Endothelial Progenitors Exist within the Kidney and Lung Mesenchyme

The renal endothelium has been debated as arising from resident hemangioblast precursors that transdifferentiate from the nephrogenic mesenchyme (vasculogenesis) and/or from invading vessels (angiogenesis). While the Foxd1-positive renal cortical stroma has been shown to differentiate into cells that support the vasculature in the kidney (including vascular smooth muscle and pericytes) it has not been considered as a source of endothelial cell progenitors. In addition, it is unclear if Foxd1-positive mesenchymal cells in other organs such as the lung have the potential to form endothelium. This study examines the potential for Foxd1-positive cells of the kidney and lung to give rise to endothelial progenitors. We utilized immunofluorescence (IF) and fluorescence-activated cell sorting (FACS) to co-label Foxd1-expressing cells (including permanently lineage-tagged cells) with endothelial markers in embryonic and postnatal mice. We also cultured FACsorted Foxd1-positive cells, performed in vitro endothelial cell tubulogenesis assays and examined for endocytosis of acetylated low-density lipoprotein (Ac-LDL), a functional assay for endothelial cells. Immunofluorescence and FACS revealed that a subset of Foxd1-positive cells from kidney and lung co-expressed endothelial cell markers throughout embryogenesis. In vitro, cultured embryonic Foxd1-positive cells were able to differentiate into tubular networks that expressed endothelial cell markers and were able to endocytose Ac-LDL. IF and FACS in both the kidney and lung revealed that lineage-tagged Foxd1-positive cells gave rise to a significant portion of the endothelium in postnatal mice. In the kidney, the stromal-derived cells gave rise to a portion of the peritubular capillary endothelium, but not of the glomerular or large vessel endothelium. These findings reveal the heterogeneity of endothelial cell lineages; moreover, Foxd1-positive mesenchymal cells of the developing kidney and lung are a source of endothelial progenitors that are likely critical to patterning the vasculature.     

Persistent anthropogenic legacies structure depth dependence of regenerating rooting systems and their functions

Biotically-mediated weathering helps to shape Earth’s surface. For example, plants expend carbon (C) to mobilize nutrients in forms whose relative abundances vary with depth. It thus is likely that trees’ nutrient acquisition strategies—their investment in rooting systems and exudates—may function differently following disturbance-induced changes in depth of rooting zones and soil nutrient stocks. These changes may persist across centuries. We test the hypothesis that plant C allocation for nutrient acquisition is depth dependent as a function of rooting system development and relative abundances of organic vs. mineral nutrient stocks. We further posit that patterns of belowground C allocation to nutrient acquisition reveal anthropogenic signatures through many decades of forest regeneration. To test this idea, we examined fine root abundances and rooting system C in organic acid exudates and exo-enzymes in tandem with depth distributions of organically- and mineral-bound P stocks. Our design permitted us to estimate C tradeoffs between organic vs. mineral nutrient benefits in paired forests with many similar aboveground traits but different ages: post-agricultural mixed-pine forests and older reference hardwoods. Fine roots were more abundant throughout the upper 2 m in reference forest soils than in regenerating stands. Rooting systems in all forests exhibited depth-dependent C allocations to nutrient acquisition reflecting relative abundances of organic vs. mineral bound P stocks. Further, organic vs. mineral stocks underwent redistribution with historic land use, producing distinct ecosystem nutritional economies. In reference forests, rooting systems are allocating C to relatively deep fine roots and low-C exudation strategies that can increase mobility of mineral-bound P stocks. Regenerating forests exhibit relatively shallower fine root distributions and more diverse exudation strategies reflecting more variable nutrient stocks. We observed these disparities in rooting systems’ depth and nutritional mechanisms even though the regenerating forests have attained aboveground biomass stocks similar to those in reference hardwood forests. These distinctions offer plausible belowground mechanisms for observations of continued C sink strength in relatively old forests, and have implications for soil C fates and soil development on timescales relevant to human lifetimes. As such, depth-dependent nutrient returns on plant C investments represent a subtle but consequential signal of the Anthropocene.

     

Palmitate-derivatized human IL-2: a potential anticancer immunotherapeutic of low systemic toxicity

Purpose and experimental design

Recombinant human IL-2 (rhIL-2) is a potent cytokine and FDA-approved anticancer drug. However, its clinical use has been limited by severe toxicity, associated primarily with systemic administration with excess protein distributing freely throughout the body. We hypothesized that rhIL-2 in alternate forms permitting more restricted localization may exert stronger antitumor efficacy and less toxicity. Here, we have tested the utility of palmitate-derivatized rhIL-2. rhIL-2 was reacted with N-hydroxysuccinimide palmitate ester. The resultant lipidated rhIL-2 (pIL-2), when mixed with cells, could spontaneously transfer from solution to cell surfaces. Next, anticancer efficacy of pIL-2 was assessed in two modalities. For adoptive T cell therapy, antitumor cytotoxic T cells (CTLs) were protein transferred (“painted”) with pIL-2 and injected into mice bearing lymphoma. For in situ therapy, pIL-2 was injected intratumorally into mice bearing melanoma. Tumor growth and IL-2-associated toxicity were determined.

Results

In the lymphoma model, painting of the antitumor CTLs with pIL-2 markedly increased their viability and titer. In the melanoma model, intratumoral injection of pIL-2, but not rhIL-2, increased the number of activated CD8⁺ T cells (IFN-γ⁺) in the spleen, reduced lung metastasis and prolonged the survival of treated mice. Moreover, while repeated intratumoral injection of rhIL-2 at an excessively high dose (10 injections of 10,000 IU/mouse) caused marked vascular leakage syndrome, the same regimen using pIL-2 caused no detectable toxicity.

Conclusions

Transferring spontaneously from solution to cell surfaces, pIL-2 may bypass the current limitations of rhIL-2 and, thus, serve as a more effective and tolerable anticancer drug.     

Generalized Subcutaneous Phaeohyphomycosis Caused by Phialophora verrucosa: Report of a Case and Review of Literature

We report a case of subcutaneous phaeohyphomycosis due to Phialophora verrucosa in a 64-year-old Chinese farmer suffering from CD4+ lymphopenia. He presented with diffuse and infiltrated plaques involving the entire face including the eyes, neck, occiput, and extending to the dorsal regions of his torso. The patient is notable for the discrete multifocal nature of the illness in the absence of disseminated infection and rarity of P. verrucosa as a cause of subcutaneous phaeohyphomycosis.     

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.     

77Se Enrichment of Proteins Expands the Biological NMR Toolbox

Sulfur, a key contributor to biological reactivity, is not amendable to investigations by biological NMR spectroscopy. To utilize selenium as a surrogate, we have developed a generally applicable ⁷⁷Se isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate ⁷⁷Se NMR spectroscopy of multiple selenocysteine and selenomethionine residues in the sulfhydryl oxidase augmenter of liver regeneration (ALR). The resonances of the active-site residues were assigned by comparing the NMR spectra of ALR bound to oxidized and reduced flavin adenine dinucleotide. An additional resonance appears only in the presence of the reducing agent and disappears readily upon exposure to air and subsequent reoxidation of the flavin. Hence, ⁷⁷Se NMR spectroscopy can be used to report the local electronic environment of reactive and structural sulfur sites, as well as changes taking place in those locations during catalysis.     

Exome Sequencing Identifies Mutations in CCDC114 as a Cause of Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ∼60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.