Development of a bead-based multiplex assay for the simultaneous detection of porcine inflammation markers using xMAP technology

Commercially available assays for the simultaneous detection of multiple inflammatory and cardiac markers in porcine blood samples are currently lacking. Therefore, this study was aimed at developing a bead-based, multiplexed flow cytometric assay to simultaneously detect porcine cytokines [interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha], chemokines (IL-8 and monocyte chemotactic protein 1), growth factors [basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and platelet-derived growth factor-bb], and injury markers (cardiac troponin-I) as well as complement activation markers (C5a and sC5b-9). The method was based on the Luminex xMAP technology, resulting in the assembly of a 6- and 11-plex from the respective individual singleplex situation. The assay was evaluated for dynamic range, sensitivity, cross-reactivity, intra-assay and interassay variance, spike recovery, and correlation between multiplex and commercially available enzyme-linked immunosorbent assay as well as the respective singleplex. The limit of detection ranged from 2.5 to 30,000 pg/ml for all analytes (6- and 11-plex assays), except for soluble C5b-9 with a detection range of 2-10,000 ng/ml (11-plex). Typically, very low cross-reactivity (<3% and <1.4% by 11- and 6-plex, respectively) between analytes was found. Intra-assay variances ranged from 4.9 to 7.4% (6-plex) and 5.3 to 12.9% (11-plex). Interassay variances for cytokines were between 8.1 and 28.8% (6-plex) and 10.1 and 26.4% (11-plex). Correlation coefficients with singleplex assays for 6-plex as well as for 11-plex were high, ranging from 0.988 to 0.997 and 0.913 to 0.999, respectively. In this study, a bead-based porcine 11-plex and 6-plex assay with a good assay sensitivity, broad dynamic range, and low intra-assay variance and cross-reactivity was established. These assays therefore represent a new, useful tool for the analysis of samples generated from experiments with pigs.

Creating a public tool to assess and promote transparency in global land deals: the experience of the Land Matrix

The Beta version of the Land Matrix (Land Matrix 2012) was launched in April 2012 as a tool to promote public participation in building a constantly evolving database on large-scale land deals, and making the data visible and understandable. The aim of the Land Matrix partnership is to promote transparency and open data in decisionmaking over land and investment, as a step towards greater accountability. Since its launch, the Land Matrix has attracted a high degree of attention, and stirred some controversy. It provides valuable lessons on the challenges and benefits of promoting open data on practices that are often shrouded in secrecy. This paper critically examines the ongoing efforts by the Land Matrix partnership to build a public tool to promote greater transparency in decision-making over land and investment at a global level. It intends to provoke discussion of the extent to which such a tool can ultimately promote greater transparency and be a step towards greater accountability and improved decision-making. It will present the Land Matrix and its value addition, before detailing the challenges it encountered related to the measurement of the largescale land acquisition phenomenon. It will then specify how it intends to address these issues in order to establish a dynamic and participatory tool for open development.

CA125/MUC16 is dispensable for mouse development and reproduction.

Cancer antigen 125 (CA125) is a blood biomarker that is routinely used to monitor the progression of human epithelial ovarian cancer (EOC) and is encoded by MUC16, a member of the mucin gene family. The biological function of CA125/MUC16 and its potential role in EOC are poorly understood. Here we report the targeted disruption of the of the Muc16 gene in the mouse. To generate Muc16 knockout mice, 6.0 kb was deleted that included the majority of exon 3 and a portion of intron 3 and replaced with a lacZ reporter cassette. Loss of Muc16 protein expression suggests that Muc16 homozygous mutant mice are null mutants. Muc16 homozygous mutant mice are viable, fertile, and develop normally. Histological analysis shows that Muc16 homozygous mutant tissues are normal. By the age of 1 year, Muc16 homozygous mutant mice appear normal. Downregulation of transcripts from another mucin gene (Muc1) was detected in the Muc16 homozygous mutant uterus. Lack of any prominent abnormal phenotype in these Muc16 knockout mice suggests that CA125/MUC16 is not required for normal development or reproduction. These knockout mice provide a unique platform for future studies to identify the role of CA125/MUC16 in organ homeostasis and ovarian cancer.

