The effects of conjugated linoleic acid isomers cis-9,trans-11 and trans-10,cis-12 on in vitro bovine embryo production and cryopreservation

Conjugated linoleic acid (CLA) isomers can affect the lipid profile and signaling of cells and thereby alter their function. A total of 5,700 bovine oocytes were used in a structured series of experiments to test the effects of CLA cis-9,trans-11 and CLA trans-10,cis-12 in vitro. In experiment 1, high doses of each CLA isomer during in vitro maturation (IVM) were compared with high or low doses during the entire in vitro culture (IVC) of parthenogenetic embryos. High doses of the CLA isomers ranged from 50 to 200 μM and low doses were 15 and 25 μM. In experiment 2, the low doses of each CLA isomer were tested during IVM/IVC on embryos produced by in vitro fertilization (IVF). Experiment 3 compared the effects of 15 μM doses of each CLA isomer during IVM or IVC of IVF embryos. In experiment 4, post-rewarming survival rates and blastomere counts were assessed for embryos supplemented with each CLA isomer during IVM or for 36 h before vitrification. In experiment 1, when either CLA isomer was provided only during IVM, we observed no effects on overall rates of maturation, cleavage, or blastocysts (92.2 ± 1.6%, 78.3 ± 4.1%, and 28.9 ± 5.1%, respectively). However, high doses of each CLA isomer, but not low doses, during the entire embryo culture period decreased blastocyst rates (5-20%) in a dose-dependent manner. Cleavage rates improved with 15 or 50 μM CLA trans-10,cis-12. Progesterone concentrations in maturation media were significantly increased by high doses of each CLA isomer compared with control, but low doses of CLA isomers had no effect. In experiment 2 with IVF embryos, low doses of each CLA isomer did not alter cleavage rates (average 84.9 ± 1.9%) and only 25 μM CLA trans-10,cis-12 during IVC reduced blastocyst rates below those of controls (25.5 ± 2.1 vs. 38.2 ± 2.3%). The lipid content of embryos was increased and relative expression of the BIRC5 (baculoviral IAP repeat containing 5) gene was depressed by CLA trans-10,cis-12. In experiment 3, low doses (15μM) of each CLA isomer during IVC significantly reduced blastocyst rates (20.6 ± 2.4% and 27.7 ± 1.2% vs. 34.18 ± 1.2% for CLA trans-10,cis-12 and CLA cis-9,trans-11 compared with control, respectively) with less effect of each CLA during IVM. In experiment 4, adding 100 μM CLA cis-9,trans-11 during the final 36 h of culture resulted in a high survival rate after rewarming and culture, and the higher embryo blastomere count was comparable to that of control embryos not undergoing vitrification. In conclusion, supplementation with either CLA isomer did not improve embryo production, but inclusion of CLA cis-9,trans-11 before vitrification improved the quality of bovine IVF embryos after rewarming and culture

Phase-ordering dynamics of the Gay-Berne nematic liquid crystal

Phase-ordering dynamics in nematic liquid crystals has been the subject of much active investigation in recent years in theory, experiments and simulations. With a rapid quench from the isotropic to nematic phase a large number of topological defects are formed and dominate the subsequent equilibration process. We present here the results of a molecular dynamics simulation of the Gay-Berne model of liquid crystals after such a quench in a system with 65536 molecules. Twist disclination lines as well as type-1 lines and monopoles were observed. Evidence of dynamical scaling was found in the behavior of the spatial correlation function and the density of disclination lines. However, the behavior of the structure factor provides a more sensitive measure of scaling, and we observed a crossover from a defect dominated regime at small values of the wavevector to a thermal fluctuation dominated regime at large wavevector.Comment: 18 pages, 16 figures, animations available at http://www.physics.brown.edu/Users/faculty/pelcovits/lc/coarsening.htm

Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Serine⁶⁵

We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser(65) within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser(65) that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser(65) in ubiquitin lies in a similar motif to Ser(65) in the Ubl domain of Parkin. Remarkably, PlNK1 directly phosphorylates Ser(65) of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser(65) (Delta Ubl-Parkin) is robustly activated by ubiquitin(Phospho-Ser65), but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser(65) (Ubl(phospho-Ser65)) can also activate Delta Ubl-Parkin similarly to ubiquitin(PhosPh-Ser65). Thirdly, we establish that ubiquitin(PhosPh-Ser65), but not non-phosphorylated ubiquitin or Ubl(PhosPh-Ser65) activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser(65) and ubiquitin at Ser(65), since only mutation of both proteins at Ser(65) completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser(65), but also by phosphorylating ubiquitin at Ser(65). We propose that phosphorylation of Parkin at Ser(65) serves to prime the E3 ligase enzyme for activation by ubiquitin(PhosPh-Ser65), suggesting that small molecules that mimic ubiquitin(PhosPh-Ser65) could hold promise as novel therapies for Parkinson's disease

