Cytological and Morphological Analyses Reveal Distinct Features of Intestinal Development during Xenopus tropicalis Metamorphosis

Background

The formation and/or maturation of adult organs in vertebrates often takes place during postembryonic development, a period around birth in mammals when thyroid hormone (T3) levels are high. The T3-dependent anuran metamorphosis serves as a model to study postembryonic development. Studies on the remodeling of the intestine during Xenopus (X.) laevis metamorphosis have shown that the development of the adult intestine involves de novo formation of adult stem cells in a process controlled by T3. On the other hand, X. tropicalis, highly related to X. laevis, offers a number of advantages for studying developmental mechanisms, especially at genome-wide level, over X. laevis, largely due to its shorter life cycle and sequenced genome. To establish X. tropicalis intestinal metamorphosis as a model for adult organogenesis, we analyzed the morphological and cytological changes in X. tropicalis intestine during metamorphosis.

Methodology/Principal Findings

We observed that in X. tropicalis, the premetamorphic intestine was made of mainly a monolayer of larval epithelial cells surrounded by little connective tissue except in the single epithelial fold, the typhlosole. During metamorphosis, the larval epithelium degenerates and adult epithelium develops to form a multi-folded structure with elaborate connective tissue and muscles. Interestingly, typhlosole, which is likely critical for adult epithelial development, is present along the entire length of the small intestine in premetamorphic tadpoles, in contrast to X. laevis, where it is present only in the anterior 1/3. T3-treatment induces intestinal remodeling, including the shortening of the intestine and the typhlosole, just like in X. laevis.

Conclusions/Significance

Our observations indicate that the intestine undergoes similar metamorphic changes in X. laevis and X. tropicalis, making it possible to use the large amount of information available on X. laevis intestinal metamorphosis and the genome sequence information and genetic advantages of X. tropicalis to dissect the pathways governing adult intestinal development.     

Field inversion gel electrophoretic separation of Cryptosporidium spp. chromosome-sized DNA

Chromosomal DNA from 5 isolates of Cryptosporidium parvum and 1 of C. baileyi were compared by field-inversion gel electrophoresis (FIGE). FIGE analyses of parasite DNA prepared from purified sporozoites versus intact oocysts showed no observable differences. Chromosomal DNA migration patterns of the 5 C. parvum isolates were indistinguishable, whereas similar but distinct differences were evident between C. baileyi and the isolates of C. parvum. Oocyst-reactive monoclonal antibodies differentiated oocysts of C. parvum from those of C. baileyi but were unable to distinguish oocysts of 1 isolate of C. parvum from another.     

Protein kinase C (PKC)-induced phosphorylation of ROMK1 is essential for the surface expression of ROMK1 channels

We carried out in vitro phosphorylation assays to determine whether ROMK1 is a substrate of protein kinase C (PKC) and used the two-electrode voltage clamp method to investigate the role of serine residues 4, 183, and 201, the three putative PKC phosphorylation sites, in the regulation of ROMK1 channel activity. Incubation of the purified His-tagged ROMK1 protein with PKC and radiolabeled ATP resulted in (32)P incorporation into ROMK1 detected by autoradiography. Moreover, the in vitro phosphorylation study of three synthesized peptides corresponding to amino acids 1-16, 174-189, and 196-211 of ROMK1 revealed that serine residues 4 and 201 of ROMK1 were the two main PKC phosphorylation sites. In contrast, (32)P incorporation of peptide 174-189 was absent. In vitro phosphorylation studies with ROMK1 mutants, R1S4/201A, R1S4/183A, and R1S183/201A, demonstrated that the phosphorylation levels of R1S4/201A were significantly lower than those of the other two mutants. Also, the Ba(2+)-sensitive K(+) current in oocytes injected with green fluorescent protein (GFP)-R1S4/201A was only 5% of that in oocytes injected with wild type GFP-ROMK1. In contrast, the K(+) current in oocytes injected with GFP-ROMK1 mutants containing either serine residue 4 or 201 was similar to those injected with wild type ROMK1. Confocal microscope imaging shows that the surface expression of the K(+) channels was significantly diminished in oocytes injected with R1S4/201A and completely absent in oocytes injected with R1S4/183/201A. Furthermore, the biotin labeling technique confirmed that the membrane fraction of ROMK channels was almost absent in HEK293 cells transfected with either R1S4/201A or R1S4/183/201A. However, when serine residues 4 and 201 were mutated to aspartate, the K(+) currents and the surface expression were completely restored. Finally, addition of calphostin C in the incubation medium significantly decreased the K(+) current in comparison with that under control conditions. Biotin labeling technique further indicated that inhibition of PKC decreases the surface ROMK1 expression in human embryonic kidney (HEK) cells transfected with ROMK1. We conclude that ROMK1 is a substrate of PKC and that serine residues 4 and 201 are the two main PKC phosphorylation sites that are essential for the expression of ROMK1 in the cell surface.     

