Colon cancer stem cells are believed to originate from a rare population of putative CD133 + intestinal stem cells. Recent publications suggest that a small subset of colon cancer cells expresses CD133, and that only these CD133 + cancer cells are capable of tumor initiation. However, the precise contribution of CD133 + tumor-initiating cells in mediating colon cancer metastasis remains unknown. Therefore, to temporally and spatially track the expression of CD133 in adult mice and during tumorigenesis, we generated a knockin lacZ reporter mouse (CD133 lacZ/+ ), in which the expression of lacZ is driven by the endogenous CD133 promoters. Using this model and immunostaining, we discovered that CD133 expression in colon is not restricted to stem cells; on the contrary, CD133 is ubiquitously expressed on differentiated colonic epithelium in both adult mice and humans. Using Il10 -/-CD133 lacZ mice, in which chronic inflammation in colon leads to adenocarcinomas, we demonstrated that CD133 is expressed on a full gamut of colonic tumor cells, which express epithelial cell adhesion molecule (EpCAM). Similarly, CD133 is widely expressed by human primary colon cancer epithelial cells, whereas the CD133 -population is composed mostly of stromal and inflammatory cells. Conversely, CD133 expression does not identify the entire population of epithelial and tumor-initiating cells in human metastatic colon cancer. Indeed, both CD133 + and CD133 -metastatic tumor subpopulations formed colonospheres in in vitro cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. Moreover, metastatic CD133 -cells form more aggressive tumors and express typical phenotypic markers of cancer-initiating cells, including CD44 (CD44 + CD24 -), whereas the CD133 + fraction is composed of CD44 low CD24 + cells. Collectively, our data suggest that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and during the metastatic transition, CD133 + tumor cells might give rise to the more aggressive CD133 -subset, which is also capable of tumor initiation in NOD/SCID mice.

Recent studies have identified a specialized subset of CD31hiEMCNhi vascular endothelium that positively regulates bone formation. However, it remains unclear how CD31hiEMCNhi endothelium levels are coupled to anabolic bone formation. Mice with an osteoblast-specific deletion of Shn3, which have markedly elevated bone formation, demonstrated an increase in CD31hiEMCNhi endothelium. Transcriptomic analysis identified SLIT3 as an osteoblast-derived, SHN3-regulated proangiogenic factor. Genetic deletion of Slit3 reduced skeletal CD31hiEMCNhi endothelium, resulted in low bone mass due to impaired bone formation and partially reversed the high bone mass phenotype of Shn3−/− mice. This coupling between osteoblasts and CD31hiEMCNhi endothelium is essential for bone healing, as shown by defective fracture repair in SLIT3-mutant mice and enhanced fracture repair in SHN3-mutant mice. Finally, administration of recombinant SLIT3 both enhanced bone-fracture healing and counteracted bone loss in a mouse model of postmenopausal osteoporosis. Thus, drugs that target the SLIT3 pathway may represent a new approach for vascular-targeted osteoanabolic therapy to treat bone loss.

Growth curves were studied for the egg, larva, and pupa of Chrysomya rufifacies (Macquart) under mean cyclic temperatures of 15.6, 21.1, 26.7, and 35.0 degrees C and a constant temperature of 25.0 degrees C. Development from egg to adult under all regimes ranged from 190 to 598 h. A constant temperature of 25 degrees C produced a range of pupation times from 134 to 162 h, with adult emergence ranging from 237 to 289 h. The maximal preferential temperature of 35.1 degrees C was determined for maggots using a gradient system. Highly predictable developmental time, minimal larval length variation, and low cohort variation emphasize the utility of this species in entomological-based postmortem interval determinations. Therefore, C. rufifacies should be of primary forensic importance when recovered alone or in conjunction with other species of Calliphoridae.

