Modification of Cul1 regulates its association with proteasomal subunits

BACKGROUND: Ubiquitylation targets proteins for degradation by the 26S proteasome. Some yeast and plant ubiquitin ligases, including the highly conserved SCF (Skp1/Cul1/F-box protein) complex, have been shown to associate with proteasomes. We sought to characterize interactions between SCF complexes and proteasomes in mammalian cells. RESULTS: We found that the binding of SCF complexes to proteasomes is conserved in higher eukaryotes. The Cul1 subunit associated with both sub-complexes of the proteasome, and high molecular weight forms of Cul1 bound to the 19S proteasome. Cul1 is ubiquitylated in vivo. Ubiquitylation of Cul1 promotes its binding to the S5a subunit of the 19S sub-complex without affecting Cul1 stability. CONCLUSION: The association of ubiquitylating enzymes with proteasomes may be an additional means to target ubiquitylated substrates for degradation

Proteasome-Mediated Degradation of p21 via N-Terminal Ubiquitinylation

AbstractWe examined the mechanism responsible for the degradation of p21, a negative regulator of the cell division cycle. We found that p21 proteolysis requires functional ubiquitin and Nedd8 systems. Ubiquitinylated forms of p21 and p21(K0), a p21 mutant missing all lysines, are detected in vivo and in vitro, showing that the presence of lysines is dispensable for p21 ubiquitinylation. Instead, the free amino group of the N-terminal methionine of p21 is a site for ubiquitinylation in vivo. Although wild-type p21 is more abundantly ubiquitinylated than p21(K0) mutant due to the presence of internal lysine residues, their rates of proteolysis are indistinguishable. These results demonstrate that proteasomal degradation of p21 is regulated by the ubiquitin pathway and suggest that the site of the ubiquitin chain is critical in making p21 a competent substrate for the proteasome

Local Food Systems Course for Extension Educators in North Carolina: Summary of an Innovative Program

Interest in local foods began in the early 2000s and has grown substantially over the past decade and a half. Although Extension is addressing local food systems in many states, training and materials in this program area are nascent. To address this circumstance, we developed a graduate course on local food systems for Extension educators. Post-course evaluations indicate increased confidence and knowledge related to local food system facilitation, implementation, and evaluation. Students cited site tours and panel presentations as the most effective course aspects and suggested improving the course by adjusting content to account for varying levels of familiarity with local food systems

Planning, Delivering, and Evaluating an Extension In-Service Training Program for Developing Local Food Systems: Lessons Learned

The social movement focused on re-localizing food systems is oriented toward recreating relationships between producers, consumers, and other community stakeholders. Sustaining community efforts to build local food systems requires preparation of county Extension educators to understand how food supply chains function as systems, facilitate community partnerships, and create equitable access to locally produced food. This paper shares how North Carolina Cooperative Extension designed, delivered, and evaluated a local foods in-service training on these three topics, as well as shares lessons learned through the process. The implications of this study are helpful for Extension educators planning, delivering, and evaluating in-service training programs that support development of local food systems

Carbon Monitoring System Flux Net Biosphere Exchange 2020 (CMS-Flux NBE 2020)

Here we present a global and regionally resolved terrestrial net biosphere exchange (NBE) dataset with corresponding uncertainties between 2010-2018: Carbon Monitoring System Flux Net Biosphere Exchange 2020 (CMS-Flux NBE 2020). It is estimated using the NASA Carbon Monitoring System Flux (CMS-Flux) top-down flux inversion system that assimilates column CO2 observations from the Greenhouse Gases Observing Satellite (GOSAT) and NASA\u27s Observing Carbon Observatory 2 (OCO-2). The regional monthly fluxes are readily accessible as tabular files, and the gridded fluxes are available in NetCDF format. The fluxes and their uncertainties are evaluated by extensively comparing the posterior CO2 mole fractions with CO2 observations from aircraft and the NOAA marine boundary layer reference sites. We describe the characteristics of the dataset as the global total, regional climatological mean, and regional annual fluxes and seasonal cycles. We find that the global total fluxes of the dataset agree with atmospheric CO2 growth observed by the surface-observation network within uncertainty. Averaged between 2010 and 2018, the tropical regions range from close to neutral in tropical South America to a net source in Africa; these contrast with the extra-tropics, which are a net sink of 2.5±0.3 Gt C/year. The regional satellite-constrained NBE estimates provide a unique perspective for understanding the terrestrial biosphere carbon dynamics and monitoring changes in regional contributions to the changes of atmospheric CO2 growth rate. The gridded and regional aggregated dataset can be accessed at http://doi.org/10.25966/4v02-c391 (Liu et al., 2020).

