Applying a targeted label-free approach using LC-MS AMT tags to evaluate changes in protein phosphorylation following phosphatase inhibition

To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation site and protein. These include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.     

Soil hydrological properties as influenced by long‐term nitrogen application and landscape positions under switchgrass seeded to a marginal cropland

Switchgrass (Panicum virgatum L.) has the potential to recover the soil hydrological properties of marginal lands. Nitrogen (N) and landscape position are the key factors in impacting these soil properties under switchgrass. The specific objective of this study was to investigate the responses of N rate (low, 0 kg N/ha and high, 112 kg N/ha) and landscape positions (shoulder and footslope) on near‐surface soil hydrological properties that included: infiltration rate (q_s), saturated hydraulic conductivity (K_sat), bulk density (ρ_b), penetration resistance (SPR), water retention (SWR), pore‐size distribution (PSD), and carbon (C) and nitrogen (N) fractions under switchgrass production. Data showed that, in general, the N and landscape position significantly influenced soil hydrological properties. Higher N rate decreased ρ_b (1.23 and 1.36 g/cm³ at 0–5 and 5–15 cm, respectively) and SPR (1.06 and 1.53 MPa at 0–5 and 5–15 cm, respectively) at both depths and increased the q_s, K_sat and Green–Ampt estimated sorptivity (S) and hydraulic conductivity (K_s) parameters, and SWR (0–5 cm depth) at 0 and ?0.4 kPa matric potentials (ψ_m). Furthermore, footslope position significantly decreased ρ_b, SPR at 0–5 and 5–15 cm depths, and increased the q_s, K_sat, S, K_s, and SWR (0–5 cm depth) at every ψ_m ranged from 0 to ?30.0 kPa. The higher N rate increased the coarse mesopores (60–1,000 μm) and total pores, whereas, footslope position increased the coarse mesopores, micropores (<60 μm), and total pores. Data from this study showed that planting switchgrass with 112 kg N/ha under footslope position helped in improving the soil hydrological properties, those can be beneficial in enhancing the biomass yield under marginal lands.     

Serine Metabolism Supports Macrophage IL-1β Production

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An Economic Assessment Model of Rural and Remote Satellite Hemodialysis Units

Background

Kidney Failure is epidemic in many remote communities in Canada. In-centre hemodialysis is provided within these settings in satellite hemodialysis units. The key cost drivers of this program have not been fully described. Such information is important in informing the design of programs aimed at optimizing efficiency in providing dialysis and preventative chronic kidney disease care in remote communities.

Design, Setting, Participants, and Measurements

We constructed a cost model based on data derived from 16 of Manitoba, Canada’s remote satellite units. We included all costs for operation of the unit, transportation, treatment, and capital costs. All costs were presented in 2013 Canadian dollars.

Results

The annual per-patient cost of providing hemodialysis in the satellite units ranged from $80,372 to $215,918 per patient, per year. The median per patient, per year cost was $99,888 (IQR $89,057—$122,640). Primary cost drivers were capital costs related to construction, human resource expenses, and expenses for return to tertiary care centres for health care. Costs related to transport considerably increased estimates in units that required plane or helicopter transfers.

Conclusions

Satellite hemodialysis units in remote areas are more expensive on a per-patient basis than hospital hemodialysis and satellite hemodialysis available in urban areas. In some rural, remote locations, better value for money may reside in local surveillance and prevention programs in addition support for home dialysis therapies over construction of new satellite hemodialysis units.     

Altered Osteocyte-Specific Protein Expression in Bone after Childhood Solid Organ Transplantation

Background

Bone fragility is common post solid organ transplantation but little is known about bone pathology on a tissue level. Abnormal osteocytic protein expression has been linked to compromised bone health in chronic kidney disease (CKD) and immunosuppressant medications may impact osteocyte function.

Methods

Transiliac bone biopsies were obtained from 22 pediatric solid organ allograft recipients (average age 15.6 years) an average of 6.3 ± 1.2 years after transplantation and from 12 pediatric pre-dialysis CKD patients (average age 13.2 years). Histomorphometry and immunohistochemistry for FGF23, DMP1, sclerostin, and osteopontin were performed on all biopsies.

Results

FGF23 and sclerostin were increased in transplant recipients relative to non-transplant CKD, regardless of the type of allograft received and despite, in the case of liver and heart recipients, a higher GFR. Bone DMP1 expression was higher in liver or heart than in kidney recipients, concomitant with higher serum phosphate values. Osteopontin expression was higher in CKD than in transplant recipients (p<0.01). Bone FGF23 and sclerostin correlated directly (r = 0.38, p<0.05); bone FGF23 expression and osteoid thickness correlated inversely (r = - 0.46, p<0.01).

Conclusions

Solid-organ transplantation is associated with increased FGF23 and sclerostin expression. The contribution of these findings to compromised bone health post transplantation warrants further evaluation.     

Nek5 promotes centrosome integrity in interphase and loss of centrosome cohesion in mitosis

Nek5 is a poorly characterized member of the NIMA-related kinase family, other members of which play roles in cell cycle progression and primary cilia function. Here, we show that Nek5, similar to Nek2, localizes to the proximal ends of centrioles. Depletion of Nek5 or overexpression of kinase-inactive Nek5 caused unscheduled separation of centrosomes in interphase, a phenotype also observed upon overexpression of active Nek2. However, separated centrosomes that resulted from Nek5 depletion remained relatively close together, exhibited excess recruitment of the centrosome linker protein rootletin, and had reduced levels of Nek2. In addition, Nek5 depletion led to loss of PCM components, including γ-tubulin, pericentrin, and Cdk5Rap2, with centrosomes exhibiting reduced microtubule nucleation. Upon mitotic entry, Nek5-depleted cells inappropriately retained centrosome linker components and exhibited delayed centrosome separation and defective chromosome segregation. Hence, Nek5 is required for the loss of centrosome linker proteins and enhanced microtubule nucleation that lead to timely centrosome separation and bipolar spindle formation in mitosis.     

Seeing the light: probing ROS in vivo using redox GFP

Reactive oxygen species (ROS) dictate biological outcomes and are linked with myriad pathologies. However, measuring ROS in vivo remains a major obstacle in the field. Here, Albrecht et al. (2011) demonstrate the efficacy of redox-sensitive GFP in measuring glutathione redox state and H(2)O(2) levels of tissues in Drosophila.