Acaricidal activity of Metarhizium anisopliae isolated from paddocks in the Mexican tropics against two populations of the cattle tick Rhipicephalus microplus

The acaricidal effects of 55 strains of Metarhizium anisopliae (Metschnikoff) Sorokin, 1883 (Hypocreales: Clavicipitaceae) isolated from paddocks of cattle farms were evaluated in two Rhipicephalus microplus (Canestrini 1887) (Ixodida: Ixodidae) populations, of which one was multi‐resistant and one was susceptible to chemical acaricides. Percentage mortality and reproductive efficiency indices in R. microplus were evaluated by adult immersion tests at a dose of 1 × 10⁸ conidia/mL for each fungal strain. Some strains were selected to calculate lethal concentrations to kill 50% (LC₅₀) and 99% (LC₉₉) of engorged ticks. Strains MaV22, MaV26 and MaV55 induced 100% mortality in R. microplus on day 14. Strains MaV05, MaV09 and MaV22 caused mortality of >90% from day 12 onward in both tick populations. The most effective acaricidal fungal strain, MaV55, inhibited egg laying by 54.86 and 55.86% in acaricide‐resistant and ‐susceptible R. microplus populations, respectively. None of the fungal strains had statistically significant effects on larval hatching. In conclusion, nine strains of M. anisopliae demonstrated high acaricidal effects against R. microplus and reduced its egg laying. No differences in acaricidal effects were observed between the two populations of ticks tested.     

Calcium uptake and membrane trafficking in response to PTH or 25(OH)D3 in polarized intestinal epithelial cells

Cell culture techniques providing retention of the polarized enterocyte morphology has allowed, for the first time, comparison of parathyroid hormone (PTH)- and 25-hydroxyvitamin D(3) [25(OH)D(3)]-induced (45)Ca uptake with membrane trafficking events discerned using confocal microscopy. Treatment of cells with 65 pM bPTH(1-34) promoted enhanced (45)Ca uptake between 1 and 10 min after peptide. The protein kinase A (PKA) antagonist, RpcAMP inhibited hormone-mediated uptake. At the microscopic level, cells labeled with the endocytic tracking dye FM1-43 revealed increased punctate staining 50-550s after hormone. Pretreatment of cells with RpcAMP abolished this pattern of staining. The calcium indicator dye fluo-3 AM revealed faint punctate labeling in controls, with increased bands of punctate labeling in the apical region of the cells after peptide hormone, and ultimately the basal region. Parallel studies conducted with the metabolite 25(OH)D(3) resulted in a slower stimulation of (45)Ca uptake 5-10 min after steroid, which was also inhibited by preincubation with RpcAMP. Cells labeled with FM1-43 and then treated with steroid showed no change in distribution of fluorescence during the 10 min incubation period. Confocal microscopy with fluo-3 revealed intense apical fluorescence--that after steroid --streamed to a perinuclear position, and ultimately the basal area. Uniformly diffuse staining, which would indicate cytoplasmic calcium transport, was observed only in controls. Membrane trafficking and compartmentalized calcium appear to be integral to agonist mediated cation transport.     

High herbivory despite high sediment loads on a fringing coral reef

Coral Reefs - Algal turfs are expected to increasingly dominate the benthos of coral reefs in the Anthropocene, becoming important sources of reef productivity. The sediments trapped within algal...     

Anti-transforming growth factor ß antibody treatment rescues bone loss and prevents breast cancer metastasis to bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p?=?0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p?=?0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.     

Anti-Transforming Growth Factor ? Antibody Treatment Rescues Bone Loss and Prevents Breast Cancer Metastasis to Bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p = 0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p = 0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.