A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B

Upon induction of autophagy, the ubiquitin-like protein LC3 is conjugated to phosphatidylethanolamine (PE) on the inner and outer membrane of autophagosomes to allow cargo selection and autophagosome formation. LC3 undergoes two processing steps, the proteolytic cleavage of pro-LC3 and the de-lipidation of LC3-PE from autophagosomes, both executed by the same cysteine protease ATG4. How ATG4 activity is regulated to co-ordinate these events is currently unknown. Here we find that ULK1, a protein kinase activated at the autophagosome formation site, phosphorylates human ATG4B on serine 316. Phosphorylation at this residue results in inhibition of its catalytic activity in vitro and in vivo. On the other hand, phosphatase PP2A-PP2R3B can remove this inhibitory phosphorylation. We propose that the opposing activities of ULK1-mediated phosphorylation and PP2A-mediated dephosphorylation provide a phospho-switch that regulates the cellular activity of ATG4B to control LC3 processing

Purification, crystallization and preliminary crystallographic analysis of soybean mature glycinin A1bB2.

Glycinin is one of the most abundant storage-protein molecules in soybean seeds and is composed of five subunits (A1aB1b, A1bB2, A2B1a, A3B4 and A5A4B3). A1bB2 was purified from a mutant soybean cultivar containing glycinin composed of only A5A4B3 and A1bB2. At 281 K the protein formed hexagonal, rectangular and rod-shaped crystals in the first [0.1 M imidazole pH 8.0, 0.2 M MgCl2, 35%(v/v) MPD], second [0.1 M sodium citrate pH 5.6, 0.2 M ammonium acetate, 30%(v/v) MPD] and third (0.1 M phosphate-citrate pH 4.2, 2.0 M ammonium sulfate) crystallization conditions, respectively. X-ray diffraction data were collected to resolutions of 1.85, 1.85 and 2.5 Å from crystals of the three different shapes. The crystals belonged to space groups P6322, P21 and P1, with unit-cell parameters a = b = 143.60, c = 84.54 Å, a = 114.54, b = 105.82, c = 116.67 Å, β = 94.99° and a = 94.45, b = 94.96, c = 100.66 Å, α = 107.02, β = 108.44, γ = 110.71°, respectively. One, six and six subunits of A1bB2 were estimated to be present in the respective asymmetric units. The three-dimensional structure of the A1bB2 hexamer is currently being determined

Polypeptide modification: an improved proglycinin design to stabilise oil-in-water emulsions

β-Conglycinin and glycinin are soybean major seed storage proteins. Previous studies have shown that adding the extension region of β-conglycinin α subunit improves the emulsifying properties of proglycinin and confers more favourable characteristics than fusing the extension region of β-conglycinin α' subunit or the hypervariable regions (A4IV) of glycinin A1aB1b subunit. To evaluate the polypeptide properties, we designed mutants of A1aB1b subunits fused with truncated versions of A4IV (A4IVcut), α (αcut) or α' (α'cut) extension regions lacking the C-terminus 25 or 31 residues (A4IVC25, αC25 or α'C31), and also A4IVcut and α'cut with αC25 residues added (A4IVcut-αC25 and α'cut-αC25). All the modified proteins displayed conformations similar to the wild type. With good solubilities, the emulsion properties of the modified proteins were much better at ionic strength μ = 0.08 than at μ = 0.5. The modified A1aB1bαcut and A1aB1bα'cut showed poorer emulsion properties than those of A1aB1bα and A1aB1bα'. Replacing the hydrophobic A4IVC25 region of A1aB1bA4IV with hydrophilic αC25 created A1aB1bA4IVcut-αC25, which had the best emulsion stability among these proglycinin mutants. We found that addition of αC25 improves the emulsifying properties of two C-terminally truncated proglycinin variants, thereby illustrating its potential general utility. Our investigation showed that in order to improve the emulsifying ability and emulsion stability of a globular protein, the introduced polypeptide should (i) be highly hydrophilic, (ii) consist of multiple hydrophobic-strong hydrophilic regions comprising at least two alpha helixes, (iii) harbour a terminal α-helix at the end of the C-terminus and (iv) have properties similar to those of αC25

High prevalence of vitamin D deficiency in Cambodian women:A common deficiency in a sunny country

