Enrichment Methodology Research Articles

An Optimized Chromatographic Strategy for Multiplexing In Parallel Reaction Monitoring Mass Spectrometry: Insights from Quantitation of Activated Kinases

Reliable quantitation of protein abundances in defined sets of cellular proteins is critical to numerous biological applications. Traditional immunodetection-based methods are limited by the quality and availability of specific antibodies, especially for site-specific post-translational modifications. Targeted proteomic methods, including the recently developed parallel reaction monitoring (PRM) mass spectrometry, have enabled accurate quantitative measurements of up to a few hundred specific target peptides. However, the degree of practical multiplexing in label-free PRM workflows remains a significant limitation for the technique. Here we present a strategy for significantly increasing multiplexing in label-free PRM that takes advantage of the superior separation characteristics and retention time stability of meter-scale monolithic silica-C18 column-based chromatography. We show the utility of the approach in quantifying kinase abundances downstream of previously developed active kinase enrichment methodology based on multidrug inhibitor beads. We examine kinase activation dynamics in response to three different MAP kinase inhibitors in colorectal carcinoma cells and demonstrate reliable quantitation of over 800 target peptides from over 150 kinases in a single label-free PRM run. The kinase activity profiles obtained from these analyses reveal compensatory activation of TGF-β family receptors as a response to MAPK blockade. The gains achieved using this label-free PRM multiplexing strategy will benefit a wide array of biological applications.

Circulating Tumor Cells: Markers and Methodologies for Enrichment and Detection.

Cancer is a leading cause of disease worldwide; however, nowadays many points of its initiation processes are unknown. In this chapter, we are focusing on the role of liquid biopsies in cancer detection and progression. CTCs are one of the main components of liquid biopsies, they represent a subset of tumor cells that have acquired the ability to disseminate from the primary tumor and intravasate to the circulatory system. The greatest challenge in the detection of CTCs is their rarity in the blood. Human blood consists of white blood cells (5-10×106/mL), red blood cells (5-9×109/mL), and platelets (2.5-4×108/mL); very few CTCs will be present even in patients with known metastatic disease, with often less than one CTC per mL of blood. CTCs are found in frequencies on the order of 1-10 CTCs per mL of whole blood in patients with metastatic disease, and it is reduced in half for non-metastatic stages. Therefore, accurate methodologies for their capture and analysis are really important. The main aim of the present chapter is to describe different methodologies for CTCs capturing and analysis.

Identification of ADP-Ribose Acceptor Sites on In Vitro Modified Proteins by Liquid Chromatography-Tandem Mass Spectrometry.

Protein ADP-ribosylation is a covalent, reversible posttranslational modification (PTM) catalyzed by ADP-ribosyltransferases (ARTs). Proteins can be either mono- or poly-ADP-ribosylated under a variety of physiological and pathological conditions. To understand the functional contribution of protein ADP-ribosylation to normal and disease/stress states, modified protein and corresponding ADP-ribose acceptor site identification is crucial. Since ADP-ribosylation is a transient and relatively low abundant PTM, systematic and accurate identification of ADP-ribose acceptor sites has only recently become feasible. This is due to the development of specific ADP-ribosylated protein/peptide enrichment methodologies, as well as technical advances in high-accuracy liquid chromatography-tandem mass spectrometry (LC-MS/MS). The standardized protocol described here allows the identification of ADP-ribose acceptor sites in in vitro ADP-ribosylated proteins and will, thus, contribute to the functional characterization of this important PTM.

Circulating Tumor Cells: Moving Biological Insights into Detection.

Circulating tumor cells (CTCs) have shown promising potential as liquid biopsies that facilitate early detection, prognosis, therapeutic target selection and monitoring treatment response. CTCs in most cancer patients are low in abundance and heterogeneous in morphological and phenotypic profiles, which complicate their enrichment and subsequent characterization. Several methodologies for CTC enrichment and characterization have been developed over the past few years. However, integrating recent advances in CTC biology into these methodologies and the selection of appropriate enrichment and characterization methods for specific applications are needed to improve the reliability of CTC biopsies. In this review, we summarize recent advances in the studies of CTC biology, including the mechanisms of their generation and their potential forms of existence in blood, as well as the current CTC enrichment technologies. We then critically examine the selection of methods for appropriately enriching CTCs for further investigation of their clinical applications.

Automatic domain-specific learning: towards a methodology for ontology enrichment

At the current rate of technological development, in a world where enormous amount of data are constantly created and in which the Internet is used as the primary means for information exchange, there exists a need for tools that help processing, analyzing and using that information. However, while the growth of information poses many opportunities for social and scientific advance, it has also highlighted the difficulties of extracting meaningful patterns from massive data. Ontologies have been claimed to play a major role in the processing of large-scale data, as they serve as universal models of knowledge representation, and are being studied as possible solutions to this. This paper presents a method for the automatic expansion of ontologies based on corpus and terminological data exploitation. The proposed “ontology enrichment method” (OEM) consists of a sequence of tasks aimed at classifying an input keyword automatically under its corresponding node within a target ontology. Results prove that the method can be successfully applied for the automatic classification of specialized units into a reference ontology.

基于网络效能分析的生境网络构建与优化——以苏锡常地区白鹭为例

PDF HTML阅读 XML下载 导出引用 引用提醒 基于网络效能分析的生境网络构建与优化——以苏锡常地区白鹭为例 DOI: 10.5846/stxb201603270551 作者: 作者单位: 南京农业大学土地管理学院,南京农业大学土地管理学院,南京农业大学土地管理学院 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金资助项目（41571176）；江苏高校哲学社会科学研究项目（2015SJD096）；中央高校基本科研业务费人文社会科学研究基金配套项目（SKPT2015018） A network efficiency analysis approach to habitat network optimization: a case study of the little egret (Egretta garzetta) in the Su-Xi-Chang area, China Author: Affiliation: College of Land Management,Nanjing Agricultural University,College of Land Management,Nanjing Agricultural University,College of Land Management,Nanjing Agricultural University Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:从网络效能分析出发研究生境网络优化具有重要意义。以快速城市化苏锡常地区为小尺度研究区、苏锡常及周边15个城市为大尺度泛研究区、白鹭为焦点物种，通过采用引入并构建网络效能概念框架、识别和构建不同尺度生境网络、调整网络结构和转换网络结构特性相结合的方法，实现了跨尺度下白鹭生境网络优化。结果表明：1）依据大尺度迁移网络完善小尺度生境网络得到的方案一和改变方案一网络特性得到的方案二均是现状生境网络的优化方案；2）方案二与方案一相比，在平均路径长度值增加、聚类系数值减少、节点度概率分布幂次关系拟合度提高，即小世界特性减弱、无标度特性增强的基础上，测度网络连接度的α、β、γ指数值得到增加，同时新增生境节点和迁移廊道数量更少，具有更好的优化成效；3）从网络效能分析出发，有益于提高生境网络跨尺度下优化方案的可行性，其分析思路既丰富了网络优化的方法论研究，也为提高上位与下位规划空间契合度的实践提供了理论支持。但从网络效能概念框架看，如网络层级分析、其他尺度情景研究、网络质量测度指标等方面还亟待丰富与完善。 Abstract:The network efficiency analysis method is used to study network systematization and integrity, which are important for the assessment of ecological networks. However, this method has rarely been applied in habitat network optimization studies. The objective of the present study was to use the network efficiency analysis method to develop a new approach to optimize habitat network analysis. A small-scale area of 1.75×105 km2 was selected in the Su-Xi-Chang region as the case study; and 12 neighboring cities, together with the Su-Xi-Chang area, covering 11.91×105 km2, were selected as the large-scale study area. The little egret (Egretta garzetta) was chosen as a regionally representative species in the study area. The method included three steps: 1) a conceptual framework for the network efficiency analysis was built to optimize the habitat network, in which the small-world and scale-free network concepts were introduced, with their network structural characteristics, which included average path length, cluster coefficients, and the probability distributions of node degrees. These structural characteristics were applied in step 3 as the assessment indicators; 2) the little egret habitat patches and small- and large-scale habitat networks were identified and simulated. The present status of the small-scale habitat network was obtained from previous research results. In the large-scale area analysis, the observation sites were obtained from a dataset for 2003-2015 on the China Bird Report website. These were regarded as the habitats and those sites where > 20 little egrets were observed simultaneously were identified and transferred to habitat patches. Then, a large-scale habitat network was simulated using the same method used in the previous research; and 3) the present status of the small-scale habitat network was optimized using the large-scale habitat network and was designated as network A. Network B was generated by adjusting the network A structure and transferring its network structural characteristics. The network connectivity indexes α, β, and γ of the present status small-scale habitat network were used to calculate and compare networks A and B. The results showed that 1) the A and B networks represented the optimized results of the present status small-scale habitat network; 2) the increased average path length value, decreased cluster coefficient value, and increased goodness-of-fit of probability distribution of node degree for network B compared to that of network A showed that the network B small-world network characteristics had decreased, but its scale-free network characteristics had increased. This suggests that network B has a better fit to the large-scale habitat network than network A; and 3) the number of newly-added habitat nodes and the migration corridor for network B were smaller than network A, but its corresponding values for the network connectivity indexes α, β, and γ were greater. This suggests that network B results were better than those for network A, and therefore are the recommended, optimized results. The present study indicated that optimizing habitat networks using the network efficiency analysis method improved effectiveness, enriched network structure optimization methodology, and provided a theoretical basis for improved goodness-of-fit when undertaking spatial planning among upper- and lower-levels. However, further studies are required to improve conceptual frameworks, including aspects of network hierarchical analysis, scaling, and network quality assessment indicators. 参考文献 相似文献 引证文献

Enzymatic synthesis of high-modified DNA

Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology.

Multispectral Imaging Analysis of Circulating Tumor Cells in Negatively Enriched Peripheral Blood Samples.

A variety of biomarkers are present on cells in peripheral blood of patients with a variety of disorders, including solid tumor malignancies. While rare, characterization of these cells for specific protein levels with the advanced technology proposed, will lead to future validation studies of blood samples as "liquid biopsies" for the evaluation of disease status and therapeutic response. While circulating tumor cells (CTCs) have been isolated in the blood samples of patients with solid tumors, the exact role of CTCs as clinically useful predictive markers is still debated. Current commercial technology has significant bias in that a positive selection technology is used that preassumes specific cell surface markers (such as EpCAM) are present on CTCs. However, CTCs with low EpCAM expression have been experimentally demonstrated to be more likely to be missed by this method. In contrast, this application uses a previously developed, technology that performs a purely negative enrichment methodology on peripheral blood, yielding highly enriched blood samples that contain CTCs as well as other, undefined cell types. The focus of this contribution is the use of multispectral imaging of epifluorescent, microscopic images of these enriched cells in order to help develop clinically relevant liquid biopsies from peripheral blood samples.

Biorefinery site selection using a stepwise biogeophysical and social analysis approach

A key factor in the production of economically viable and environmentally sustainable biofuels is biorefinery site selection. Facility location analysis has traditionally been driven by access to feedstock, proximity to customers, and local incentives. While economic constraints will always be major factors in site selection, incorporating social metrics may further reduce the cost of constructing a biorefinery. A community's disposition toward a biorefinery project may significantly impact implementation success: grassroots support can lower implementation costs while opposition may increase the costs of permitting blockages and other scale-up delays. The proposed biorefinery siting tool improves upon previous research by incorporating site-specific biogeophysical measures and more complete and comprehensive social measures of community innovation and capacity for collective action. A refined biogeophysical analysis assesses pulp mills for their potential as repurposed biorefineries. The social asset components of site selection are greatly improved by enhancing and disaggregating key metrics through the use of multiple indicators of community collective action capacity and propensity for change. The refined measures are integrated into a biorefinery site-selection tool. Pulp mills that rank highly in both the biogeophysical and social asset measures may be considered more suitable candidates for repurpose into a biorefinery. This enriched methodology has been applied to biorefinery siting decisions in the U.S. Pacific Northwest region; however, it is suitable for applications to infrastructure development projects in any region of the U.S.

