Senescence‐associated tissue microenvironment promotes colon cancer formation through the secretory factor GDF15

The risk of colorectal cancer (CRC) varies between people, and the cellular mechanisms mediating the differences in risk are largely unknown. Senescence has been implicated as a causative cellular mechanism for many diseases, including cancer, and may affect the risk for CRC. Senescent fibroblasts that accumulate in tissues secondary to aging and oxidative stress have been shown to promote cancer formation via a senescence‐associated secretory phenotype (SASP). In this study, we assessed the role of senescence and the SASP in CRC formation. Using primary human colon tissue, we found an accumulation of senescent fibroblasts in normal tissues from individuals with advanced adenomas or carcinomas in comparison with individuals with no polyps or CRC. In in vitro and ex vivo model systems, we induced senescence using oxidative stress in colon fibroblasts and demonstrated that the senescent fibroblasts secrete GDF15 as an essential SASP factor that promotes cell proliferation, migration, and invasion in colon adenoma and CRC cell lines as well as primary colon organoids via the MAPK and PI3K signaling pathways. In addition, we observed increased mRNA expression of GDF15 in primary normal colon tissue from people at increased risk for CRC in comparison with average risk individuals. These findings implicate the importance of a senescence‐associated tissue microenvironment and the secretory factor GDF15 in promoting CRC formation.     

Adaptive management assists reintroduction as higher tides threaten an endangered salt marsh plant

In theory, extirpated plant species can be reintroduced and managed to restore sustainable populations. However, few reintroduced plants are known to persist for more than a few years. Our adaptive‐management case study illustrates how we restored the endangered hemiparasitic annual plant, Chloropyron maritimum subsp. maritimum (salt marsh bird's beak), to Sweetwater Marsh, San Diego Bay National Wildlife Refuge, California, United States, and used monitoring and experimentation to identify the factors limiting the reintroduced population. After extirpation in 1988, reintroduction starting that year led to a resilient, genetically diverse population in 2016 (a “boom” of approximately 14,000) that rebounded from a “bust” (62 in 2014). Multiple regressions attributed 82% of the variation in population counts to tidal amplitude, rainfall, and temperature. Populations of salt marsh bird's beak crashed when the diurnal tide range peaked during the 18.6‐year lunar nodal cycle (a rarely considered factor that periodically added approximately 12 cm to tidal ranges). We explain booms as follows: During smaller tidal amplitudes, above‐average rainfall could desalinize upper intertidal soils and stimulate salt marsh bird's beak germination. Then, moderate temperature in May favors growth to reproduction in June. In addition, salt marsh bird's beak needs a short and open canopy of native perennial plants, with roots to parasitize (not non‐native annual grass pseudohosts) and nearby upland soil for a preferred pollinator, ground‐burrowing bees. Although our reintroduced salt marsh bird's beak population is an exceptional case of persistence, this rare species‐specific environmental and biological requirement makes it vulnerable to rising sea levels and global warming.     

Vocal divergence is concordant with genomic evidence for strong reproductive isolation in grasshopper mice (Onychomys)

Behavioral barriers to gene flow often evolve faster than intrinsic incompatibilities and can eliminate the opportunity for hybridization between interfertile species. While acoustic signal divergence is a common driver of premating isolation in birds and insects, its contribution to speciation in mammals is less studied. Here we characterize the incidence of, and potential barriers to, hybridization among three closely related species of grasshopper mice (genus Onychomys). All three species use long‐distance acoustic signals to attract and localize mates; Onychomys arenicola and Onychomys torridus are acoustically similar and morphologically cryptic whereas Onychomys leucogaster is larger and acoustically distinct. We used genotyping‐by‐sequencing (GBS) to test for evidence of introgression in 227 mice from allopatric and sympatric localities in the western United States and northern Mexico. We conducted laboratory mating trials for all species pairs to assess reproductive compatibility, and recorded vocalizations from O. arenicola and O. torridus in sympatry and allopatry to test for evidence of acoustic character displacement. Hybridization was rare in nature and, contrary to prior evidence for O. torridus/O. arenicola hybrids, only involved O. leucogaster and O. arenicola. In contrast, laboratory crosses between O. torridus and O. arenicola produced litters whereas O. leucogaster and O. arenicola crosses did not. Call fundamental frequency in O. torridus and O. arenicola was indistinguishable in allopatry but significantly differentiated in sympatry, a pattern consistent with reproductive character displacement. These results suggest that assortative mating based on a long‐distance signal is an important isolating mechanism between O. torridus and O. arenicola and highlight the importance of behavioral barriers in determining the permeability of species boundaries.     

Characterization of glycosomal RING finger proteins of trypanosomatids

The glycosomes of trypanosomatids are essential organelles that are evolutionarily related to peroxisomes of other eukaryotes. The peroxisomal RING proteins-PEX2, PEX10 and PEX12-comprise a network of integral membrane proteins that function in the matrix protein import cycle. Here, we describe PEX10 and PEX12 in Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi. We expressed GFP fusions of each T. brucei coding region in procyclic form T. brucei, where they localized to glycosomes and behaved as integral membrane proteins. Despite the weak transmembrane predictions for TbPEX12, protease protection assays demonstrated that both the N and C termini are cytosolic, similar to mammalian PEX12. GFP fusions of T. cruzi PEX10 and L. major PEX12 also localized to glycosomes in T. brucei indicating that glycosomal membrane protein targeting is conserved across trypanosomatids.     

