Inflammation and Proliferation Act Together to Mediate Intestinal Cell Fusion

Cell fusion between circulating bone marrow-derived cells (BMDCs) and non-hematopoietic cells is well documented in various tissues and has recently been suggested to occur in response to injury. Here we illustrate that inflammation within the intestine enhanced the level of BMDC fusion with intestinal progenitors. To identify important microenvironmental factors mediating intestinal epithelial cell fusion, we performed bone marrow transplantation into mouse models of inflammation and stimulated epithelial proliferation. Interestingly, in a non-injury model or in instances where inflammation was suppressed, an appreciable baseline level of fusion persisted. This suggests that additional mediators of cell fusion exist. A rigorous temporal analysis of early post-transplantation cellular dynamics revealed that GFP-expressing donor cells first trafficked to the intestine coincident with a striking increase in epithelial proliferation, advocating for a required fusogenic state of the host partner. Directly supporting this hypothesis, induction of augmented epithelial proliferation resulted in a significant increase in intestinal cell fusion. Here we report that intestinal inflammation and epithelial proliferation act together to promote cell fusion. While the physiologic impact of cell fusion is not yet known, the increased incidence in an inflammatory and proliferative microenvironment suggests a potential role for cell fusion in mediating the progression of intestinal inflammatory diseases and cancer.     

Genetic Characterization of Venezuelan Equine Encephalitis Virus from Bolivia, Ecuador and Peru: Identification of a New Subtype ID Lineage

Venezuelan equine encephalitis virus (VEEV) has been responsible for hundreds of thousands of human and equine cases of severe disease in the Americas. A passive surveillance study was conducted in Peru, Bolivia and Ecuador to determine the arboviral etiology of febrile illness. Patients with suspected viral-associated, acute, undifferentiated febrile illness of <7 days duration were enrolled in the study and blood samples were obtained from each patient and assayed by virus isolation. Demographic and clinical information from each patient was also obtained at the time of voluntary enrollment. In 2005–2007, cases of Venezuelan equine encephalitis (VEE) were diagnosed for the first time in residents of Bolivia; the patients did not report traveling, suggesting endemic circulation of VEEV in Bolivia. In 2001 and 2003, VEE cases were also identified in Ecuador. Since 1993, VEEV has been continuously isolated from patients in Loreto, Peru, and more recently (2005), in Madre de Dios, Peru. We performed phylogenetic analyses with VEEV from Bolivia, Ecuador and Peru and compared their relationships to strains from other parts of South America. We found that VEEV subtype ID Panama/Peru genotype is the predominant one circulating in Peru. We also demonstrated that VEEV subtype ID strains circulating in Ecuador belong to the Colombia/Venezuela genotype and VEEV from Madre de Dios, Peru and Cochabamba, Bolivia belong to a new ID genotype. In summary, we identified a new major lineage of enzootic VEEV subtype ID, information that could aid in the understanding of the emergence and evolution of VEEV in South America.     

Microhabitat characteristics of Akodon montensis,a reservoir for hantavirus,and hantaviral seroprevalence in an Atlantic forest site in eastern Paraguay

Hantaviruses may cause serious disease when transmitted to humans by their rodent hosts. Since their emergence in the Americas in 1993, there have been extensive efforts to understand the role of environmental factors on the presence of these viruses in their host rodent populations. HPS outbreaks have been linked to precipitation, but climatic factors alone have not been sufficient to predict the spatial‐temporal dynamics of the environment‐reservoir‐virus system. Using a series of mark‐recapture sampling sites located at the Mbaracayú Biosphere Reserve, an Atlantic Forest site in eastern Paraguay, we investigated the hypothesis that microhabitat might also influence the prevalence of Jaborá hantavirus within populations of its reservoir species, Akodon montensis. Seven trapping sessions were conducted during 2005‐2006 at four sites chosen to capture variable microhabitat conditions within the study site. Analysis of microhabitat preferences showed that A. montensis preferred areas with little forest overstory and denser vegetation cover on and near the ground. Moreover, there was a significant difference in the microhabitat occupied by antibody‐positive vs antibody‐negative rodents, indicating that microhabitats with greater overstory cover may promote transmission and maintenance of hantavirus in A. montensis.     