Dicer is required for female reproductive tract development and fertility in the mouse.

Dicer encodes a riboendonuclease required for microRNA biosynthesis. Dicer was inactivated in Müllerian duct mesenchyme-derived tissues of the reproductive tract of the mouse, using an Amhr2-Cre allele. Although Amhr2-Cre; Dicer conditional mutant males appeared normal and were fertile, mutant females were infertile. In adult mutant females, there was a reduction in the size of the oviducts and uterine horns. The oviducts were less coiled compared to controls and cysts formed at the isthmus near the uterotubal junction. Unfertilized, degenerate oocytes were commonly found within these cysts, indicating a defect in embryo transit. Beads transferred into the mutant oviduct failed to migrate into the uterus. In addition, blastocysts transferred directly into the mutant uterus did not result in pregnancy. Histological analysis demonstrated that the mutant uterus contained less glandular tissue and often the few glands that remained were found within the myometrium, an abnormal condition known as adenomyosis. In adult mutants, there was ectopic expression of Wnt4 and Wnt5a in the luminal epithelium (LE) and glandular epithelium (GE) of the uterus, and Wnt11 was ectopically expressed in GE. These results demonstrate that Dicer is necessary for postnatal differentiation of Müllerian duct mesenchyme-derived tissues of the female reproductive tract, suggesting that microRNAs are important regulators of female reproductive tract development and fertility.

Eomesodermin, a target gene of Pou4f2, is required for retinal ganglion cell and optic nerve development in the mouse.

The mechanisms regulating retinal ganglion cell (RGC) development are crucial for retinogenesis and for the establishment of normal vision. However, these mechanisms are only vaguely understood. RGCs are the first neuronal lineage to segregate from pluripotent progenitors in the developing retina. As output neurons, RGCs display developmental features very distinct from those of the other retinal cell types. To better understand RGC development, we have previously constructed a gene regulatory network featuring a hierarchical cascade of transcription factors that ultimately controls the expression of downstream effector genes. This has revealed the existence of a Pou domain transcription factor, Pou4f2, that occupies a key node in the RGC gene regulatory network and that is essential for RGC differentiation. However, little is known about the genes that connect upstream regulatory genes, such as Pou4f2 with downstream effector genes responsible for RGC differentiation. The purpose of this study was to characterize the retinal function of eomesodermin (Eomes), a T-box transcription factor with previously unsuspected roles in retinogenesis. We show that Eomes is expressed in developing RGCs and is a mediator of Pou4f2 function. Pou4f2 directly regulates Eomes expression through a cis-regulatory element within a conserved retinal enhancer. Deleting Eomes in the developing retina causes defects reminiscent of those in Pou4f2(-/-) retinas. Moreover, myelin ensheathment in the optic nerves of Eomes(-/-) embryos is severely impaired, suggesting that Eomes regulates this process. We conclude that Eomes is a crucial regulator positioned immediately downstream of Pou4f2 and is required for RGC differentiation and optic nerve development.

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure.

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

Taxonomy development and knowledge representation of nurses' personal cognitive artifacts.

Nurses prepare knowledge representations, or summaries of patient clinical data, each shift. These knowledge representations serve multiple purposes, including support of working memory, workload organization and prioritization, critical thinking, and reflection. This summary is integral to internal knowledge representations, working memory, and decision-making. Study of this nurse knowledge representation resulted in development of a taxonomy of knowledge representations necessary to nursing practice.This paper describes the methods used to elicit the knowledge representations and structures necessary for the work of clinical nurses, described the development of a taxonomy of this knowledge representation, and discusses translation of this methodology to the cognitive artifacts of other disciplines. Understanding the development and purpose of practitioner's knowledge representations provides important direction to informaticists seeking to create information technology alternatives. The outcome of this paper is to suggest a process template for transition of cognitive artifacts to an information system.

A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.

The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life, called hereafter bone formation, are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation we generated transgenic mice overexpressing Cbfa1 DNA-binding domain (DeltaCbfa1) in differentiated osteoblasts only postnatally. DeltaCbfa1 has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. DeltaCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that DeltaCbfa1-expressing osteoblasts were less active than wild-type osteoblasts. We also show that Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally.

