Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. Identifying novel regulators of mitochondrial bioenergetics will broaden our understanding of regulatory checkpoints that coordinate complex metabolic pathways. We previously showed that Nur77, an orphan nuclear receptor of the NR4A family, regulates the expression of genes linked to glucose utilization. Here we demonstrate that expression of Nur77 in skeletal muscle also enhances mitochondrial function. We generated MCK-Nur77 transgenic mice that express wild-type Nur77 specifically in skeletal muscle. Nur77-overexpressing muscle had increased abundance of oxidative muscle fibers and mitochondrial DNA content. Transgenic muscle also exhibited enhanced oxidative metabolism, suggestive of increased mitochondrial activity. Metabolomic analysis confirmed that Nur77 transgenic muscle favored fatty acid oxidation over glucose oxidation, mimicking the metabolic profile of fasting. Nur77 expression also improved the intrinsic respiratory capacity of isolated mitochondria, likely due to the increased abundance of complex I of the electron transport chain. These changes in mitochondrial metabolism translated to improved muscle contractile function ex vivo and improved cold tolerance in vivo. Our studies outline a novel role for Nur77 in the regulation of oxidative metabolism and mitochondrial activity in skeletal muscle.     

Diversity,equity, and inclusion in the melanoma research community

The inaugural Diversity and Inclusion in Science Session was held during the 2021 Society for Melanoma Research (SMR) congress. The goal of the session was to discuss diversity, equity, and inclusion in the melanoma research community and strategies to promote the advancement of underrepresented melanoma researchers. An international survey was conducted to assess the diversity, equity, and inclusion (DEI) climate among researchers and clinicians within the Society for Melanoma Research (SMR). The findings suggest there are feelings and experiences of inequity, bias, and harassment within the melanoma community that correlate with one's gender, ethnic/racial group, and/or geographic location. Notably, significant reports of inequity in opportunity, discrimination, and sexual harassment demonstrate there is much work remaining to ensure all scientists in our community experience an academic workplace culture built on mutual respect, fair access, inclusion, and equitable opportunity.     

A highly efficient method for porcine cloning by nuclear transfer using in vitro-matured oocytes

To date, the efficiency of pig cloning by nuclear transfer of somatic cell nuclei has been extremely low, with less than 1% of transferred embryos surviving to term. Even the utilization of complex procedures such as two rounds of nuclear transfer has not resulted in greater overall efficiencies. As a result, the applicability of the technology for the generation of transgenic and cloned animals has not moved forward rapidly. We report here a simple nuclear transfer protocol, utilizing commercially available in vitro-matured oocytes, that results in greater than 5% overall cloning efficiency. Of five recipients receiving nuclear transfer embryos produced with a fetal fibroblast cell line as nuclear donor, all five established pregnancies by day 28 (100%), and 4/5 (80%) went to term. Efficiencies for each transfer were 7% (9 piglets/128 doublets transferred), 5% (5/100), 12% (7/59), and 6.6% (7/106). The overall efficiency in all recipients was 5.5% and in pregnant recipients 7.7%, with a total of 28 cloned piglets produced. With the average fusion rate being 58%, the percentage of fused doublets producing a live piglet approached 12%. The method described here can be undertaken by a single micromanipulator at a reasonable cost, and should facilitate the broad utilization of porcine cloning technology in transgenic and nontransgenic applications.     

Relaxation-optimised Hartmann–Hahn transfer using a specifically Tailored MOCCA-XY16 mixing sequence for carbonyl–carbonyl correlation spectroscopy in <Superscript>13</Superscript>C direct detection NMR experiments

Isotropic mixing sequences are one of the key methods to achieve efficient coherence transfer. Among them, the MOCCA-XY16, which keeps the magnetization longitudinal for a significant amount of time, is characterised by favourable relaxation properties. We show here that its adapted version is particularly suited for carbonyl–carbonyl correlations in ¹³C direct detection NMR experiments.     

Contribution of Position α4S336 on Functional Expression and Up-regulation of α4β2 Neuronal Nicotinic Receptors

Phosphorylation of the nicotinic acetylcholine receptor (nAChR) is believed to play a critical role in its nicotine-induced desensitization and up-regulation. We examined the contribution of a consensus PKC site in the α4 M3/M4 intracellular loop (α4S336) on the desensitization and up-regulation of α4β2 nAChRs expressed in oocytes. Position α4S336 was replaced with either alanine to abolish potential phosphorylation at this site or with aspartic acid to mimic phosphorylation at this same site. Mutations α4S336A and α4S336D displayed a threefold increase in the ACh-induced response and an increase in ACh EC₅₀. Epibatidine binding revealed a three and sevenfold increase in surface expression for the α4S336A and α4S336D mutations, respectively, relative to wild-type, therefore, both mutations enhanced expression of the α4β2 nAChR. Interestingly, the EC₅₀’s and peak currents for nicotine activation remained unaffected in both mutants. Both mutations abolished the nicotine-induced up-regulation that is normally observed in the wild-type. The present data suggest that adding or removing a negative charge at this phosphorylation site cannot be explained by a simple straightforward on-and-off mechanism; rather a more complex mechanism(s) may govern the functional expression of the α4β2 nAChR. Along the same line, our data support the idea that phosphorylation at multiple consensus sites in the α4 subunit could play a remarkable role on the regulation of the functional expression of the α4β2 nAChR.     

Formation of Thick Dense Yttrium Iron Garnet Films Using Aerosol Deposition

Aerosol deposition (AD) is a thick-film deposition process that can produce layers up to several hundred micrometers thick with densities greater than 95% of the bulk. The primary advantage of AD is that the deposition takes place entirely at ambient temperature; thereby enabling film growth in material systems with disparate melting temperatures. This report describes in detail the processing steps for preparing the powder and for performing AD using the custom-built system. Representative characterization results are presented from scanning electron microscopy, profilometry, and ferromagnetic resonance for films grown in this system. As a representative overview of the capabilities of the system, focus is given to a sample produced following the described protocol and system setup. Results indicate that this system can successfully deposit 11 µm thick yttrium iron garnet films that are  > 90% of the bulk density during a single 5 min deposition run. A discussion of methods to afford better control of the aerosol and particle selection for improved thickness and roughness variations in the film is provided.