Evolutionary Patterns of Eastern Equine Encephalitis Virus in North versus South America Suggest Ecological Differences and Taxonomic Revision

The eastern equine encephalitis (EEE) complex consists of four distinct genetic lineages: one that circulates in North America (NA EEEV) and the Caribbean and three that circulate in Central and South America (SA EEEV). Differences in their geographic, pathogenic, and epidemiologic profiles prompted evaluation of their genetic diversity and evolutionary histories. The structural polyprotein open reading frames of all available SA EEEV and recent NA EEEV isolates were sequenced and used in evolutionary and phylogenetic analyses. The nucleotide substitution rate per year for SA EEEV (1.2 × 10⁻⁴) was lower and more consistent than that for NA EEEV (2.7 × 10⁻⁴), which exhibited considerable rate variation among constituent clades. Estimates of time since divergence varied widely depending upon the sequences used, with NA and SA EEEV diverging ca. 922 to 4,856 years ago and the two main SA EEEV lineages diverging ca. 577 to 2,927 years ago. The single, monophyletic NA EEEV lineage exhibited mainly temporally associated relationships and was highly conserved throughout its geographic range. In contrast, SA EEEV comprised three divergent lineages, two consisting of highly conserved geographic groupings that completely lacked temporal associations. A phylogenetic comparison of SA EEEV and Venezuelan equine encephalitis viruses (VEEV) demonstrated similar genetic and evolutionary patterns, consistent with the well-documented use of mammalian reservoir hosts by VEEV. Our results emphasize the evolutionary and genetic divergences between members of the NA and SA EEEV lineages, consistent with major differences in pathogenicity and ecology, and propose that NA and SA EEEV be reclassified as distinct species in the EEE complex.Eastern equine encephalitis virus (EEEV) is an important veterinary and human pathogen belonging to one of seven antigenic complexes in the Alphavirus genus, family Togaviridae (32). Isolated throughout the Americas, EEEV is classified as the only species in the eastern equine encephalitis (EEE) complex (9, 10), which was originally divided into North and South American varieties based on antigenic properties (11). However, additional antigenic and phylogenetic analyses have refined its classification to include four subtypes that correspond to four major genetic lineages (I to IV) (7, 55). North American EEEV (NA EEEV) strains and most strains from the Caribbean comprise subtype/lineage I, while subtypes/lineages II to IV include South and Central American EEEV (SA EEEV) strains. The EEEV genome consists of a nonsegmented, single-stranded, positive-sense RNA of approximately 11.7 kb, which includes a 5′ cap and a 3′ poly(A) tail. The 5′ end of the genome encodes four nonstructural proteins (nsP1 to -4), while a subgenomic RNA (26S) is encoded by the 3′ end and ultimately produces three main structural proteins: capsid and envelope glycoproteins E1 and E2 (46).Despite considerable nucleotide sequence divergence between NA and SA EEEV lineages, NA EEEV is highly conserved throughout its geographic and temporal spectra. Multiple robust analyses have demonstrated less than 2% nucleotide sequence divergence among NA EEEV strains isolated between 1933 and 2007 (5, 7, 64, 68, 69). An overall temporal trend of genetic conservation is also maintained, with newer isolates differing most from ancestral strains at the base of the North American clade (7, 64). In contrast, SA EEEV is highly divergent both between and among the three lineages/subtypes. Although less robust than previous NA EEEV phylogenetic analyses, those of SA EEEV show a tendency for geographic clustering of isolates rather than temporal relationships (7). Differing patterns of genetic conservation between NA and SA EEEV may be the result of differences in their ecology and adaptation to different mosquito and vertebrate hosts (65).Transmission of NA EEEV occurs in an enzootic cycle involving the ornithophilic mosquito vector Culiseta