presence of MHV68 in distinct populations of splenic B cells harboring MHV68. Notably, these analyses also revealed that markers used to discriminate between newly formed, follicular and marginal zone B cells may not be reliable for phenotyping B cells harboring MHV68 since virus infection appears to modulate cell surface expression levels of CD21 and CD23. However, as expected, we observed that the overwhelming majority of latently infected B cells at the peak of latency exhibited a germinal center phenotype. These analyses also demonstrated that a significant percentage of MHV68-infected splenocytes at the peak of viral latency are plasma cells (ca. 15% at day 14 and ca. 8% at day 18). Notably, the frequency of virus-infected plasma cells correlated well with the frequency of splenocytes that spontaneously reactivate virus upon explant. Finally, we observed that the efficiency of marking latently infected B cells with the MHV68-YFP recombinant virus declined at later times postinfection, likely due to shut down of transgene expression, and indicating that the utility of this marking strategy is currently limited to the early stages of virus infection.Gammaherpesviruses are characterized by their ability to establish life-long infection in lymphocytes of their host as well as their oncogenic potential. The human gammaherpesviruses, Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8; also known as Kaposi''s sarcoma-associated herpesvirus [KSHV]), are associated with a variety of neoplasms. EBV has been implicated in Burkitt''s lymphoma, nasopharyngeal carcinoma, and non-Hodgkin''s lymphoma (15, 27, 33). HHV-8 has been associated with Kaposi''s sarcoma, primary effusion lymphoma, and multicentric Castleman''s disease (4, 5, 7, 24).Research on the human gammaherpesvirus is hindered by their strict species specificity, and thus has been limited mostly to in vitro analyses. Murine gammaherpesvirus 68 (MHV68) is a closely related gammaherpesvirus that naturally infects rodents and provides a useful small animal model to study aspects of gammaherpesvirus pathogenesis that cannot be addressed for the human herpesviruses (3, 22, 25). In addition, the viral genome has been cloned as a bacterial artificial chromosome (BAC) and can readily be manipulated in Escherichia coli (1) and, coupled with the availability of numerous transgenic and knockout strains of mice, MHV68 infection of laboratory mice has provided a powerful small animal model for characterizing basic aspects of gammaherpesvirus pathogenesis in vivo.Like the human gammaherpesviruses, MHV68 establishes long-term latency in B cells, although at early time points after infection latency can also be detected in macrophages and dendritic cells (11, 26, 30). Acute infection is cleared around 2 to 3 weeks postinfection, and by days 16 to 18 postinfection the frequency of viral genome-positive cells in the spleen is ca. 1 in 100 splenocytes (19, 31). This is the peak of splenic latency, and the frequency of infected cells begins to decline significantly until it reaches a steady-state level of ca. 1 in 10,000 splenocytes by 3 months postinfection. Previous analyses have shown that latency is mainly established in germinal center (GC) and memory B cells (12, 19, 31). At early time points during the establishment of latency, the GC fraction has been shown to have the highest percentage of infected cells (ca. 60 to 80% of MHV68-infected B cells) (12). However, even in this population, only around 10% of total GC cells are infected (12). This low frequency limits detailed molecular analyses that can be performed on infected cells (e.g., analysis of virus-induced changes in cellular gene expression).Until now, there has not been an efficient way to directly detect or purify/enrich for MHV68-infected cells harvested from the spleens of infected mice. Because of these issues, we sought to develop a method to efficiently mark infected cells that would allow easy detection, as well as isolation, of infected cells. To this end, we created a transgenic virus that expresses the enhanced yellow fluorescent protein (YFP) from a neutral locus in the viral genome located between open reading frames (ORFs) 27 and 29b. We have previously used this locus to introduce other transgenes (Cre-recombinase and IκBαM expression cassettes) and have shown that this locus tolerates the insertion of transgene expression cassettes (14, 20). We show here that the MHV68-YFP recombinant virus is capable of efficiently marking infected cells, that highly enriched populations of infected cells can easily be isolated based of YFP expression, and that direct detection of infected cells provides a powerful tool for phenotypic analysis of infected cell populations.     

Assessment of Trading Partners for China's Rare Earth Exports Using a Decision Analytic Approach

Chinese rare earth export policies currently result in accelerating its depletion. Thus adopting an optimal export trade selection strategy is crucial to determining and ultimately identifying the ideal trading partners. This paper introduces a multi-attribute decision-making methodology which is then used to select the optimal trading partner. In the method, an evaluation criteria system is established to assess the seven top trading partners based on three dimensions: political relationships, economic benefits and industrial security. Specifically, a simple additive weighing model derived from an additive utility function is utilized to calculate, rank and select alternatives. Results show that Japan would be the optimal trading partner for Chinese rare earths. The criteria evaluation method of trading partners for China''s rare earth exports provides the Chinese government with a tool to enhance rare earth industrial policies.     