Hsp90 nuclear accumulation in quiescence is linked to chaperone function and spore development in yeast.

The 90-kDa heat-shock protein (Hsp90) operates in the context of a multichaperone complex to promote maturation of nuclear and cytoplasmic clients. We have discovered that Hsp90 and the cochaperone Sba1/p23 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells. Hsp90 nuclear accumulation was unaffected in sba1Delta cells, demonstrating that Hsp82 translocates independently of Sba1. Translocation of both chaperones was dependent on the alpha/beta importin SRP1/KAP95. Hsp90 nuclear retention was coincident with glucose exhaustion and seems to be a starvation-specific response, as heat shock or 10% ethanol stress failed to elicit translocation. We generated nuclear accumulation-defective HSP82 mutants to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to retain Hsp90 in the cytoplasm in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.

The role of peroxisome proliferator-activated receptors in the development and physiology of gametes and preimplantation embryos.

In several species, a family of nuclear receptors, the peroxisome proliferator-activated receptors (PPARs) composed of three isotypes, is expressed in somatic cells and germ cells of the ovary as well as the testis. Invalidation of these receptors in mice or stimulation of these receptors in vivo or in vitro showed that each receptor has physiological roles in the gamete maturation or the embryo development. In addition, synthetic PPAR gamma ligands are recently used to induce ovulation in women with polycystic ovary disease. These results reveal the positive actions of PPAR in reproduction. On the other hand, xenobiotics molecules (in herbicides, plasticizers, or components of personal care products), capable of activating PPAR, may disrupt normal PPAR functions in the ovary or the testis and have consequences on the quality of the gametes and the embryos. Despite the recent data obtained on the biological actions of PPARs in reproduction, relatively little is known about PPARs in gametes and embryos. This review summarizes the current knowledge on the expression and the function of PPARs as well as their partners, retinoid X receptors (RXRs), in germ cells and preimplantation embryos. The effects of natural and synthetic PPAR ligands will also be discussed from the perspectives of reproductive toxicology and assisted reproductive technology.

Influence of host interleukin-10 polymorphisms on development of traveler's diarrhea due to heat-labile enterotoxin-producing Escherichia coli in travelers from the United States who are visiting Mexico.

Up to 60% of U.S. visitors to Mexico develop traveler's diarrhea (TD), mostly due to enterotoxigenic Escherichia coli (ETEC) strains that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Distinct single-nucleotide polymorphisms (SNPs) within the interleukin-10 (IL-10) promoter have been associated with high, intermediate, or low production of IL-10. We conducted a prospective study to investigate the association of SNPs in the IL-10 promoter and the occurrence of TD in ETEC LT-exposed travelers. Sera from U.S. travelers to Mexico collected on arrival and departure were studied for ETEC LT seroconversion by using cholera toxin as the antigen. Pyrosequencing was performed to genotype IL-10 SNPs. Stools from subjects who developed diarrhea were also studied for other enteropathogens. One hundred twenty-one of 569 (21.3%) travelers seroconverted to ETEC LT, and among them 75 (62%) developed diarrhea. Symptomatic seroconversion was more commonly seen in subjects who carried a genotype producing high levels of IL-10; it was seen in 83% of subjects with the GG genotype versus 54% of subjects with the AA genotype at IL-10 gene position -1082 (P, 0.02), in 71% of those with the CC genotype versus 33% of those with the TT genotype at position -819 (P, 0.005), and in 71% of those with the CC genotype versus 38% of those with the AA genotype at position -592 (P, 0.02). Travelers with the GCC haplotype were more likely to have symptomatic seroconversion than those with the ATA haplotype (71% versus 38%; P, 0.002). Travelers genetically predisposed to produce high levels of IL-10 were more likely to experience symptomatic ETEC TD.