Recent studies have shown that in spite of being generally close to the equator; vitamin D deficiency is common in South East Asian countries. In order to quantify micronutrient status for women and children in Cambodia; a nationally-representative survey was conducted in 2014 linked to the Cambodian Demographic Health Survey. The countrywide median of 25(OH) D was, respectively, 64.9 and 91.1 nmol/L for mothers and children. Based on The Endocrine Society cutoffs (>50<75 nmol/L = insufficiency; <= 50 nmol/L = deficiency); 64.6% of mothers and 34.8% of their children had plasma vitamin D concentrations indicating insufficiency or deficiency. For deficiency alone, 29% of the mothers were found to be vitamin D deficient, but only 13.4% of children. Children who live in urban areas had a 43% higher rate of vitamin D insufficiency versus those who live in rural areas (OR; 1.434; 95% CI: 1.007; 2.041). However, such differences were not observed in their mothers. The high prevalence of vitamin D deficiency is likely in part due to lifestyle choices, including sun avoidance, increasingly predominant indoor work, and covered transport. These survey findings support the need for a broader national Cambodian study incorporating testing of adult men, adolescents and the elderly, and encompassing other parameters such as skeletal health. However, the data presented in this study already show significant deficiencies which need to be addressed and we discuss the benefit of establishing nationally-mandated food fortification programs to enhance the intake of vitamin D

Persistence and selection of an expanded B-cell clone in the setting of rituximab therapy for Sjögren's syndrome

Introduction: Subjects with primary Sjögren's syndrome (SjS) have an increased risk of developing B-cell lymphoma and may harbor monoclonal B-cell expansions in the peripheral blood. Expanded B-cell clones could be pathogenic, and their persistence could exacerbate disease or predispose toward the development of lymphoma. Therapy with anti-CD20 (rituximab) has the potential to eliminate expanded B-cell clones and thereby potentially ameliorate disease. This study was undertaken to identify and track expanded B-cell clones in the blood of subjects with primary SjS who were treated with rituximab.Methods: To determine whether circulating B-cell clones in subjects with primary SjS emerge or remain after B cell-depleting therapy with rituximab, we studied the antibody heavy-chain repertoire. We performed single-memory B-cell and plasmablast sorting and antibody heavy-chain sequencing in six rituximab-treated SjS subjects over the course of a 1-year follow-up period.Results: Expanded B-cell clones were identified in four out of the six rituximab-treated SjS subjects, based upon the independent amplification of sequences with identical or highly similar VH, DH, and JH gene segments. We identified one SjS subject with a large expanded B-cell clone that was present prior to therapy and persisted after therapy. Somatic mutations in the clone were numerous but did not increase in frequency over the course of the 1-year follow-up, suggesting that the clone had been present for a long period of time. Intriguingly, a majority of the somatic mutations in the clone were silent, suggesting that the clone was under chronic negative selection.Conclusions: For some subjects with primary SjS, these data show that (a) expanded B-cell clones are readily identified in the peripheral blood, (b) some clones are not eliminated by rituximab, and (c) persistent clones may be under chronic negative selection or may not be antigen-driven. The analysis of sequence variation among members of an expanded clone may provide a novel means of measuring the chronicity and selection of expanded B-cell populations in humans. © 2014 Hershberg et al.; licensee BioMed Central Ltd

Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in Cells

Autophagy protease ATG4B is a key regulator of the LC3/GABARAP conjugation system required for autophagosome formation, maturation and closure. Members of the ATG4 and the LC3/GABARAP family have been implicated in various diseases including cancer, and targeting the ATG4B protease has been suggested as a potential therapeutic anti-cancer strategy. Recently, it has been demonstrated that ATG4B is regulated by multiple post-translational modifications, including phosphorylation and de-phosphorylation. In order to identify regulators of ATG4B activity, we optimized a cell-based luciferase assay based on ATG4B-dependent release of Gaussia luciferase. We applied this assay in a proof-of-concept small molecule compound screen and identified activating compounds that increase cellular ATG4B activity. Next, we performed a high-throughput screen to identify kinases and phosphatases that regulate cellular ATG4B activity using siRNA mediated knockdown and cDNA overexpression. Of these, we provide preliminary evidence that the kinase AKT2 enhances ATG4B activity in cells. We provide all raw and processed data from the screens as a resource for further analysis. Overall, our findings provide novel insights into the regulation of ATG4B and highlight the importance of post-translational modifications of ATG4B

Low-dose thiamine supplementation of lactating Cambodian mothers improves human milk thiamine concentrations: a randomized controlled trial.