Nitric oxide produced by inducible nitric oxide synthase (iNOS) contributes to ischemic brain injury, but the cell types expressing iNOS and mediating tissue damage have not been elucidated. To examine the relative contribution of iNOS in resident brain cells and peripheral leukocytes infiltrating the ischemic brain, we used bone marrow (BM) chimeric mice in which the middle cerebral artery was occluded and infarct volume was determined 3 days later. iNOS−/− mice engrafted with iNOS+/+ BM exhibited larger infarcts (44±2 mm3; n=13; Mean±SE) compared to autologous transplanted iNOS−/− mice (24±3 mm3; n=10; p<0.01), implicating blood-borne leukocytes in the damage. Furthermore, iNOS+/+ mice transplanted with iNOS−/− BM had large infarcts (39±6 mm3; n=13), similar to those of autologous transplanted iNOS+/+ mice (39±4 mm3; n=14), indicating the resident brain cells also play a role. Flow cytometry and cell sorting revealed that iNOS is highly expressed in neutrophils and endothelium, but not microglia. Surprisingly, post-ischemic iNOS expression was enhanced in the endothelium of iNOS+/+ mice transplanted with iNOS−/− BM, and in leukocytes of iNOS−/− mice with iNOS+/+ BM, suggesting that endothelial iNOS suppresses iNOS expression in leukocytes and vice-versa. To provide independent evidence that neutrophils mediate brain injury, neutrophils were isolated and transferred to mice 24 hours after stroke. Consistent with the result in chimeric mice, transfer of iNOS+/+, but not iNOS−/−, neutrophils into iNOS−/−mice increased infarct volume. The findings establish that iNOS both in neutrophils and endothelium mediates tissue damage and identify these cell types as putative therapeutic targets for stroke injury.

SummaryRecent evidence points to the embryonic emergence of some tissue-resident innate immune cells, such as B-1a lymphocytes, prior to and independently of hematopoietic stem cells (HSCs). However, whether the full hematopoietic repertoire of embryonic HSCs initially includes these unique lineages of innate immune cells has been difficult to assess due to lack of clonal assays that identify and assess HSC precursor (pre-HSC) potential. Here, by combining index sorting of single embryonic hemogenic precursors with in vitro HSC maturation and transplantation assays, we analyze emerging pre-HSCs at the single-cell level, revealing their unique stage-specific properties and clonal lineage potential. Remarkably, clonal pre-HSCs detected between E9.5 and E11.5 contribute to the complete B cell repertoire, including B-1a lymphocytes, revealing a previously unappreciated common precursor for all B cell lineages at the pre-HSC stage and a second embryonic origin for B-1a lymphocytes.

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Four extant subspecies of Terrapene carolina in eastern North America, Terrapene carolina bauri, Terrapene carolina carolina, Terrapene carolina triunguis, and Terrapene carolina major, are recognized based on morphological studies. A fifth subspecies, Terrapene carolina putnami, has been described from Pleistocene deposits but is very similar morphologically to T. c. major. Questions concerning the relationship of the Gulf Coast box turtle (T. c. major) to other box turtles have been pervasive ever since it was described. We used a combined morphological and genetic analysis to address the status of T. c. major and other T. carolina lineages. Terrapene c. bauri, T. c. carolina, and T. c. triunguis are distinct based on a discriminate function analysis of 25 morphological characters, including characters traditionally used to assign subspecies. The results of the present study confirm that box turtles phenotypically diagnosed as T. c. bauri, T. c. carolina, and T. c. triunguis all occur within the hypothesized range of T. c. major, and that the latter does not possess a diagnosable morphology. The three morphological lineages also possess divergent mitochondrial haplotypes that are present within the hypothesized range of T. c. major. In addition, a fourth distinct mtDNA lineage co-occurs within the putative range of T. c. major. This unique lineage may include mitochondrial DNA variation from the Pleistocene T. c. putnami. Analysis of nine nuclear DNA microsatellites revealed no population structure in box turtles currently assigned to T. c. major from the Florida Panhandle, suggesting a complete admixture of lineages in this region. The results of the present study indicate that box turtles traditionally assigned to T. c. major based on phenotype are the result of introgression between eastern extant (predominantly T. c. carolina) and an extinct subspecies, T.

Growth curves were studied for the egg, larva, and pupa of the secondary screwworm, Cochliomyia macellaria (F), under mean cyclic temperatures of 15.6, 21.1, 26.7, 32.2, and a constant 25.0 degrees C. Development from egg to adult under all regimes ranged from 170 to 588 h. A constant temperature regime of 25 degrees C produced a range of pupation times from 92 to 142 h, with adult emergence ranging from 196 to 261 h. The maximal preferential temperature of 39 degrees C was determined for maggots by using a gradient system. The minimal variation in the range of larval lengths and development time makes this species suitable for establishing relatively narrow confidence intervals when constructing a postmortem interval estimate for use in legal investigations.