Ultrasonic energy enhanced the efficiency of advance extraction methodology for enrichment of trace level of copper in serum samples of patients having neurological disorders

An innovative dual dispersive ionic liquid based on ultrasound assisted microextraction (UDIL-μE), for the enrichment of trace levels of copper ion (Cu2+), in serum (blood) of patients suffering from different neurological disorders. The enriched metal ions were subjected to flame atomic absorption spectrometry (FAAS). In the UDIL-μE method, the extraction solvent, ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate [C4mim][PF6], was dispersed into the aqueous samples using an ultrasonic bath. The(PAN) 1-(2-pyridylazo)-2-naphthol was used as ligand for the complexation of Cu ion in IL (as extracting solvent). The various variables such as sonication time, pH, concentration of complexing agent, time and rate of centrifugation, IL volume that affect the extraction process were optimized. The enhancement factor (EF) and detection limit (LOD) was found under favorable condition was 31 and 0.36μgL−1, respectively. Reliability of the proposed method was checked by relative standard deviation (%RSD), which was found to be <5%. The accuracy of developed procedure was assured by using certified reference material (CRM) of blood serum. The developed procedure was applied successfully to the analysis of concentration of Cu ion in blood serum of different neurological disorders subjects and referents of same age group. It was observed that the levels of Cu ion was two folds higher in serum samples of neurological disorders patients as related to normal referents of same age group.

Comparative proteomic assessment of matrisome enrichment methodologies.

The matrisome is a complex and heterogeneous collection of extracellular matrix (ECM) and ECM-associated proteins that play important roles in tissue development and homeostasis. While several strategies for matrisome enrichment have been developed, it is currently unknown how the performance of these different methodologies compares in the proteomic identification of matrisome components across multiple tissue types. In the present study, we perform a comparative proteomic assessment of two widely used decellularisation protocols and two extraction methods to characterise the matrisome in four murine organs (heart, mammary gland, lung and liver). We undertook a systematic evaluation of the performance of the individual methods on protein yield, matrisome enrichment capability and the ability to isolate core matrisome and matrisome-associated components. Our data find that sodium dodecyl sulphate (SDS) decellularisation leads to the highest matrisome enrichment efficiency, while the extraction protocol that comprises chemical and trypsin digestion of the ECM fraction consistently identifies the highest number of matrisomal proteins across all types of tissue examined. Matrisome enrichment had a clear benefit over non-enriched tissue for the comprehensive identification of matrisomal components in murine liver and heart. Strikingly, we find that all four matrisome enrichment methods led to significant losses in the soluble matrisome-associated proteins across all organs. Our findings highlight the multiple factors (including tissue type, matrisome class of interest and desired enrichment purity) that influence the choice of enrichment methodology, and we anticipate that these data will serve as a useful guide for the design of future proteomic studies of the matrisome.

Comparison of simple sucrose and percoll based methodologies for synaptosome enrichment

Synaptosomes are isolated nerve terminals. They represent an extremely attractive in vitro model system to study synaptic physiology since they preserve morphological and functional characteristics of the synapse. As such they have been used to investigate synaptic dysfunctions associated with neuropathologies like Alzheimer's disease.In the present work two simple methodologies for isolating synaptosomal-enriched fractions were compared for the first time. The starting points of both protocols were rat cortical or hippocampal homogenized tissues that underwent several differential centrifugation steps followed by a final purification of synaptosomal-enriched fractions using either a Percoll gradient or a Sucrose gradient. Comparison of the fractions obtained was carried out, using both biochemical and electron microscopy approaches.In the biochemical analysis the protein levels of pre-synaptic, post-synaptic, nuclear and mitochondrial markers were evaluated. Additional characterization of the synaptosomal-enriched fractions was performed using transmission electron microscopy. In summary, the results indicate that under the conditions tested the Sucrose based protocol is more efficient for the isolation of synaptosomal-enriched fractions from both neuronal tissues, being particularly efficient for hippocampus that is a less abundant brain tissue. Further, the sucrose protocol apparently results in a higher yield of viable synaptosomes suitable for further assays, including structural and functional studies of synapses; making this an attractive procedure to study processes associated with neuropathologies.