BACKGROUND: Infantile beriberi-related mortality is still common in South and Southeast Asia. Interventions to increase maternal thiamine intakes, and thus human milk thiamine, are warranted; however, the required dose remains unknown. OBJECTIVES: We sought to estimate the dose at which additional maternal intake of oral thiamine no longer meaningfully increased milk thiamine concentrations in infants at 24 wk postpartum, and to investigate the impact of 4 thiamine supplementation doses on milk and blood thiamine status biomarkers. METHODS: In this double-blind, 4-parallel arm randomized controlled dose-response trial, healthy mothers were recruited in Kampong Thom, Cambodia. At 2 wk postpartum, women were randomly assigned to consume 1 capsule, containing 0, 1.2 (estimated average requirement), 2.4, or 10 mg of thiamine daily from 2 through 24 weeks postpartum. Human milk total thiamine concentrations were measured using HPLC. An Emax curve was plotted, which was estimated using a nonlinear least squares model in an intention-to-treat analysis. Linear mixed-effects models were used to test for differences between treatment groups. Maternal and infant blood thiamine biomarkers were also assessed. RESULTS: In total, each of 335 women was randomly assigned to1 of the following thiamine-dose groups: placebo (n = 83), 1.2 mg (n = 86), 2.4 mg (n = 81), and 10 mg (n = 85). The estimated dose required to reach 90% of the maximum average total thiamine concentration in human milk (191 µg/L) is 2.35 (95% CI: 0.58, 7.01) mg/d. The mean ± SD milk thiamine concentrations were significantly higher in all intervention groups (183 ± 91, 190 ± 105, and 206 ± 89 µg/L for 1.2, 2.4, and 10 mg, respectively) compared with the placebo group (153 ± 85 µg/L; P < 0.0001) and did not significantly differ from each other. CONCLUSIONS: A supplemental thiamine dose of 2.35 mg/d was required to achieve a milk total thiamine concentration of 191 µg/L. However, 1.2 mg/d for 22 wk was sufficient to increase milk thiamine concentrations to similar levels achieved by higher supplementation doses (2.4 and 10 mg/d), and comparable to those of healthy mothers in regions without beriberi. This trial was registered at clinicaltrials.gov as NCT03616288

The decline and rise of neighbourhoods: the importance of neighbourhood governance

There is a substantial literature on the explanation of neighbourhood change. Most of this literature concentrates on identifying factors and developments behind processes of decline. This paper reviews the literature, focusing on the identification of patterns of neighbourhood change, and argues that the concept of neighbourhood governance is a missing link in attempts to explain these patterns. Including neighbourhood governance in the explanations of neighbourhood change and decline will produce better explanatory models and, finally, a better view about what is actually steering neighbourhood change

Transport of Explosive Residue Surrogates in Saturated Porous Media

Department of Defense operational ranges may become contaminated by particles of explosives residues (ER) as a result of low-order detonations of munitions. The goal of this study was to determine the extent to which particles of ER could migrate through columns of sandy sediment, representing model aquifer materials. Transport experiments were conducted in saturated columns (2 × 20 cm) packed with different grain sizes of clean sand or glass beads. Fine particles (approximately 2 to 50 μm) of 2,6-dinitrotoluene (DNT) were used as a surrogate for ER. DNT particles were applied to the top 1 cm of sand or beads in the columns, and the columns were subsequently leached with artificial groundwater solutions. DNT migration occurred as both dissolved and particulate phases. Concentration differences between unfiltered and filtered samples indicate that particulate DNT accounted for up to 41% of the mass recovered in effluent samples. Proportionally, more particulate than dissolved DNT was recovered in effluent solutions from columns with larger grain sizes, while total concentrations of DNT in effluent were inversely related to grain size. Of the total DNT mass applied to the uppermost layer of the column, <3% was recovered in the effluent with the bulk remaining in the top 2 cm of the column. Our results suggest there is some potential for subsurface migration of ER particles and that most of the particles will be retained over relatively short transport distances

A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B

Upon induction of autophagy, the ubiquitin-like protein LC3 is conjugated to phosphatidylethanolamine (PE) on the inner and outer membrane of autophagosomes to allow cargo selection and autophagosome formation. LC3 undergoes two processing steps, the proteolytic cleavage of pro-LC3 and the de-lipidation of LC3-PE from autophagosomes, both executed by the same cysteine protease ATG4. How ATG4 activity is regulated to co-ordinate these events is currently unknown. Here we find that ULK1, a protein kinase activated at the autophagosome formation site, phosphorylates human ATG4B on serine 316. Phosphorylation at this residue results in inhibition of its catalytic activity in vitro and in vivo. On the other hand, phosphatase PP2A-PP2R3B can remove this inhibitory phosphorylation. We propose that the opposing activities of ULK1-mediated phosphorylation and PP2A-mediated dephosphorylation provide a phospho-switch that regulates the cellular activity of ATG4B to control LC3 processing