Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCF^Slmb degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

The effects of dietary concentrations of minerals, source of protein, amino acids and antibiotics on the growth of and digestibility of amino acids by broiler chickens

The effects of dietary calcium, available phosphorus, amino acid and antibiotic supplements on the digestibilities of amino acids and growth of broiler chickens were investigated in two experiments. 2. The performance of chickens fed on diets containing high concentrations of calcium and available phosphorus was poorer with meat meal-based diets than with soyabean-based diets. Methionine supplementation improved the performance of chickens fed meat meal-based diets. 3. A high dietary content of calcium (25.9 vs 11.8 g/kg) reduced chick performance and the digestibility of glutamic acid, leucine and phenylalanine but increased the digestibility of lysine and histidine. 4. High dietary contents of calcium and available phosphorus (24.3 and 13.0 vs 11.8 and 4.0 g/kg) reduced chick performance and the digestibilities of most amino acids. 5. Antibiotic supplementation did not improve the performance of chickens, but increased the digestibilities of most amino acids in chickens fed on diets with a high calcium or high calcium and available phosphorus contents. 6. It was concluded that excess dietary calcium alone, or calcium and phosphorus together, reduced chick performance and the digestibilities of most amino acids. Growing chickens tolerated excess dietary calcium and available phosphorus better in well-balanced amino acid diets, such as soyabean meal or methionine-supplemented meat meal diets, than in poorly balanced amino acid diets, such as unsupplemented meat meal diets

Effects of dietary calcium:available phosphorus ratio on calcium tolerance of broiler chickens

The effects of dietary levels of available phosphorus (AP) on calcium (Ca) tolerance of growing chickens were investigated in 3 experiments. In the first experiment, increasing dietary Ca to 21.2 g/kg with AP at 5 g/kg significantly reduced tibia P, plasma P and increased plasma total Ca concentration without any significant effect on performance. With dietary Ca at 25.7 g/kg and AP at 5 g/kg, growth and plasma P were reduced and plasma total Ca was increased, whilst Ca at 30 g/kg diet reduced growth, plasma P, tibia P and increased feed conversion ratio (FCR) and plasma total Ca. Increasing dietary AP to 10 g/kg diet with Ca level at 10.6 g/kg reduced growth and increased FCR without affecting any of the other parameters. Experiment 2 was a 3-dimensional composite design with a central basal diet having Ca and AP concentrations of 12.7 and 4.6 g/kg, The first dimension was 4 dietary concentrations of Ca from 15 to 30 g/kg; the second dimension was 4 dietary concentrations of AP from 6.3 to 12.4 g/kg; and the third dimension was 4 dietary Ca : AP combinations spanning the above ranges but with the Ca: AP ratio maintained at approximately 2.5 (i.e. 15.8 and 6.3; 20.5 and 8.2; 25.5 and 10.2; 30 and 12.1 g/kg for Ca and AP, respectively). A dietary Ca concentration of 25.4 g/kg increased FCR, whilst 30 g Ca/kg reduced growth and increased FCR. Dietary AP of 12.4 g/kg reduced growth and increased FCR. Growth and FCR were less affected when dietary Ca:AP ratio was kept constant at approximately 2.5 than when the ratio was altered by increasing either mineral alone. This relationship was used to develop a linear regression relationship between chicken growth and dietary Ca: AP ratio. In experiment 3, the effects of excessive dietary levels of Ca (12.7, 25.5 and 33.1 g/kg) and AP (9 and 13 g/kg) were investigated in sexed chickens in a factorial design. Ca at 33.1 g/kg significantly reduced growth, tibia P and plasma P, whilst FCR, plasma total Ca and excreta moisture were significantly increased. Dietary Ca at 25.5 g/kg reduced tibia P and increased excreta Ca. High dietary AP significantly reduced growth and tibia Ca and increased tibia ash, excreta moisture and excreta Ca. Significant interactions between dietary levels of Ca and AP were found for growth, tibia ash, excreta moisture and excreta Ca, and for tibia P and plasma total Ca. Significant interactions between sex and either dietary Ca or AP for tibia ash were found. A Ca x sex interaction was found for tibia P, excreta moisture and excreta P. It was concluded that high dietary levels of Ca (up to 21.2 g/kg) can be tolerated by growing chickens without any significant effect on performance, providing the corresponding level of AP is also high

Effects of dietary calcium, available phosphorus and vitamin D on growth rate, food utilisation, plasma and bone constituents and calcium and phosphorus retention of commercial broiler strains

