Expanding the evo-devo toolkit: generation of 3D mammary tissue from diverse mammals.

The varying pathways of mammary gland development across species and evolutionary history are underexplored, largely due to a lack of model systems. Recent progress in organoid technology holds the promise of enabling in-depth studies of the developmental adaptations that have occurred throughout the evolution of different species, fostering beneficial phenotypes. The practical application of this technology for mammary glands has been mostly confined to rodents and humans. In the current study, we have successfully created next-generation 3D mammary gland organoids from eight eutherian mammals and the first branched organoid of a marsupial mammary gland. Using mammary organoids, we identified a role for ROCK protein in regulating branching morphogenesis, a role that manifests differently in organoids from different mammals. This finding demonstrates the utility of the 3D organoid model for understanding the evolution and adaptations of signaling pathways. These achievements highlight the potential for organoid models to expand our understanding of mammary gland biology and evolution, and their potential utility in studies of lactation or breast cancer.

Experimental Evolution of Campylobacter jejuni Leads to Loss of Motility, rpoN (σ54) Deletion and Genome Reduction.

Evolution experiments in the laboratory have focused heavily on model organisms, often to the exclusion of clinically relevant pathogens. The foodborne bacterial pathogen Campylobacter jejuni belongs to a genus whose genomes are small compared to those of its closest genomic relative, the free-living genus Sulfurospirillum, suggesting genome reduction during the course of evolution to host association. In an in vitro experiment, C. jejuni serially passaged in rich medium in the laboratory exhibited loss of flagellar motility-an essential function for host colonization. At early time points the motility defect was often reversible, but after 35 days of serial culture, motility was irreversibly lost in most cells in 5 independently evolved populations. Population re-sequencing revealed disruptive mutations to genes in the flagellar transcriptional cascade, rpoN (σ54)-therefore disrupting the expression of the genes σ54 regulates-coupled with deletion of rpoN in all evolved lines. Additional mutations were detected in virulence-related loci. In separate in vivo experiments, we demonstrate that a phase variable (reversible) motility mutant carrying an adenine deletion within a homopolymeric tract resulting in truncation of the flagellar biosynthesis gene fliR was deficient for colonization in a C57BL/6 IL-10-/- mouse disease model. Re-insertion of an adenine residue partially restored motility and ability to colonize mice. Thus, a pathogenic C. jejuni strain was rapidly attenuated by experimental laboratory evolution and demonstrated genomic instability during this evolutionary process. The changes observed suggest C. jejuni is able to evolve in a novel environment through genome reduction as well as transition, transversion, and slip-strand mutations.

Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis.

Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E(125) with D(125), or E(125) and C(127) with D(125) and F(127) within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.

Hemocyanin-derived phenoloxidase activity with broad temperature stability extending into the cold environment in hemocytes of the hair crab Erimacrus isenbeckii.

Phenoloxidase (PO) activity is a major component of the innate immune response in arthropods. In this study, we characterized PO activity from the hair crab Erimacrus isenbeckii, which inhabits very cold regions (2.4-3.4°C) of the Bering Sea. Hemocyte lysate supernatant (HLS) prepared from E. isenbeckii was inactive HLS until activated by nonspecific agents such as sodium dodecyl sulfate and trypsin, and elicitors such as lipopolysaccharide and lipoteichoic acid from the cell wall constituent of bacteria. The PO activity was maximal at 4°C, decreased slightly at temperatures up to 60°C, and fell rapidly at 80°C. Both L-DOPA and catechol were efficient substrates for the PO (EC 1.10.3.1), with K(m) values of 0.96 and 1.15mM, respectively, whereas tyrosine and hydroquinone were not. We isolated a protein fraction from HLS as a hexamer of 75kDa units with 216.7-fold higher PO activity than that of the HLS. The N-terminal amino acid analysis of an isolated protein revealed 80% sequence identity to hemocyanins from other crabs. These results suggest that cold-adapted hemocyanin-derived PO activity is important to the survival of these crabs. This is the first report of a crab PO activity with broad temperature stability extending into the cold environment.

A novel lectin isolated from the hemolymph of the marine hair crab Erimacrus isenbeckii.

A lectin that induces hemagglutination activity in mouse and rabbit erythrocytes has been purified from the hemolymph of the marine hair crab Erimacrus isenbeckii. The results of SDS-PAGE, gel-filtration, affinity and anion-exchange chromatography indicate that this lectin, designated EIL (E. isenbeckii lectin), was successfully purified as a single protein, and comprises a mixture of a major (90%) dimeric and a minor (10%) oligomeric protein with a molecular mass of 116 kDa, with covalent linking between two subunits of 62 and 54 kDa. The activity was maximal at pH 5.6-8.0 and at temperatures below 50 degrees C. The N-terminal amino acid sequences were determined, and these differed greatly from those of other reported lectins from invertebrates, vertebrates, or plants. EIL binds with high specificities to both the O-acetylsialic acid and mannose that are present in bacterial pathogens, which suggests that EIL can act as a defense protein against infection in this crab.

A sialic acid-specific lectin from the mushroom Paecilomyces Japonica that exhibits hemagglutination activity and cytotoxicity.

The mushroom Paecilomyces japonica, grown on the silkworm larvae, has been used in Asia as a nutraceutical, tea, and Chinese medicine. In the present study, a sialic acid-specific lectin has been purified from the mushroom P. japonica using affinity chromatography on a fetuin-agarose column. Electrophoretical analyses indicated that this lectin, designated P. japonica agglutinin (PJA), is an acidic protein with a molecular mass of 16 kDa, and has no intermolecular disulfide bonds. PJA induced hemagglutination activity in human ABO, mouse, rat, and rabbit erythrocytes. This activity was inhibited by sialic acid and sialoglycoproteins, but not by any other carbohydrates. PJA was stable at pH 4.0-8.0, and at temperatures below 55 degrees C. The activity of PJA was independent of EDTA and divalent cations. In addition, PJA exerts cytotoxic effects on the following cancer cell lines: human stomach cancer SNU-1, human pancreas cancer AsPc-1, and human breast cancer MDA-MB-231.