Cauli: A Mouse Strain with an Ift140 Mutation That Results in a Skeletal Ciliopathy Modelling Jeune Syndrome

Cilia are architecturally complex organelles that protrude from the cell membrane and have signalling, sensory and motility functions that are central to normal tissue development and homeostasis. There are two broad categories of cilia; motile and non-motile, or primary, cilia. The central role of primary cilia in health and disease has become prominent in the past decade with the recognition of a number of human syndromes that result from defects in the formation or function of primary cilia. This rapidly growing class of conditions, now known as ciliopathies, impact the development of a diverse range of tissues including the neural axis, craniofacial structures, skeleton, kidneys, eyes and lungs. The broad impact of cilia dysfunction on development reflects the pivotal position of the primary cilia within a signalling nexus involving a growing number of growth factor systems including Hedgehog, Pdgf, Fgf, Hippo, Notch and both canonical Wnt and planar cell polarity. We have identified a novel ENU mutant allele of Ift140, which causes a mid-gestation embryonic lethal phenotype in homozygous mutant mice. Mutant embryos exhibit a range of phenotypes including exencephaly and spina bifida, craniofacial dysmorphism, digit anomalies, cardiac anomalies and somite patterning defects. A number of these phenotypes can be attributed to alterations in Hedgehog signalling, although additional signalling systems are also likely to be involved. We also report the identification of a homozygous recessive mutation in IFT140 in a Jeune syndrome patient. This ENU-induced Jeune syndrome model will be useful in delineating the origins of dysmorphology in human ciliopathies.     

Endothelin Receptor A Blockade Is an Ineffective Treatment for Adriamycin Nephropathy

Endothelin is a vasoconstricting peptide that plays a key role in vascular homeostasis, exerting its biologic effects via two receptors, the endothelin receptor A (ETA) and endothelin receptor B (ETB). Activation of ETA and ETB has opposing actions, in which hyperactive ETA is generally vasoconstrictive and pathologic. Selective ETA blockade has been shown to be beneficial in renal injuries such as diabetic nephropathy and can improve proteinuria. Atrasentan is a selective pharmacologic ETA blocker that preferentially inhibits ETA activation. In this study, we evaluated the efficacy of ETA blockade by atrasentan in ameliorating proteinuria and kidney injury in murine adriamycin nephropathy, a model of human focal segmental glomerulosclerosis. We found that ETA expression was unaltered during the course of adriamycin nephropathy. Whether initiated prior to injury in a prevention protocol (5 mg/kg/day, i.p.) or after injury onset in a therapeutic protocol (7 mg/kg or 20 mg/kg three times a week, i.p.), atrasentan did not significantly affect the initiation and progression of adriamycin-induced albuminuria (as measured by urinary albumin-to-creatinine ratios). Indices of glomerular damage were also not improved in atrasentan-treated groups, in either the prevention or therapeutic protocols. Atrasentan also failed to improve kidney function as determined by serum creatinine, histologic damage, and mRNA expression of numerous fibrosis-related genes such as collagen-I and TGF-β1. Therefore, we conclude that selective blockade of ETA by atrasentan has no effect on preventing or ameliorating proteinuria and kidney injury in adriamycin nephropathy.     

Distinguishing the effects of vegetation restoration on runoff and sediment generation on simulated rainfall on the hillslopes of the loess plateau of China

Aims

Since the 1970s, extensive croplands were converted to forest and pasture lands to control severe soil erosion on the Loess Plateau of China. We quantify the direct and indirect effects of vegetation restoration on runoff and sediment yield on hillslopes in the field to improve environmental governance.

Methods

An artificial rainfall experiment at a rainfall intensity of 120 mm h⁻¹ and a slope gradient of 22° were used to distinguish the effects of vegetation restoration on runoff and sediment yield.

Results

Compared to the farmland slopes, vegetation restoration directly prolonged the time-to-runoff by 140%, reduced the runoff rate by 20%, and increased the soil infiltration capacity by 15%. Vegetation restoration indirectly delayed the time-to-runoff by 120%, reduced the runoff rate and sediment yield rate by 50% and 94%, respectively, and increased the soil infiltration capacity by 58% on the hillslopes with vegetation restoration.

Conclusions

The direct effects of vegetation restoration on runoff and sediment yield were lower than its indirect impacts. Vegetation cover, decreases in soil bulk density, and increases in belowground root biomasses and > 0.25 mm aggregate stability were the primary causes of runoff and sediment yield reduction on the slopes with vegetation restoration.

     

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20–25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.     

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.