DYSREGULATION OF MEOX2 FOLLOWING WT1 MUTATION IN KIDNEY DEVELOPMENT AND WILMS TUMORIGENESIS

Wilms tumor (WT) is a childhood tumor of the kidney and a productive model for understanding the role of genetic alteration and interactions in tumorigenesis. The Wilms tumor gene 1 (WT1) is a transcriptional factor and one of the few genes known to have genetic alterations in WT and has been shown be inactivated in 20% of WTs. However, the mechanisms of how WT1 mutations lead to Wilms tumorigenesis and its influence on downstream genes are unknown. Since it has been established that WT1 is a transcriptional regulator, it has been hypothesized that the loss of WT1 leads to the dysregulation of downstream genes, in turn result in the formation of WTs. To identify the dysregulated downstream genes following WT1 mutations, an Affymetrix GeneChip Human Genome Array was previously conducted to assess the differentially expressed genes in the WT1-wildtype human and WT1-mutant human WTs. Approximately 700 genes were identified as being significantly dysregulated. These genes were further prioritized based on their statistical significance, fold change, chromosomal region, spatial pattern of gene expression and known or putative cellular functions. Mesenchyme homeobox 2 (MEOX2) was one of the most significantly upregulated genes in WT1-mutant WT. MEOX2 is known to play a role in cell proliferation, apoptosis, and differentiation. In addition to its biological roles, it is expressed during early kidney development in the condensed mesenchyme similar to WT1. Furthermore, the use of the Match® web-based tool from the BIOBASE Biological Data base identified a significant predicted WT1 binding site within the first intron of MEOX2. The similarity in spatial gene expression in the developing kidney and the significant predicted WT1 binding site found in the first intron of MEOX2 lead to the development of my hypothesis that MEOX2 is upregulated via a WT1-dependent manner. Here as a part of my master’s work, I have validated the Affymetrix GeneChip Human Genome Array data using an independent set of Wilms tumors. MEOX2 remained upregulated in the mutant WT1 Wilms tumor by 41-fold. Wt1 and Meox2 gene expression were assessed in murine newborn kidney; both Wt1 and Meox2 were expressed in the condensed, undifferentiated metanephric mesenchyme. I have shown that the in vivo ablation of Wt1 during embryonic development at embryonic day (E) 13.5 resulted in the slight increase of Meox2 gene expression by two fold. In order to functionally demonstrate the effect of the loss of Wt1 on Meox2 gene expression in undifferentiated metanephric mesenchyme, I have generated a kidney mesenchymal cell line to genetically ablate Wt1 in vitro by adenoviral infection. The ablation of Wt1 in the kidney mesenchymal cell line resulted in the upregulation of Meox2 by 61-fold. Moreover, the upregulation of Meox2 resulted in the significant induction of p21 and Itgb5. In addition to the dysregulation of these genes the ablation of Wt1 in the kidney mesenchymal cells resulted in decrease in cell growth and loss of cellular adherence. However, it is uncertain whether the upregulation of Meox2 caused this particular cellular phenotype. Overall, I have demonstrated that the upregulation of Meox2 is Wt1-dependent during early kidney development.

Development of a rapid bioassessment method and index of biotic integrity for estuaries located along the northwest Gulf of Mexico

Recently it has been proposed that the evaluation of effects of pollutants on aquatic organisms can provide an early warning system of potential environmental and human health risks (NRC 1991). Unfortunately there are few methods available to aquatic biologists to conduct assessments of the effects of pollutants on aquatic animal community health. The primary goal of this research was to develop and evaluate the feasibility of such a method. Specifically, the primary objective of this study was to develop a prototype rapid bioassessment technique similar to the Index of Biotic Integrity (IBI) for the upper Texas and Northwestern Gulf of Mexico coastal tributaries. The IBI consists of a series of "metrics" which describes specific attributes of the aquatic community. Each of these metrics are given a score which is then subtotaled to derive a total assessment of the "health" of the aquatic community. This IBI procedure may provide an additional assessment tool for professionals in water quality management.^ The experimental design consisted primarily of compiling previously collected data from monitoring conducted by the Texas Natural Resource Conservation Commission (TNRCC) at five bayous classified according to potential for anthropogenic impact and salinity regime. Standardized hydrological, chemical, and biological monitoring had been conducted in each of these watersheds. The identification and evaluation of candidate metrics for inclusion in the estuarine IBI was conducted through the use of correlation analysis, cluster analysis, stepwise and normal discriminant analysis, and evaluation of cumulative distribution frequencies. Scores of each included metric were determined based on exceedances of specific percentiles. Individual scores were summed and a total IBI score and rank for the community computed.^ Results of these analyses yielded the proposed metrics and rankings listed in this report. Based on the results of this study, incorporation of an estuarine IBI method as a water quality assessment tool is warranted. Adopted metrics were correlated to seasonal trends and less so to salinity gradients observed during the study (0-25 ppt). Further refinement of this method is needed using a larger more inclusive data set which includes additional habitat types, salinity ranges, and temporal variation. ^