The effects of different dietary concentrations of calcium (Ca), available phosphorus (AP) and vitamin D (D) on 5- to 16-day growth performance, and aspects of calcium and phosphorus (P) metabolism of chickens from three commercial strains were studied in two experiments. 2. Increasing dietary Ca reduced weight gain, tibia Ca and P content and increased plasma total Ca, Ca consumption and excretion, whilst dietary Ca at 32 g/kg increased tibia Ca:P ratio, plasma ionized calcium and reduced plasma P, tibia ash, P excretion, excreta moisture and Ca retention. 3. Increasing dietary AP reduced plasma total and ionized Ca and excreta moisture and increased P consumption and excretion, plasma P and tibia ash. 4. The addition of vitamin D increased plasma total and ionized Ca, tibia Ca:P ratio and reduced plasma sodium and P concentrations. 5. Strains differed in their tibia contents of Ca and Ca:P ratios, in response to Ca, AP and vitamin D diets whilst they differed in Ca excretion and excreta moisture caused by feeding either dietary Ca or AP. 6. It was concluded that dietary Ca, AP, vitamin D and strain of broiler chickens influenced the metabolism of Ca and P and that, as a consequence, the tolerance to high dietary Ca. A lean strain of chickens tolerated high dietary calcium better than its fat counterpart

Effects of ultrasonic waves on eggshell strength and hatchability of layer-type breeder eggs

Two trials were conducted to investigate the effects of exposing layer-type breeder eggs before incubation to ultrasonic waves (ULT). Eggs were subjected to ULT of 117 volts at 40 kHz for up to 15 minutes. Eggshell breaking force (EBF), hatchability and chick hatching weight (CHW) of Balady breeder eggs (Trial 1), and egg weight loss, embryo weight, hatchability and CHW of Leghorn hen eggs (Trial 2) were measured. In Trial 1 the eggs were subjected to seven treatments: non-dipped (control), and dipped for 5, 10 and 15 minutes in a water bath (W5, W10 and W15) or a ULT bath (ULT5, ULT10 and ULT15). In Trial 2 the eggs were subjected to four treatments, a control (ULT0) and the ultrasonic treatments, ULT5, ULT10 and ULT15. In treatments ULT10 and ULT15 the EBF of Balady eggs was significantly reduced while the hatchability of the Balady and Leghorn eggs was significantly reduced by the ULT15 and W15 treatments, compared with the control. Orthogonal contrast (control vs. ULT) indicated that ULT exposure of eggs reduced their EBF significantly. Percentage egg weight loss, embryo weight and CHW was not affected by the treatments. It was concluded that exposure of eggs to ULT of 117 volts at 40 kHz for 15 minutes reduced EBF and hatchability of layer-type breeder eggs without altering egg weight loss, embryo weight or CHW.Keywords: Balady breeders, Leghorn hens, egg weight loss, embryo, hatchabilit

In ovo feeding of carbohydrates and incubated at a high incubation temperature on hatchability and glycogen status of chicks

Eggs from a meat-type breeder flock at 29 and 35 weeks of age were used in two trials to investigate the effects of in ovo feeding of carbohydrates (CHO) and high incubation temperature (37.5 vs. 38.5 °C (HIT)) during days 16 to 21 of incubation on hatchability traits, chick weight at hatch as an absolute value or as a percentage of egg weight (CWTP), hatching time, glycogen concentration in the liver and pectoral muscle, and glycogen index of hatched chicks. The treatments were a non-injected control, a positive control where saline was injected, or saline with a CHO mixture at 50, 100, 150, 200 and 250 mg/egg. The CHO mixture was maltose, sucrose and dextrin in a proportion of 1 : 1 : 8 by weight. As a result of this study, in ovo feeding of CHO increased CWTP without altering hatchability traits. Hatched chicks from eggs injected with 250 mg CHO/egg had a higher liver glycogen content and glycogen index than those of the control treatments. The high incubation temperature reduced chick weight, hatching time, liver glycogen and glycogen index of the hatched chicks. In ovo feeding of 100 and above mg CHO/egg overcame the negative effects of HIT. Hatched chicks from older hens had a lower concentration of liver glycogen and glycogen index than those of younger hens. It was concluded that in ovo feeding of CHO improved the weight and glycogen index of hatched chicks and those of the HIT treatment, and older hens negatively affected the glycogen index of hatched chicks.Keywords: Broiler eggs, glycogen, incubation, liver, pectoral muscl