Crystal Structure of TDRD3 and Methyl-Arginine Binding Characterization of TDRD3, SMN and SPF30

SMN (Survival motor neuron protein) was characterized as a dimethyl-arginine binding protein over ten years ago. TDRD3 (Tudor domain-containing protein 3) and SPF30 (Splicing factor 30 kDa) were found to bind to various methyl-arginine proteins including Sm proteins as well later on. Recently, TDRD3 was shown to be a transcriptional coactivator, and its transcriptional activity is dependent on its ability to bind arginine-methylated histone marks. In this study, we systematically characterized the binding specificity and affinity of the Tudor domains of these three proteins quantitatively. Our results show that TDRD3 preferentially recognizes asymmetrical dimethylated arginine mark, and SMN is a very promiscuous effector molecule, which recognizes different arginine containing sequence motifs and preferentially binds symmetrical dimethylated arginine. SPF30 is the weakest methyl-arginine binder, which only binds the GAR motif sequences in our library. In addition, we also reported high-resolution crystal structures of the Tudor domain of TDRD3 in complex with two small molecules, which occupy the aromatic cage of TDRD3

Small Scattered Fragments Do Not a Dwarf Make: Biological and Archaeological Data Indicate that Prehistoric Inhabitants of Palau Were Normal Sized

Current archaeological evidence from Palau in western Micronesia indicates that the archipelago was settled around 3000–3300 BP by normal sized populations; contrary to recent claims, they did not succumb to insular dwarfism

Genomic epidemiology reveals multiple introductions of Zika virus into the United States

Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital abnormalities. In July 2016, mosquito-borne ZIKV transmission was reported in the continental United States; since then, hundreds of locally acquired infections have been reported in Florida. To gain insights into the timing, source, and likely route(s) of ZIKV introduction, we tracked the virus from its first detection in Florida by sequencing ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We show that at least 4 introductions, but potentially as many as 40, contributed to the outbreak in Florida and that local transmission is likely to have started in the spring of 2016-several months before its initial detection. By analysing surveillance and genetic data, we show that ZIKV moved among transmission zones in Miami. Our analyses show that most introductions were linked to the Caribbean, a finding corroborated by the high incidence rates and traffic volumes from the region into the Miami area. Our study provides an understanding of how ZIKV initiates transmission in new regions

Large-scale sequencing of SARS-CoV-2 genomes from one region allows detailed epidemiology and enables local outbreak management.

The COVID-19 pandemic has spread rapidly throughout the world. In the UK, the initial peak was in April 2020; in the county of Norfolk (UK) and surrounding areas, which has a stable, low-density population, over 3200 cases were reported between March and August 2020. As part of the activities of the national COVID-19 Genomics Consortium (COG-UK) we undertook whole genome sequencing of the SARS-CoV-2 genomes present in positive clinical samples from the Norfolk region. These samples were collected by four major hospitals, multiple minor hospitals, care facilities and community organizations within Norfolk and surrounding areas. We combined clinical metadata with the sequencing data from regional SARS-CoV-2 genomes to understand the origins, genetic variation, transmission and expansion (spread) of the virus within the region and provide context nationally. Data were fed back into the national effort for pandemic management, whilst simultaneously being used to assist local outbreak analyses. Overall, 1565 positive samples (172 per 100 000 population) from 1376 cases were evaluated; for 140 cases between two and six samples were available providing longitudinal data. This represented 42.6 % of all positive samples identified by hospital testing in the region and encompassed those with clinical need, and health and care workers and their families. In total, 1035 cases had genome sequences of sufficient quality to provide phylogenetic lineages. These genomes belonged to 26 distinct global lineages, indicating that there were multiple separate introductions into the region. Furthermore, 100 genetically distinct UK lineages were detected demonstrating local evolution, at a rate of ~2 SNPs per month, and multiple co-occurring lineages as the pandemic progressed. Our analysis: identified a discrete sublineage associated with six care facilities; found no evidence of reinfection in longitudinal samples; ruled out a nosocomial outbreak; identified 16 lineages in key workers which were not in patients, indicating infection control measures were effective; and found the D614G spike protein mutation which is linked to increased transmissibility dominates the samples and rapidly confirmed relatedness of cases in an outbreak at a food processing facility. The large-scale genome sequencing of SARS-CoV-2-positive samples has provided valuable additional data for public health epidemiology in the Norfolk region, and will continue to help identify and untangle hidden transmission chains as the pandemic evolves.The sequencing costs were funded by the COVID-19 Genomics UK (COG-UK) Consortium which is supported by funding from the Medical Research Council (MRC) part of UK Research and Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute

Investigating Bacterial Sources of Toxicity as an Environmental Contributor to Dopaminergic Neurodegeneration

Parkinson disease (PD) involves progressive neurodegeneration, including loss of dopamine (DA) neurons from the substantia nigra. Select genes associated with rare familial forms of PD function in cellular pathways, such as the ubiquitin-proteasome system (UPS), involved in protein degradation. The misfolding and accumulation of proteins, such as α-synuclein, into inclusions termed Lewy Bodies represents a clinical hallmark of PD. Given the predominance of sporadic PD among patient populations, environmental toxins may induce the disease, although their nature is largely unknown. Thus, an unmet challenge surrounds the discovery of causal or contributory neurotoxic factors that could account for the prevalence of sporadic PD. Bacteria within the order Actinomycetales are renowned for their robust production of secondary metabolites and might represent unidentified sources of environmental exposures. Among these, the aerobic genera, Streptomyces, produce natural proteasome inhibitors that block protein degradation and may potentially damage DA neurons. Here we demonstrate that a metabolite produced by a common soil bacterium, S. venezuelae, caused DA neurodegeneration in the nematode, Caenorhabditis elegans, which increased as animals aged. This metabolite, which disrupts UPS function, caused gradual degeneration of all neuronal classes examined, however DA neurons were particularly vulnerable to exposure. The presence of DA exacerbated toxicity because neurodegeneration was attenuated in mutant nematodes depleted for tyrosine hydroxylase (TH), the rate-limiting enzyme in DA production. Strikingly, this factor caused dose-dependent death of human SH-SY5Y neuroblastoma cells, a dopaminergic line. Efforts to purify the toxic activity revealed that it is a highly stable, lipophilic, and chemically unique small molecule. Evidence of a robust neurotoxic factor that selectively impacts neuronal survival in a progressive yet moderate manner is consistent with the etiology of age-associated neurodegenerative diseases. Collectively, these data suggest the potential for exposures to the metabolites of specific common soil bacteria to possibly represent a contributory environmental component to PD

Rad21-Cohesin Haploinsufficiency Impedes DNA Repair and Enhances Gastrointestinal Radiosensitivity in Mice

Approximately half of cancer-affected patients receive radiotherapy (RT). The doses delivered have been determined upon empirical experience based upon average radiation responses. Ideally higher curative radiation doses might be employed in patients with genuinely normal radiation responses and importantly radiation hypersensitive patients would be spared the consequences of excessive tissue damage if they were indentified before treatment. Rad21 is an integral subunit of the cohesin complex, which regulates chromosome segregation and DNA damage responses in eukaryotes. We show here, by targeted inactivation of this key cohesin component in mice, that Rad21 is a DNA-damage response gene that markedly affects animal and cell survival. Biallelic deletion of Rad21 results in early embryonic death. Rad21 heterozygous mutant cells are defective in homologous recombination (HR)-mediated gene targeting and sister chromatid exchanges. Rad21+/− animals exhibited sensitivity considerably greater than control littermates when challenged with whole body irradiation (WBI). Importantly, Rad21+/− animals are significantly more sensitive to WBI than Atm heterozygous mutant mice. Since supralethal WBI of mammals most typically leads to death via damage to the gastrointestinal tract (GIT) or the haematopoietic system, we determined the functional status of these organs in the irradiated animals. We found evidence for GIT hypersensitivity of the Rad21 mutants and impaired bone marrow stem cell clonogenic regeneration. These data indicate that Rad21 gene dosage is critical for the ionising radiation (IR) response. Rad21 mutant mice thus represent a new mammalian model for understanding the molecular basis of irradiation effects on normal tissues and have important implications in the understanding of acute radiation toxicity in normal tissues