THE DESIGN AND DEVELOPMENT OF A SURVEY INSTRUMENT FOR THE ASSESSMENT OF OCCUPATIONAL STRESS

Using stress and coping as a unifying theoretical concept, a series of five models was developed in order to synthesize the survey questions and to classify information. These models identified the question, listed the research study, described measurements, listed workplace data, and listed industry and national reference data.^ A set of 38 instrument questions was developed within the five coping correlate categories. In addition, a set of 22 stress symptoms was also developed. The study was conducted within two groups, police and professors, on a large university campus. The groups were selected because their occupations were diverse, but they were a part of the same macroenvironment. The premise was that police officers would be more highly stressed than professors.^ Of a total study group of 80, there were 37 respondents. The difference in the mean stress responses was observable between the two groups. Not only were the responses similar within each group, but the stress level of response was also similar within each group. While the response to the survey instrument was good, only 3 respondents answered the stress symptom survey properly. It was determined that none of the 37 respondents believed that they were ill. This perception of being well was also evidenced by the grand mean of the stress scores of 2.76 (3.0 = moderate stress). This also caused fewer independent variables to be entered in the multiple regression model.^ The survey instrument was carefully designed to be universal. Universality is the ability to transcend occupational or regional definitions as applied to stress. It is the ability to measure responses within broad categories such as physiological, emotional, behavioral, social, and cognitive functions without losing the ability to measure the detail within the individual questions, or the relationships between questions and categories.^ Replication is much easier to achieve with standardized categories, questions, and measurement procedures such as those developed for the universal survey instrument. Because the survey instrument is universal it can be used as an analytical device, an assessment device, a basic tool for planning and a follow-up instrument to measure individual response to planned reductions in occupational stress. (Abstract shortened with permission of author.) ^

Responses to rapid warming at Termination 1a at Gerzensee (Central Europe): Primary succession, albedo, soils, lake development, and ecological interactions

The transition from the Oldest Dryas to the Bølling around 14,685 cal yr BP was a period of extremely rapid climatic warming. From a single core of lake marl taken at Gerzensee (Switzerland) we studied the transition in stable isotopes of oxygen and carbon on bulk sediment and charophyte remains, as well as on monospecific samples of ostracods, after Pisidium a; in addition pollen, chironomids, and Cladocera were analyzed. The δ18O record serves as an estimate of mean air temperature, and by correlation to the one from NGRIP in Greenland it provides a timescale. The timing of responses: The statistically significant zone boundaries of the biostratigraphies are telescoped at the rapid increase of about 3‰ in δ18O at the onset of Bølling. Biotic responses may have occurred within sampling resolution (8 to 16 years), although younger zone boundaries are less synchronous. Gradual and longer-lasting responses include complex processes such as primary or secular succession. During the late-glacial interstadial of Bølling and Allerød, two stronger and two weaker cool phases were found. Biological processes involved in the responses occurred on levels of individuals (e.g. pollen productivity), of populations (increases or decreases, immigration, or extinction), and on the ecosystem level (species interactions such as facilitation or competition). Abiotic and biotic interactions include pedogenesis, nitrogen-fixation, nutrient cycling, catchment hydrology, water chemistry of the lake and albedo (controlled by the transition from tundra to forest). For the Swiss Plateau this major change in vegetation induced a change in the mammal fauna, which in turn led to changes in the tool-making by Paleolithic people.