Leptin interacts with glucagon-like peptide-1 neurons to reduce food intake and body weight in rodents

AbstractThe adipose tissue hormone, leptin, and the neuropeptide glucagon-like peptide-1 (7–36) amide (GLP-1) both reduce food intake and body weight in rodents. Using dual in situ hybridization, long isoform leptin receptor (OB-Rb) was localized to GLP-1 neurons originating in the nucleus of the solitary tract. ICV injection of the specific GLP-1 receptor antagonist, exendin(9–39), at the onset of dark phase, did not affect feeding in saline pre-treated controls, but blocked the reduction in food intake and body weight of leptin pre-treated rats. These findings suggest that GLP-1 neurons are a potential target for leptin in its control of feeding

Splicing and the Evolution of Proteins in Mammals

It is often supposed that a protein's rate of evolution and its amino acid content are determined by the function and anatomy of the protein. Here we examine an alternative possibility, namely that the requirement to specify in the unprocessed RNA, in the vicinity of intron–exon boundaries, information necessary for removal of introns (e.g., exonic splice enhancers) affects both amino acid usage and rates of protein evolution. We find that the majority of amino acids show skewed usage near intron–exon boundaries, and that differences in the trends for the 2-fold and 4-fold blocks of both arginine and leucine show this to be owing to effects mediated at the nucleotide level. More specifically, there is a robust relationship between the extent to which an amino acid is preferred/avoided near boundaries and its enrichment/paucity in splice enhancers. As might then be expected, the rate of evolution is lowest near intron–exon boundaries, at least in part owing to splice enhancers, such that domains flanking intron–exon junctions evolve on average at under half the rate of exon centres from the same gene. In contrast, the rate of evolution of intronless retrogenes is highest near the domains where intron–exon junctions previously resided. The proportion of sequence near intron–exon boundaries is one of the stronger predictors of a protein's rate of evolution in mammals yet described. We conclude that after intron insertion selection favours modification of amino acid content near intron–exon junctions, so as to enable efficient intron removal, these changes then being subject to strong purifying selection even if nonoptimal for protein function. Thus there exists a strong force operating on protein evolution in mammals that is not explained directly in terms of the biology of the protein

MRI-guided focal laser ablation of prostate cancer: a prospective single-arm, single-center trial with 3 years of follow-up

PURPOSEWe aimed to assess post-interventional and 36-month follow-up results of a single-center, single-arm, in-bore phase I trial of focal laser ablation (FLA) guided by multiparametric magnetic resonance imaging (mpMRI).METHODSFLA procedures were done in-bore MRI using a transperineal approach. Primary endpoints were feasibility and safety expressed as lack of grade 3 complications. Secondary endpoints were changes in international prostate symptom score (IPSS), sexual health inventory for men (SHIM), quality of life (QoL) scores, and serum prostate specific antigen (PSA) levels. Treatment outcomes were assessed by combined mpMRI-ultrasound fusion-guided and extended sextant systematic biopsy after 12, 24, and optionally after 36 months.RESULTSFifteen participants were included. Seven patients (46.67%) had Gleason 3+3 and 8 patients (53.33%) had Gleason 3+4 cancer. All patients tolerated the procedure well, and no grade 3/4 complications occurred. All grade 1 and 2 complications were transient and resolved completely. There was no significant change in mean IPSS from baseline (-1, p = 0.460) and QoL (0, p = 0.441) scores following FLA but there was a significant drop in mean SHIM scores (-2, p = 0.010) compared to pretreatment baselines. Mean PSA significantly decreased after FLA (-2.5, p < 0.001). Seven out of 15 patients (46.67%) had residual cancer in, adjacent, or in close proximity to the treatment area (1 × 4+3=7, 1 × 3+4=7, and 5 × 3+3=6). Four out of 15 patients (26.67%) underwent salvage therapy (2 repeat FLA, 2 radical prostatectomy).CONCLUSIONAfter 3 years of follow-up we conclude focal laser ablation is safe and feasible without significant complications

Deconvoluting Post-Transplant Immunity: Cell Subset-Specific Mapping Reveals Pathways for Activation and Expansion of Memory T, Monocytes and B Cells

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO+CD62L− effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant