Novel Cell- and Tissue-Based Assays for Detecting Misfolded and Aggregated Protein Accumulation Within Aggresomes and Inclusion Bodies

Aggresomes and related inclusion bodies appear to serve as storage depots for misfolded and aggregated proteins within cells, which can potentially be degraded by the autophagy pathway. A homogenous fluorescence-based assay was devised to detect aggregated proteins inside aggresomes and inclusion bodies within an authentic cellular context. The assay employs a novel red fluorescent molecular rotor dye, which is essentially nonfluorescent until it binds to structural features associated with the aggregated protein cargo. Aggresomes and related structures were generated within cultured cells using various potent, cell permeable, proteasome inhibitors: MG-132, lactacystin, epoxomicin and bortezomib, and then selectively detected with the fluorescent probe. Employing the probe in combination with various fluorescein-labeled primary antibodies facilitated co-localization of key components of the autophagy system (ubiquitin, p62, and LC3) with aggregated protein cargo by fluorescence microscopy. Furthermore, cytoplasmic aggregates were highlighted in SK-N-SH human neuroblastoma cells incubated with exogenously supplied amyloid beta peptide 1–42. SMER28, a small molecule modulator of autophagy acting via an mTOR-independent mechanism, prevented the accumulation of amyloid beta peptide within these cells. The described assay allows assessment of the effects of protein aggregation directly in cells, without resorting to the use of non-physiological protein mutations or genetically engineered cell lines. With minor modification, the assay was also adapted to the analysis of frozen or formalin-fixed, paraffin-embedded tissue sections, with demonstration of co-localization of aggregated cargo with β-amyloid and tau proteins in brain tissue sections from Alzheimer’s disease patients

Widespread Endogenization of Genome Sequences of Non-Retroviral RNA Viruses into Plant Genomes

Non-retroviral RNA virus sequences (NRVSs) have been found in the chromosomes of vertebrates and fungi, but not plants. Here we report similarly endogenized NRVSs derived from plus-, negative-, and double-stranded RNA viruses in plant chromosomes. These sequences were found by searching public genomic sequence databases, and, importantly, most NRVSs were subsequently detected by direct molecular analyses of plant DNAs. The most widespread NRVSs were related to the coat protein (CP) genes of the family Partitiviridae which have bisegmented dsRNA genomes, and included plant- and fungus-infecting members. The CP of a novel fungal virus (Rosellinia necatrix partitivirus 2, RnPV2) had the greatest sequence similarity to Arabidopsis thaliana ILR2, which is thought to regulate the activities of the phytohormone auxin, indole-3-acetic acid (IAA). Furthermore, partitivirus CP-like sequences much more closely related to plant partitiviruses than to RnPV2 were identified in a wide range of plant species. In addition, the nucleocapsid protein genes of cytorhabdoviruses and varicosaviruses were found in species of over 9 plant families, including Brassicaceae and Solanaceae. A replicase-like sequence of a betaflexivirus was identified in the cucumber genome. The pattern of occurrence of NRVSs and the phylogenetic analyses of NRVSs and related viruses indicate that multiple independent integrations into many plant lineages may have occurred. For example, one of the NRVSs was retained in Ar. thaliana but not in Ar. lyrata or other related Camelina species, whereas another NRVS displayed the reverse pattern. Our study has shown that single- and double-stranded RNA viral sequences are widespread in plant genomes, and shows the potential of genome integrated NRVSs to contribute to resolve unclear phylogenetic relationships of plant species

The 3-Hydroxy-2-Butanone Pathway Is Required for Pectobacterium carotovorum Pathogenesis

Pectobacterium species are necrotrophic bacterial pathogens that cause soft rot diseases in potatoes and several other crops worldwide. Gene expression data identified Pectobacterium carotovorum subsp. carotovorum budB, which encodes the α-acetolactate synthase enzyme in the 2,3-butanediol pathway, as more highly expressed in potato tubers than potato stems. This pathway is of interest because volatiles produced by the 2,3-butanediol pathway have been shown to act as plant growth promoting molecules, insect attractants, and, in other bacterial species, affect virulence and fitness. Disruption of the 2,3-butanediol pathway reduced virulence of P. c. subsp. carotovorum WPP14 on potato tubers and impaired alkalinization of growth medium and potato tubers under anaerobic conditions. Alkalinization of the milieu via this pathway may aid in plant cell maceration since Pectobacterium pectate lyases are most active at alkaline pH

Effects of ammonia exposure on carcass traits and fatty acid composition of broiler meat

We aimed to study the effects of ammonia on carcass traits, organ indices and fatty acid composition of broilers. Four hundred 21-d-old male Arbor Acres broilers with initial weight 563.52 ± 2.82 g were randomly allotted to 1 of 4 groups treated with ammonia at <3 mg/kg (control), 25 ± 3, 50 ± 3, and 75 ± 3 mg/kg concentrations. Each group consisted of 4 replicates of 25 birds. Broilers from 21 to 42 d were reared on the net floor in the respiration-metabolism chambers where similar environmental conditions were maintained. At 32 and 42 d of age, carcass traits and organ indices were determined for 4 birds per pen. At 42 d of age, fatty acid composition in the breast and thigh muscle of broilers was measured. Results showed as follows: 1) At 32 d, the dressing percentage of broilers exposed to 25 and 75 mg/kg ammonia were lower than those in the control group (P < 0.05); eviscerated yield percentage of broilers in the 25 mg/kg ammonia group was also lower (P < 0.05). At 42 d, the dressing percentage of broilers in the ammonia treatments and the thigh muscle percentage of broilers in the 50 and 75 mg/kg ammonia groups were lower (P < 0.05) than those in the control. Breast muscle percentage of broilers exposed to 25 and 50 mg/kg ammonia and eviscerated yield percentage exposed to 50 mg/kg ammonia were lower than those in the control (P < 0.05). 2) The kidney index of broilers (d 32) exposed to ammonia was greater (P < 0.05) than that of the control. At 42 d, hepatic index of broilers exposed to ammonia was increased (P < 0.05), and spleen index was decreased (P < 0.05). 3) At 42 d, stearic (C18:0) and saturated fatty acids (SFA) in the thigh muscle of broilers were higher, while the unsaturated fatty acid:saturated fatty acid (U:F) ratio and unsaturated fatty acid (UFA) were lower in the 50 mg/kg ammonia treatment than in the control group (P < 0.05). In conclusion, ammonia over 25 mg/kg could decline carcass traits and immune organ indices and increase the kidney and hepatic indices. Further, exposure to 50 mg/kg ammonia could also decrease breast and thigh muscle yield percentage while increasing SFA content and decreasing UFA content in the thigh muscle of broilers

Transcriptome Profile Analysis of Breast Muscle Tissues from High or Low Levels of Atmospheric Ammonia Exposed Broilers (Gallus gallus).

Atmospheric ammonia is a common problem in poultry industry. High concentrations of aerial ammonia cause great harm to broilers' health and production. For the consideration of human health, the limit exposure concentration of ammonia in houses is set at 25 ppm. Previous reports have shown that 25 ppm is still detrimental to livestock, especially the gastrointestinal tract and respiratory tract, but the negative relationship between ammonia exposure and the tissue of breast muscle of broilers is still unknown. In the present study, 25 ppm ammonia in poultry houses was found to lower slaughter performance and breast yield. Then, high-throughput RNA sequencing was utilized to identify differentially expressed genes in breast muscle of broiler chickens exposed to high (25 ppm) or low (3 ppm) levels of atmospheric ammonia. The transcriptome analysis showed that 163 genes (fold change ≥ 2 or ≤ 0.5; P-value < 0.05) were differentially expressed between Ammonia25 (treatment group) and Ammonia3 (control group), including 96 down-regulated and 67 up-regulated genes. qRT-PCR analysis validated the transcriptomic results of RNA sequencing. Gene Ontology (GO) functional annotation analysis revealed potential genes, processes and pathways with putative involvement in growth and development inhibition of breast muscle in broilers caused by aerial ammonia exposure. This study facilitates understanding of the genetic architecture of the chicken breast muscle transcriptome, and has identified candidate genes for breast muscle response to atmospheric ammonia exposure

Proteome changes in the small intestinal mucosa of growing pigs with dietary supplementation of non-starch polysaccharide enzymes

Abstract Background Non-starch polysaccharide enzymes (NSPEs) have long been used in monogastric animal feed production to degrade non-starch polysaccharides (NSPs) to oligosaccharides in order to promote growth performance and gastrointestinal (GI) tract health. However, the precise molecular mechanism of NSPEs in the improvement of the mammalian small intestine remains unknown. Methods In this study, isobaric tags were applied to investigate alterations of the small intestinal mucosa proteome of growing pigs after 50\ua0days of supplementation with 0.6% NSPEs (mixture of xylanase, \u3b2-glucanase and cellulose) in the diet. Bioinformatics analysis including gene ontology annotation was performed to determine the differentially expressed proteins. A protein fold-change of\u2009\u2265\u20091.2 and a P -value of\u2009<\u20090.05 were selected as thresholds. Results Dietary supplementation of NSPEs improved the growth performance of growing pigs. Most importantly, a total of 90 proteins were found to be differentially abundant in the small intestinal mucosa between a control group and the NSPE group. Up-regulated proteins were related to nutrient metabolism (energy, lipids, protein and mineral), immunity, redox homeostasis, detoxification and the cell cytoskeleton. Down-regulated proteins were primarily related to transcriptional and translational regulation. Our results indicate that the effect of NSPEs on the increase of nutrient availability in the intestinal lumen facilitates the efficiency of nutrient absorption and utilization, and the supplementation of NSPEs in growing pigs also modulates redox homeostasis and enhances immune response during simulating energy metabolism due to a higher uptake of nutrients in the small intestine. Conclusions These findings have important implications for understanding the mechanisms of NSPEs on the small intestine of pigs, which provides new information for the better utilization of this feed additive in the future

Effects of Phytase Source and Dose on Its Stability during Pelleting Process under Different Conditioning Temperatures

Phytase activity can be impaired during pelleting because of extreme thermal conditions. This study investigated the effects of dose and source of phytase on phytase activity during the conditioning, pelleting, and cooling process. A split-plot design was used in two experiments, with five phytase doses (Exp. 1; 7560, 14310, 33830, 43590 and 61500 FTU/kg) or eight phytase sources (Exp. 2) as the main plot and steam conditioning temperatures (Exp. 1 and 2; 75 and 85 °C) as the subplot. Each treatment processed four batches, one batch per replicate. The results of Exp. 1 showed phytase dose in diets had no effect (p > 0.05) on the recovery rate of phytase activity after the conditioning, pelleting, or cooling process. The recovery rate of phytase activity in each process was higher (p p < 0.05) the recovery rate of phytase activity and had varied appearances of structure. In conclusion, the structure, phytase activity, and phytase recovery after steam conditioning–pelleting significantly varied across sources, but the stability of phytase was not affected by dose

Additional file 2: Table S1. of Proteome changes in the small intestinal mucosa of growing pigs with dietary supplementation of non-starch polysaccharide enzymes

qPCR primers used for verification of the differentially expressed genes of the small intestinal mucosa in growing pigs. (DOCX 14 kb

Additional file 1: of Proteome changes in the small intestinal mucosa of growing pigs with dietary supplementation of non-starch polysaccharide enzymes

The detailed description of the experiment methods, including mass spectrometric analysis procedures and parameters, bioinformatics analysis softwares, websites and real-time qPCR procedures. (DOCX 15 kb

Additional file 4: Table S3. of Proteome changes in the small intestinal mucosa of growing pigs with dietary supplementation of non-starch polysaccharide enzymes

List of all differentially expressed proteins (n = 104) identified in the study. (XLSX 31 kb