A phase 1/2 clinical trial of enzyme replacement in fabry disease: pharmacokinetic, substrate clearance, and safety studies

Fabry disease results from deficient alpha-galactosidase A (alpha-Gal A) activity and the pathologic accumulation of the globotriaosylceramide (GL-3) and related glycosphingolipids, primarily in vascular endothelial lysosomes. Treatment is currently palliative, and affected patients generally die in their 40s or 50s. Preclinical studies of recombinant human alpha-Gal A (r-halphaGalA) infusions in knockout mice demonstrated reduction of GL-3 in tissues and plasma, providing rationale for a phase 1/2 clinical trial. Here, we report a single-center, open-label, dose-ranging study of r-halphaGalA treatment in 15 patients, each of whom received five infusions at one of five dose regimens. Intravenously administered r-halphaGalA was cleared from the circulation in a dose-dependent manner, via both saturable and non-saturable pathways. Rapid and marked reductions in plasma and tissue GL-3 were observed biochemically, histologically, and/or ultrastructurally. Clearance of plasma GL-3 was dose-dependent. In patients with pre- and posttreatment biopsies, mean GL-3 content decreased 84% in liver (n=13), was markedly reduced in kidney in four of five patients, and after five doses was modestly lowered in the endomyocardium of four of seven patients. GL-3 deposits were cleared to near normal or were markedly reduced in the vascular endothelium of liver, skin, heart, and kidney, on the basis of light- and electron-microscopic evaluation. In addition, patients reported less pain, increased ability to sweat, and improved quality-of-life measures. Infusions were well tolerated; four patients experienced mild-to-moderate reactions, suggestive of hypersensitivity, that were managed conservatively. Of 15 patients, 8 (53%) developed IgG antibodies to r-halphaGalA; however, the antibodies were not neutralizing, as indicated by unchanged pharmacokinetic values for infusions 1 and 5. This study provides the basis for a phase 3 trial of enzyme-replacement therapy for Fabry disease.     

In Organello and in Vivo Evidence of the Importance of the Regulatory Sulfhydryl/Disulfide System and Pyruvate for Alternative Oxidase Activity in Tobacco

After isolation of tobacco (Nicotiana tabacum) leaf mitochondria, alternative oxidase (AOX) is predominantly present as the disulfide-linked, less-active “oxidized” form. In an in organello assay, significant AOX activity was dependent upon both the reduction of the regulatory disulfide bond (such as occurs by dithiothreitol) and upon the presence of the activator pyruvate. However, AOX activity in these assays was substantially affected when mitochondria were isolated in the presence of pyruvate. First, pyruvate protects against the oxidation of the regulatory sulfhydryl during isolation, such that subsequent in organello AOX activity is not dependent upon dithiothreitol. Second, pyruvate stabilizes AOX activity, such that mitochondria kept in the presence of pyruvate have higher maximum rates of AOX activity than mitochondria kept for some time in the absence of pyruvate. The ability of pyruvate to protect against AOX oxidation was exploited to assess the in vivo status of the regulatory sulfhydryl/disulfide system. In both tobacco suspension cells and tobacco leaves with high levels of AOX protein, the protein is predominantly present as the “reduced” active form in vivo under a range of respiratory conditions. Experiments also indicate that, while the presence of reduced protein may be a necessary prerequisite for significant AOX activity, it is not sufficient for activity and other factors must also be critical.     

Identification and biochemical characterization of a novel nortriterpene inhibitor of the human lymphocyte voltage-gated potassium channel, Kv1.3

A novel nortriterpene, termed correolide, purified from the tree Spachea correae, inhibits Kv1.3, a Shaker-type delayed rectifier potassium channel present in human T lymphocytes. Correolide inhibits 86Rb+ efflux through Kv1.3 channels expressed in CHO cells (IC50 86 nM; Hill coefficient 1) and displays a defined structure-activity relationship. Potency in this assay increases with preincubation time and with time after channel opening. Correolide displays marked selectivity against numerous receptors and voltage- and ligand-gated ion channels. Although correolide is most potent as a Kv1.3 inhibitor, it blocks all other members of the Kv1 family with 4-14-fold lower potency. C20-29-[3H]dihydrocorreolide (diTC) was prepared and shown to bind in a specific, saturable, and reversible fashion (Kd = 11 nM) to a single class of sites in membranes prepared from CHO/Kv1.3 cells. The molecular pharmacology and stoichiometry of this binding reaction suggest that one diTC site is present per Kv1.3 channel tetramer. This site is allosterically coupled to peptide and potassium binding sites in the pore of the channel. DiTC binding to human brain synaptic membranes identifies channels composed of other Kv1 family members. Correolide depolarizes human T cells to the same extent as peptidyl inhibitors of Kv1.3, suggesting that it is a candidate for development as an immunosuppressant. Correolide is the first potent, small molecule inhibitor of Kv1 series channels to be identified from a natural product source and will be useful as a probe for studying potassium channel structure and the physiological role of such channels in target tissues of interest.