Prediction of Candidate Primary Immunodeficiency Disease Genes Using a Support Vector Machine Learning Approach

Screening and early identification of primary immunodeficiency disease (PID) genes is a major challenge for physicians. Many resources have catalogued molecular alterations in known PID genes along with their associated clinical and immunological phenotypes. However, these resources do not assist in identifying candidate PID genes. We have recently developed a platform designated Resource of Asian PDIs, which hosts information pertaining to molecular alterations, protein–protein interaction networks, mouse studies and microarray gene expression profiling of all known PID genes. Using this resource as a discovery tool, we describe the development of an algorithm for prediction of candidate PID genes. Using a support vector machine learning approach, we have predicted 1442 candidate PID genes using 69 binary features of 148 known PID genes and 3162 non-PID genes as a training data set. The power of this approach is illustrated by the fact that six of the predicted genes have recently been experimentally confirmed to be PID genes. The remaining genes in this predicted data set represent attractive candidates for testing in patients where the etiology cannot be ascribed to any of the known PID genes.     

A New Stage in the Performance of a Human Immunogram: A Visual Streptavidin-Biotin Approach

The production of a reagent kit has been recently organized by DAKO, Immunotekh and other companies, for phenotyping of lymphocytes by the streptavidin–biotin method. The method needs no sophisticated equipment, is highly sensitive, and allows rapid staining of different lymphocyte subpopulations in capillary blood smears and subsequent observation of them under a light microscope. We have modified this method for staining leukocytes in the monolayer prepared with a plate cytorotor. Not decreasing the above-mentioned advantages of the method, this modification significantly cheapens and simplifies the staining procedure; the blood cells of 16 subjects can be stained concurrently, and the staining can be performed by a technician. The streptavidin–biotin method of lymphocyte phenotyping can be mastered in every immunological laboratory, thus improving its technical level.     

Cost-Effectiveness of Clinical Decision Support System in Improving Maternal Health Care in Ghana

ObjectiveThis paper investigated the cost-effectiveness of a computer-assisted Clinical Decision Support System (CDSS) in the identification of maternal complications in Ghana.MethodsA cost-effectiveness analysis was performed in a before- and after-intervention study. Analysis was conducted from the provider’s perspective. The intervention area was the Kassena- Nankana district where computer-assisted CDSS was used by midwives in maternal care in six selected health centres. Six selected health centers in the Builsa district served as the non-intervention group, where the normal Ghana Health Service activities were being carried out.ResultsComputer-assisted CDSS increased the detection of pregnancy complications during antenatal care (ANC) in the intervention health centres (before-intervention= 9 /1,000 ANC attendance; after-intervention= 12/1,000 ANC attendance; P-value=0.010). In the intervention health centres, there was a decrease in the number of complications during labour by 1.1%, though the difference was not statistically significant (before-intervention =107/1,000 labour clients; after-intervention= 96/1,000 labour clients; P-value=0.305). Also, at the intervention health centres, the average cost per pregnancy complication detected during ANC (cost –effectiveness ratio) decreased from US$17,017.58 (before-intervention) to US$15,207.5 (after-intervention). Incremental cost –effectiveness ratio (ICER) was estimated at US$1,142. Considering only additional costs (cost of computer-assisted CDSS), cost per pregnancy complication detected was US$285.ConclusionsComputer –assisted CDSS has the potential to identify complications during pregnancy and marginal reduction in labour complications. Implementing computer-assisted CDSS is more costly but more effective in the detection of pregnancy complications compared to routine maternal care, hence making the decision to implement CDSS very complex. Policy makers should however be guided by whether the additional benefit is worth the additional cost.     

Development of a recA Gene-Based Identification Approach for the Entire Burkholderia Genus

Burkholderia is an important bacterial genus containing species of ecological, biotechnological, and pathogenic interest. With their taxonomy undergoing constant revision and the phenotypic similarity of several species, correct identification of Burkholderia is difficult. A genetic scheme based on the recA gene has greatly enhanced the identification of Burkholderia cepacia complex species. However, the PCR developed for the latter approach was limited by its specificity for the complex. By alignment of existing and novel Burkholderia recA sequences, we designed new PCR primers and evaluated their specificity by testing a representative panel of Burkholderia strains. PCR followed by restriction fragment length polymorphism analysis of an 869-bp portion of the Burkholderia recA gene was not sufficiently discriminatory. Nucleotide sequencing followed by phylogenetic analysis of this recA fragment differentiated both putative and known Burkholderia species and all members of the B. cepacia complex. In addition, it enabled the design of a Burkholderia genus-specific recA PCR that produced a 385-bp amplicon, the sequence of which was also able to discriminate all species examined. Phylogenetic analysis of 188 novel recA genes enabled clarification of the taxonomic position of several important Burkholderia strains and revealed the presence of four novel B. cepacia complex recA lineages. Although the recA phylogeny could not be used as a means to differentiate B. cepacia complex strains recovered from clinical infection versus the natural environment, it did facilitate the identification of clonal strain types of B. cepacia, B. stabilis, and B. ambifaria capable of residing in both niches.     