melanura and passerine birds in hardwood swamp habitats (32, 43). The broad geographic distribution and distinctly ornithophagic behavior of Cs. melanura result in a close relationship between NA EEEV and avian vertebrate hosts, which is one proposed mechanism for its highly conserved genetic nature. Infected birds provide for efficient geographic dispersal and the mixing of strains with distant origins. While genetic drift tends to have less impact on large, panmictic populations, competition and natural selection may periodically constrain genetic diversity in the NA EEEV population, resulting in the antigenic and genetic conservation observed (64, 66). Transmission of NA EEEV by bridge vectors probably does not impact viral evolution; however, it does result in sporadic outbreaks of severe disease in humans, equids, and other domestic animals, including game birds, swine, and dogs that are considered dead-end hosts (22, 23, 43, 50).Although they are associated with equine disease, SA strains of EEEV are not clearly associated with human disease (4, 17, 18, 40). This lack of human pathogenicity has limited research to expand our epidemiologic and ecologic understanding of SA strains. EEEV isolations from Culex (Melanoconion) spp. in the Spissipes section (Culex pedroi in South America and Culex taeniopus in Central America) suggest that they are the primary enzootic, and potentially epizootic, vectors (28, 33, 53, 58). Movement of these vectors beyond their tropical forest habitat is typically limited (29), which may influence the focality of transmission. However, these species are relatively catholic in their feeding behavior, which broadens the potential transmission cycles used by SA EEEV. Greater vector diversity in tropical regions may also contribute to genetic diversity among the SA EEEV lineages, although vector competence data are limited.The vertebrate ecology of SA EEEV is not well described, with serological associations including wild birds, ground-dwelling rodents, marsupials, and reptiles (12, 17, 31, 45, 56, 57, 58). The observed genetic divergence and geographic clustering of the SA EEEV phylogeny could reflect the use of ground-dwelling mammals as primary hosts for enzootic transmission (43, 65). With limited mobility, these vector and vertebrate species may restrict the distribution of SA EEEV to geographically defined regions, thus limiting competition among distant strains and allowing for the independent evolution of genetic lineages (65). Geographically delineated transmission foci may also be more susceptible to the impacts of genetic drift, thus constraining genetic diversity locally. Venezuelan equine encephalitis viruses (VEEV), which also utilize Culex (Melanoconion) sp. vectors and small mammals as primary vertebrate hosts (15, 42, 51, 52, 59, 67), exhibit a similar genetic pattern of independent evolution and multiple, cocirculating subtypes in Central and South America (60). However, a robust comparison of the evolutionary patterns between SA EEEV and VEEV has not been conducted.Elucidation of patterns of enzootic transmission and dispersal of zoonotic, arboviral pathogens is critical for understanding and predicting the risk to human health. Therefore, we studied the evolutionary progression of the EEE complex to clarify the extent of divergence between NA and SA EEEV. Because previous analyses of SA EEEV were either limited in their geographic scope or utilized only partial, concatenated sequences, conclusions regarding the genetic relationships of members within and among EEEV lineages were limited. In addition, previous analyses utilized linear regression and were based on few representatives of a single SA EEEV lineage. Here we exploited contemporary techniques to sequence and analyze the structural protein open reading frames (ORFs) of all available SA EEEV and additional NA EEEV isolates and phylogenetically compared SA EEEV and VEEV. Our results support evolutionary and ecological diversity between NA and SA EEEV and suggest that NA and SA lineages be considered independent species in the EEE complex.     

Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells

Background

Wnt/β-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.

Methodology/Principal Findings

We tested the hypothesis that Wnt/β-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/β-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for β-myosin heavy chain (β-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/β-catenin signaling is required for Smad1 activation by BMP4.

Conclusions/Significance

Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/β-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.     

Comprehensive evaluation of isoprenoid biosynthesis regulation in Saccharomyces cerevisiae utilizing the Genome Reporter Matrix

Gene expression profiling is rapidly becoming a mainstay of functional genomic studies. However, there have been relatively few studies of how the data from expression profiles integrate with more classic approaches to examine gene expression. This study used gene expression profiling of a portion of the genome of Saccharomyces cerevisiae to explore the impact of blocks in the isoprenoid biosynthetic pathway on the expression of genes and the regulation of this pathway. Approximately 50% of the genes whose expression was altered by blocks in isoprenoid biosynthesis were genes previously known to participate in the pathway. In contrast to this simple correspondence, the regulatory patterns revealed by different blocks, and in particular by antifungal azoles, was complex in a manner not anticipated by earlier studies.     

Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma

Although microdialysis is widely used to sample endogenous and exogenous substances in vivo, interpretation of the results obtained by this technique remains controversial. The goal of the present study was to examine recent criticism of microdialysis in the specific case of dopamine (DA) measurements in the brain extracellular microenvironment. The apparent steady-state basal extracellular concentration and extraction fraction of DA were determined in anesthetized rat striatum by the concentration difference (no-net-flux) microdialysis technique. A rate constant for extracellular clearance of DA calculated from the extraction fraction was smaller than the previously determined estimate by fast-scan cyclic voltammetry for cellular uptake of DA. Because the relatively small size of the voltammetric microsensor produces little tissue damage, the discrepancy between the uptake rate constants may be a consequence of trauma from microdialysis probe implantation. The trauma layer has previously been identified by histology and proposed to distort measurements of extracellular DA levels by the no-net-flux method. To address this issue, an existing quantitative mathematical model for microdialysis was modified to incorporate a traumatized tissue layer interposed between the probe and surrounding normal tissue. The tissue layers are hypothesized to differ in their rates of neurotransmitter release and uptake. A post-implantation traumatized layer with reduced uptake and no release can reconcile the discrepancy between DA uptake measured by microdialysis and voltammetry. The model predicts that this trauma layer would cause the DA extraction fraction obtained from microdialysis in vivo calibration techniques, such as no-net-flux, to differ from the DA relative recovery and lead to an underestimation of the DA extracellular concentration in the surrounding normal tissue.     

Rap1 couples cAMP signaling to a distinct pool of p42/44MAPK regulating excitability,synaptic plasticity,learning, and memory

Learning-induced synaptic plasticity commonly involves the interaction between cAMP and p42/44MAPK. To investigate the role of Rap1 as a potential signaling molecule coupling cAMP and p42/44MAPK, we expressed an interfering Rap1 mutant (iRap1) in the mouse forebrain. This expression selectively decreased basal phosphorylation of a membrane-associated pool of p42/44MAPK, impaired cAMP-dependent LTP in the hippocampal Schaffer collateral pathway induced by either forskolin or theta frequency stimulation, decreased complex spike firing, and reduced the p42/44MAPK-mediated phosphorylation of the A-type potassium channel Kv4.2. These changes correlated with impaired spatial memory and context discrimination. These results indicate that Rap1 couples cAMP signaling to a selective membrane-associated pool of p42/44MAPK to control excitability of pyramidal cells, the early and late phases of LTP, and the storage of spatial memory.     

VEGF: a modifier of the del22q11 (DiGeorge) syndrome?

Hemizygous deletion of chromosome 22q11 (del22q11) causes thymic, parathyroid, craniofacial and life-threatening cardiovascular birth defects in 1 in 4,000 infants. The del22q11 syndrome is likely caused by haploinsufficiency of TBX1, but its variable expressivity indicates the involvement of additional modifiers. Here, we report that absence of the Vegf164 isoform caused birth defects in mice, reminiscent of those found in del22q11 patients. The close correlation of birth and vascular defects indicated that vascular dysgenesis may pathogenetically contribute to the birth defects. Vegf interacted with Tbx1, as Tbx1 expression was reduced in Vegf164-deficient embryos and knocked-down vegf levels enhanced the pharyngeal arch artery defects induced by tbx1 knockdown in zebrafish. Moreover, initial evidence suggested that a VEGF promoter haplotype was associated with an increased risk for cardiovascular birth defects in del22q11 individuals. These genetic data in mouse, fish and human indicate that VEGF is a modifier of cardiovascular birth defects in the del22q11 syndrome.     

Two Copies of form I RuBisCO genes in Acidithiobacillus ferrooxidans ATCC 23270

Acidithiobacillus ferrooxidans ATCC 23270 possesses two copies of form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The nucleotide sequence identity between the two large and two small subunit peptides was 75% and 58%, respectively. It is proposed that the two copies resulted from lateral gene transfer. Received: 27 October 2000 / Accepted 7 December 2001