Uterine-Derived CD11b Cells Significantly Increase Vasculogenesis and Promote Myocardial Healing in Ischemic Cardiomyopathy.

Ischemic heart disease is the leading cause of mortality in industrialized countries. Cell transplantation could restore function of the ischemic heart likely through the mechanism of cell-induced angiogenesis. We have previously shown that cells isolated from uteri increase angiogenesis and alleviate cardiac dysfunction when transplanted after MI. However, which uterine cell type contributes to angiogenesis is unknown. Here we report that uterine-derived CD11b cells significantly increase vasculogenesis and promote myocardial healing in ischemic cardiomyopathy. We have established a novel and simple methodology for uterine CD11b cell isolation and enrichment and demonstrate that this technique can be used for purifying and establishing viable CD11b cell cultures in rats. The isolated fresh CD11b cells were transplanted into ischemic rat hearts 5 days after injury. Following transplantation, vasculogenesis significantly increased in ischemic cardiac tissue, which reduced infarct size and restored myocardial function and perfusion compared with controls. Thus, uterine CD11b cells have the potential to promote functional healing when implanted after ischemic cardiomyopathy. Importantly, we have demonstrated a novel means by which CD11b cells can be easily purified and cultured for cell transplantation.

Abstract 169: Drug resistance gene expression pathways in primary refractory breast cancer

Abstract Purpose/Objective: Breast cancer is a collection of diseases with distinct molecular subtypes with differing prognoses. Clinically, there is a group of therapy resistant breast cancers characterized by resistance to conventional chemo- and radiotherapy with short overall survival and poor prognosis, which we termed primary refractory breast cancer (PRBC). We hypothesized that PRBC is characterized by specific genomic changes that may contribute to therapy resistance. The aim of this study was to identify changes in gene expression profiles of PRBC compared to triple negative (TNBC) and ER+/-PR positive (HR) breast cancer cohorts. Methods: PRBC was defined as patients with an initial diagnosis of Stage I-III breast cancer who either I) developed metastatic disease within 6 months of completing treatment or II) died of breast cancer within two years of initial diagnosis. Formalin-fixed paraffin-embedded (FFPE) tissue was obtained under an IRB approved protocol 01-13-43c. RNA was extracted from macro-dissected FFPE tumor tissue. Gene expression profiling was performed with Affymetrix Human Transcriptome Array 2.0 and gene signatures were analyzed using a single sample gene set enrichment methodology. Gene signatures of PRBC patients were compared to signatures of two TNBC and HR cohorts with gene expression data generated by identical methodology. Results: A total of nineteen samples from eight patients had sufficient tumor material to obtain RNA. The median age at diagnosis was 44.3 years, 62.5% were triple negative. At initial diagnosis the stage ranged from IA to IIIC. Sixty-three percent received neoadjuvant chemotherapy. The median time to relapse was 8.4 months and median time to death 1.7 years. Genomic analysis identified highly significant (p&amp;lt;10-6) increased expression of ABC transporters, hypoxia, interferon, DNA repair and AKT signatures compared to control cohorts. Conclusions: PRBC is characterized by significant upregulation of multiple gene expression signatures important in drug resistance including ABC transporters, hypoxia, AKT and DNA repair pathways. Confirmation of these results would allow the development of alternative treatment strategies for PRBC to help improve the devastatingly poor prognosis of this subgroup of patients. Citation Format: Charlene Kan, Shikha Parsai, Stefanie Avril, Janice Lyons, Hannah Gilmore, Junran Zhang, Lyndsay Harris. Drug resistance gene expression pathways in primary refractory breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 169.

The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity.