Identification and Comparative Analysis of the Tegillarca granosa Haemocytes MicroRNA Transcriptome in Response to Cd Using a Deep Sequencing Approach

Background

MicroRNAs (miRNAs) are endogenous non-coding small RNAs (sRNAs) that can base pair with their target mRNAs, which represses their translation or induces their degradation in various biological processes. To identify miRNAs regulated by heavy metal stress, we constructed two sRNA libraries for the blood clam Tegillarca granosa: one for organisms exposed to toxic levels of cadmium (Cd) and one for a control group.

Results

Sequencing of the two libraries and subsequent analysis revealed 215 conserved and 39 new miRNAs. Most of the new miRNAs in T. granosa were up- or down-regulated in response to Cd exposure. There were significant differences in expression between the Cd and control groups for 16 miRNAs. Of these, five miRNAs were significantly up-regulated and 11 were significantly down-regulated in the Cd stress library. Potential targets were predicted for the 16 differential miRNAs in pre-miRNAs identified according to sequence homology. Some of the predicted miRNA targets are associated with regulation of the response to stress induced by heavy metals. Five differentially expressed miRNAs (Tgr-nmiR-8, Tgr-nmiR-21, Tgr-miR-2a, Tgr-miR-10a-5p, and Tgr-miR-184b) were validated by qRT-PCR.

Conclusion

Our study is the first large-scale identification of miRNAs in T. granosa haemocytes. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in the responses of this species to environmental heavy metal stresses.     

Identification of Candidate Genes Related to Stem Development in Brassica napus Using RNA-Seq

Plant Molecular Biology Reporter - Plant stems are involved in supporting the entire plant body, thus having an important effect on the yield of oilseed rape. The current understanding of the...     

Development of a System to Monitor Laryngeal Movement during Swallowing Using a Bend Sensor

Background

Swallowing dysfunction (also known as dysphagia), which results in a deterioration of nutritional intake, slows rehabilitation and causes aspiration pneumonia, is very common following neurological impairments. Although videofluorographic (VF) examination is widely used for detecting aspiration, an objective and non-invasive method for assessing swallowing function has yet to be established because of a lack of adequate devices and protocols. In this paper, a bend sensor whose resistance is altered by bending was introduced to monitor swallowing-related laryngeal movement.

Methods

Six healthy male volunteers were recruited in the present study. Specific time points on the signal waveform produced by the bend sensor were defined to describe laryngeal movement by differential analysis. Additionally, the physiological significance of the obtained waveform was confirmed by analyzing the sequential correlations between the signal waveform from the bend sensor and hyoid bone kinetics simultaneously recorded by VF.

Results

Seven time points were successfully defined on the signal waveform to reference laryngeal movement. Each time point was well correlated with certain VF events, with evidence of no significant time lags, and there were positive correlations between waveform time points and matched VF events. Furthermore, obvious similarities were noticed between the duration of each phase on the signal waveform and the duration of the matched hyoid bone activity.

Conclusions

The present monitoring system using a bend sensor might be useful for observing the temporal aspects of laryngeal movement during swallowing, and it was well coordinated with hyoid bone movement.     

Insights into Persistence Mechanisms of a Zoonotic Virus in Bat Colonies Using a Multispecies Metapopulation Model

Rabies is a worldwide zoonosis resulting from Lyssavirus infection. In Europe, Eptesicus serotinus is the most frequently reported bat species infected with Lyssavirus, and thus considered to be the reservoir of European bat Lyssavirus type 1 (EBLV-1). To date, the role of other bat species in EBLV-1 epidemiology and persistence remains unknown. Here, we built an EBLV-1−transmission model based on local observations of a three-cave and four-bat species (Myotis capaccinii, Myotis myotis, Miniopterus schreibersii, Rhinolophus ferrumequinum) system in the Balearic Islands, for which a 1995–2011 serological dataset indicated the continuous presence of EBLV-1. Eptesicus serotinus was never observed in the system during the 16-year follow-up and therefore was not included in the model. We used the model to explore virus persistence mechanisms and to assess the importance of each bat species in the transmission dynamics. We found that EBLV-1 could not be sustained if transmission between M. schreibersii and other bat species was eliminated, suggesting that this species serves as a regional reservoir. Global sensitivity analysis using Sobol''s method revealed that following the rate of autumn−winter infectious contacts, M. schreibersii''s incubation- and immune-period durations, but not the infectious period length, were the most relevant factors driving virus persistence.