Autophagy gene expression profiling identifies a defective microtubule-associated protein light chain 3A mutant in cancer.

The cellular stress response autophagy has been implicated in various diseases including neuro-degeneration and cancer. The role of autophagy in cancer is not clearly understood and both tumour promoting and tumour suppressive effects of autophagy have been reported, which complicates the design of therapeutic strategies based on targeting the autophagy pathway. Here, we have systematically analyzed gene expression data for 47 autophagy genes for deletions, amplifications and mutations in various cancers. We found that several cancer types have frequent autophagy gene amplifications, whereas deletions are more frequent in prostate adenocarcinomas. Other cancer types such as glioblastoma and thyroid carcinoma show very few alterations in any of the 47 autophagy genes. Overall, individual autophagy core genes are altered at low frequency in cancer, suggesting that cancer cells require functional autophagy. Some autophagy genes show frequent single base mutations, such as members of the ULK family of protein kinases. Furthermore, we found hotspot mutations in the arginine-rich stretch in MAP1LC3A resulting in reduced cleavage of MAP1LC3A by ATG4B both in vitro and in vivo, suggesting a functional implication of this gene mutation in cancer development

Increased Ipsilateral M1 Activation after Incomplete Spinal Cord Injury Facilitates Motor Performance

Incomplete spinal cord injury (SCI) may result in muscle weakness and difficulties with force gradation. Although these impairments arise from the injury and subsequent changes at spinal levels, changes have also been demonstrated in the brain. Blood-oxygen-level dependent (BOLD) imaging was used to investigate these changes in brain activation in the context of unimanual contractions with the first dorsal interosseous muscle. BOLD- and force data were obtained in 19 individuals with SCI (AISA Impairment Scale [AIS] C/D, level C4-C8) and 24 able-bodied controls during maximal voluntary contractions (MVCs). To assess force modulation, participants performed 12 submaximal contractions with each hand (at 10, 30, 50, and 70% MVC) by matching their force level to a visual target. MVCs were weaker in the SCI group (both hands p < 0.001), but BOLD activation did not differ between SCI and control groups. For the submaximal contractions, force (as %MVC) was similar across groups. However, SCI participants showed increased activity of the ipsilateral motor cortex and contralateral cerebellum across all contractions, with no differential effect of force level. Activity of ipsilateral M1 was best explained by force of the target hand (vs. the non-target hand). In conclusion, the data suggest that after incomplete cervical SCI, individuals remain capable of producing maximal supraspinal drive and are able to modulate this drive adequately. Activity of the ipsilateral motor network appears to be task related, although it remains uncertain how this activity contributes to task performance and whether this effect could potentially be harnessed to improve motor functioning

RS rearrangement frequency as a marker of receptor editing in lupus and type 1 diabetes

Continued antibody gene rearrangement, termed receptor editing, is an important mechanism of central B cell tolerance that may be defective in some autoimmune individuals. We describe a quantitative assay for recombining sequence (RS) rearrangement that we use to estimate levels of antibody light chain receptor editing in various B cell populations. RS rearrangement is a recombination of a noncoding gene segment in the κ antibody light chain locus. RS rearrangement levels are highest in the most highly edited B cells, and are inappropriately low in autoimmune mouse models of systemic lupus erythematosus (SLE) and type 1 diabetes (T1D), including those without overt disease. Low RS rearrangement levels are also observed in human subjects with SLE or T1D

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

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

Optimising Strategies for Plasmodium falciparum Malaria Elimination in Cambodia: Primaquine, Mass Drug Administration and Artemisinin Resistance

Malaria elimination requires a variety of approaches individually optimized for different transmission settings. A recent field study in an area of low seasonal transmission in South West Cambodia demonstrated dramatic reductions in malaria parasite prevalence following both mass drug administration (MDA) and high treatment coverage of symptomatic patients with artemisinin-piperaquine plus primaquine. This study employed multiple combined strategies and it was unclear what contribution each made to the reductions in malaria.A mathematical model fitted to the trial results was used to assess the effects of the various components of these interventions, design optimal elimination strategies, and explore their interactions with artemisinin resistance, which has recently been discovered in Western Cambodia. The modelling indicated that most of the initial reduction of P. falciparum malaria resulted from MDA with artemisinin-piperaquine. The subsequent continued decline and near elimination resulted mainly from high coverage with artemisinin-piperaquine treatment. Both these strategies were more effective with the addition of primaquine. MDA with artemisinin combination therapy (ACT) increased the proportion of artemisinin resistant infections, although much less than treatment of symptomatic cases with ACT, and this increase was slowed by adding primaquine. Artemisinin resistance reduced the effectiveness of interventions using ACT when the prevalence of resistance was very high. The main results were robust to assumptions about primaquine action, and immunity.The key messages of these modelling results for policy makers were: high coverage with ACT treatment can produce a long-term reduction in malaria whereas the impact of MDA is generally only short-term; primaquine enhances the effect of ACT in eliminating malaria and reduces the increase in proportion of artemisinin resistant infections; parasite prevalence is a better surveillance measure for elimination programmes than numbers of symptomatic cases; combinations of interventions are most effective and sustained efforts are crucial for successful elimination