BackgroundThe ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions. While emerging sequencing platforms are capable of producing several kilobases-long reads, the fragment sizes generated by current DNA target enrichment technologies remain a limiting factor, producing DNA fragments generally shorter than 1 kbp. The DNA enrichment methodology described herein, Region-Specific Extraction (RSE), produces DNA segments in excess of 20 kbp in length. Coupling this enrichment method to appropriate sequencing platforms will significantly enhance the ability to generate complete and accurate sequence characterization of any genomic region without the need for reference-based assembly.ResultsRSE is a long-range DNA target capture methodology that relies on the specific hybridization of short (20-25 base) oligonucleotide primers to selected sequence motifs within the DNA target region. These capture primers are then enzymatically extended on the 3’-end, incorporating biotinylated nucleotides into the DNA. Streptavidin-coated beads are subsequently used to pull-down the original, long DNA template molecules via the newly synthesized, biotinylated DNA that is bound to them. We demonstrate the accuracy, simplicity and utility of the RSE method by capturing and sequencing a 4 Mbp stretch of the major histocompatibility complex (MHC). Our results show an average depth of coverage of 164X for the entire MHC. This depth of coverage contributes significantly to a 99.94 % total coverage of the targeted region and to an accuracy that is over 99.99 %.ConclusionsRSE represents a cost-effective target enrichment method capable of producing sequencing templates in excess of 20 kbp in length. The utility of our method has been proven to generate superior coverage across the MHC as compared to other commercially available methodologies, with the added advantage of producing longer sequencing templates amenable to DNA sequencing on recently developed platforms. Although our demonstration of the method does not utilize these DNA sequencing platforms directly, our results indicate that the capture of long DNA fragments produce superior coverage of the targeted region.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2836-6) contains supplementary material, which is available to authorized users.

Molecularly imprinted polymers as the extracted sorbents of clenbuterol ahead of liquid chromatographic determination.

A pre-treatment methodology for clenbuterol hydrochloride (CLEN) isolation and enrichment in a complex matrix environment was developed through exploiting molecularly imprinted polymers (MIPs). CLEN-imprinted polymers were synthesized by the combined use of ally-β-cyclodextrin (ally-β-CD) and methacrylic acid (MAA), allyl-β-CD and acrylonitrile (AN), and allyl-β-CD and methyl methacrylate (MMA) as the binary functional monomers. MAA-linked allyl-β-CD MIPs (M-MAA) were characterized by Fourier transform-infrared (FT-IR) spectroscopy and a scanning electron microscope (SEM). Based upon the results, M-MAA polymers generally proved to be an excellent selective extraction compared to its references: AN-linked allyl-β-CD MIPs (M-AN) and MMA-linked allyl-β-CD MIPs (M-MMA). M-MAA polymers were eventually chosen to run through a molecularly imprinted solid-phase extraction (MISPE) micro-column to enrich CLEN residues spiked in pig livers. A high recovery was achieved, ranging from 91.03% to 96.76% with relative standard deviation (RSD) ≤4.45%.

Comparative analysis of various targeted enrichment methodologies to identify actionable mutations in lung cancer.

e13110Background: The recent expansion of knowledge about genetic changes associated with various diseases including cancers, using whole genome or exome sequencing has led to the development of fo...

Integrative Phosphoproteomics Links IL-23R Signaling with Metabolic Adaptation in Lymphocytes.

Interleukin (IL)-23 mediated signal transduction represents a major molecular mechanism underlying the pathology of inflammatory bowel disease, Crohn’s disease and ulcerative colitis. In addition, emerging evidence supports the role of IL-23-driven Th17 cells in inflammation. Components of the IL-23 signaling pathway, such as IL-23R, JAK2 and STAT3, have been characterized, but elements unique to this network as compared to other interleukins have not been readily explored. In this study, we have undertaken an integrative phosphoproteomics approach to better characterise downstream signaling events. To this end, we performed and compared phosphopeptide and phosphoprotein enrichment methodologies after activation of T lymphocytes by IL-23. We demonstrate the complementary nature of the two phosphoenrichment approaches by maximizing the capture of phosphorylation events. A total of 8202 unique phosphopeptides, and 4317 unique proteins were identified, amongst which STAT3, PKM2, CDK6 and LASP-1 showed induction of specific phosphorylation not readily observed after IL-2 stimulation. Interestingly, quantitative analysis revealed predominant phosphorylation of pre-existing STAT3 nuclear subsets in addition to translocation of phosphorylated STAT3 within 30 min after IL-23 stimulation. After IL-23R activation, a small subset of PKM2 also translocates to the nucleus and may contribute to STAT3 phosphorylation, suggesting multiple cellular responses including metabolic adaptation.

Strategies for Efficient Microbial Fuel Cell Operation

Microbial fuel cells (MFCs) are bioelectrochemical systems that exploit microbial respiration for the conversion of organic compounds into electrical energy. Within a typical system, electrochemically active microorganisms extracellularly transfer electrons released during the oxidation of organic compounds to the anode surface. The electrons travel from the anode, across an external load to the cathode, where they react with the terminal electron acceptor (e.g. oxygen from air). MFCs are a promising means for wastewater treatment and as a source of renewable energy. The operational mode of the MFC can directly control microbial metabolism and respiration and thus define the wastewater treatment rate, energy recovery and final products from wastewater treatment. Despite the advantages, MFCs are not yet a viable solution for large-scale wastewater treatment and energy generation. Extensive research must still be conducted to optimize the system including MFC start-up time, stability and treatment rates. MFC start-up time is widely variable depending on external conditions, inoculum source and substrate. There are challenges associated with stability and reproducibility of the system. Finally, wastewater treatment rates using MFCs still are not comparable to conventional wastewater treatment methods, and the magnitude of energy recovery is not at a meaningful scale. In this study, we evaluated different enrichment and operational strategies to optimize MFC performance. At the beginning of the experiment, during the enrichment phase, the anode potential was modulated either by an external resistor or an applied voltage. It has been previously shown that the anodic community composition and biomass formation changes according to the external resistor or potential applied [1,2]. Under such operational conditions, a faster start up time can also be achieved [3]. In addition, start-up time and overall performance will also be impacted by inoculum source and substrate selection. Previous reports addressed MFC performance as a function of operation and startup time using either a defined media and single carbon substrate [2] or a single enrichment strategy with primary clarifier effluent as the single inoculum and substrate [1]. Therefore our evaluations addressed a highly diverse inoculum source including lagoon sediment and swine waste and different methodologies for enrichment to expand the knowledge base about what enrichment strategies may be best for a given wastewater treatment application. Varying ranges of external resistors and applied voltages were tested. A periodically induced open circuit condition was also evaluated to study its effect on MFC output. With these experimental conditions, we are aiming to identify an optimal strategy for MFC start-up and operation, and evaluate the long-term effects on MFC performance including the electrochemical performance, community taxonomic dynamics, biomass formation, and wastewater treatment rate.

Clinical and Environmental Surveillance for Vibrio cholerae in Resource Constrained Areas: Application During a 1-Year Surveillance in the Far North Region of Cameroon.

Biological confirmation of the presence of Vibrio cholerae in clinical and environmental samples is often constrained due to resource- and labor-intensive gold standard methods. To develop low-cost, simple, and sustainable surveillance techniques, we modified previously published specimen sampling and culture techniques and applied the use of enriched dipstick testing in conjunction with the use of filter paper for DNA specimen preservation during clinical and environmental surveillance in the Far North of Cameroon from August 2013 to October 2014. The enriched dipstick methodology during routine use in a remote setting demonstrated a specificity of 99.8% compared with polymerase chain reaction (PCR). The novel application of filter paper as a preservation method for cholera DNA specimens reduced the need for cold chain storage and allowed for PCR characterization and confirmation of V. cholerae. The application of basic technologies such as the enriched dipstick, the use of simplified gauze filtration for environmental sample collection, and the use of filter paper for sample preservation enabled early case identification with reduced logistics and supply cost while reporting minimal false-positive results. Simplified laboratory and epidemiological methodologies can improve the feasibility of cholera surveillance in rural and resource-constrained areas, facilitating early case detection and rapid response implementation.