Pdx1 Is Post-Translationally Modified In vivo and Serine 61 Is the Principal Site of Phosphorylation

Maintaining sufficient levels of Pdx1 activity is a prerequisite for proper regulation of blood glucose homeostasis and beta cell function. Mice that are haploinsufficient for Pdx1 display impaired glucose tolerance and lack the ability to increase beta cell mass in response to decreased insulin signaling. Several studies have shown that post-translational modifications are regulating Pdx1 activity through intracellular localization and binding to co-factors. Understanding the signaling cues converging on Pdx1 and modulating its activity is therefore an attractive approach in diabetes treatment. We employed a novel technique called Nanofluidic Proteomic Immunoassay to characterize the post-translational profile of Pdx1. Following isoelectric focusing in nano-capillaries, this technology relies on a pan specific antibody for detection and it therefore allows the relative abundance of differently charged protein species to be examined simultaneously. In all eukaryotic cells tested we find that the Pdx1 protein separates into four distinct peaks whereas Pdx1 protein from bacteria only produces one peak. Of the four peaks in eukaryotic cells we correlate one of them to a phosphorylation Using alanine scanning and mass spectrometry we map this phosphorylation to serine 61 in both Min6 cells and in exogenous Pdx1 over-expressed in HEK293 cells. A single phosphorylation is also present in cultured islets but it remains unaffected by changes in glucose levels. It is present during embryogenesis but is not required for pancreas development

Gene Expression Profile of Neuronal Progenitor Cells Derived from hESCs: Activation of Chromosome 11p15.5 and Comparison to Human Dopaminergic Neurons

BACKGROUND: We initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription. METHODOLOGY: Dopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined. PRINCIPAL FINDINGS: A number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture. CONCLUSIONS: The present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons

Conserved Genes Act as Modifiers of Invertebrate SMN Loss of Function Defects

Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species

DNA methylation and methyl-CpG binding proteins: developmental requirements and function

DNA methylation is a major epigenetic modification in the genomes of higher eukaryotes. In vertebrates, DNA methylation occurs predominantly on the CpG dinucleotide, and approximately 60% to 90% of these dinucleotides are modified. Distinct DNA methylation patterns, which can vary between different tissues and developmental stages, exist on specific loci. Sites of DNA methylation are occupied by various proteins, including methyl-CpG binding domain (MBD) proteins which recruit the enzymatic machinery to establish silent chromatin. Mutations in the MBD family member MeCP2 are the cause of Rett syndrome, a severe neurodevelopmental disorder, whereas other MBDs are known to bind sites of hypermethylation in human cancer cell lines. Here, we review the advances in our understanding of the function of DNA methylation, DNA methyltransferases, and methyl-CpG binding proteins in vertebrate embryonic development. MBDs function in transcriptional repression and long-range interactions in chromatin and also appear to play a role in genomic stability, neural signaling, and transcriptional activation. DNA methylation makes an essential and versatile epigenetic contribution to genome integrity and function

Evidence-based Kernels: Fundamental Units of Behavioral Influence

This paper describes evidence-based kernels, fundamental units of behavioral influence that appear to underlie effective prevention and treatment for children, adults, and families. A kernel is a behavior–influence procedure shown through experimental analysis to affect a specific behavior and that is indivisible in the sense that removing any of its components would render it inert. Existing evidence shows that a variety of kernels can influence behavior in context, and some evidence suggests that frequent use or sufficient use of some kernels may produce longer lasting behavioral shifts. The analysis of kernels could contribute to an empirically based theory of behavioral influence, augment existing prevention or treatment efforts, facilitate the dissemination of effective prevention and treatment practices, clarify the active ingredients in existing interventions, and contribute to efficiently developing interventions that are more effective. Kernels involve one or more of the following mechanisms of behavior influence: reinforcement, altering antecedents, changing verbal relational responding, or changing physiological states directly. The paper describes 52 of these kernels, and details practical, theoretical, and research implications, including calling for a national database of kernels that influence human behavior

Breast-cancer-secreted miR-122 reprograms glucose metabolism in premetastatic niche to promote metastasis

Reprogrammed glucose metabolism as a result of increased glycolysis and glucose uptake is a hallmark of cancer. Here we show that cancer cells can suppress glucose uptake by non-tumour cells in the pre-metastatic niche, by secreting vesicles that carry high levels of the miR-122 microRNA. High miR-122 levels in the circulation have been associated with metastasis in breast cancer patients and we show that cancer-cell-secreted miR-122 facilitates metastasis by increasing nutrient availability in the pre-metastatic niche. Mechanistically cancer-cell-derived miR-122 suppresses glucose uptake by niche cells in vitro and in vivo by downregulating the glycolytic enzyme pyruvate kinase (PKM). In vivo inhibition of miR-122 restores glucose uptake in distant organs, including brain and lungs, and decreases the incidence of metastasis. These results demonstrate that by modifying glucose utilization by recipient pre-metastatic niche cells, cancer-derived extracellular miR-122 is able to reprogram systemic energy metabolism to facilitate disease progression

Assessing transmissibility of SARS-CoV-2 lineage B.1.1.7 in England

The SARS-CoV-2 lineage B.1.1.7, designated variant of concern (VOC) 202012/01 by Public Health England1, was first identified in the UK in late summer to early autumn 20202. Whole-genome SARS-CoV-2 sequence data collected from community-based diagnostic testing for COVID-19 show an extremely rapid expansion of the B.1.1.7 lineage during autumn 2020, suggesting that it has a selective advantage. Here we show that changes in VOC frequency inferred from genetic data correspond closely to changes inferred by S gene target failures (SGTF) in community-based diagnostic PCR testing. Analysis of trends in SGTF and non-SGTF case numbers in local areas across England shows that B.1.1.7 has higher transmissibility than non-VOC lineages, even if it has a different latent period or generation time. The SGTF data indicate a transient shift in the age composition of reported cases, with cases of B.1.1.7 including a larger share of under 20-year-olds than non-VOC cases. We estimated time-varying reproduction numbers for B.1.1.7 and co-circulating lineages using SGTF and genomic data. The best-supported models did not indicate a substantial difference in VOC transmissibility among different age groups, but all analyses agreed that B.1.1.7 has a substantial transmission advantage over other lineages, with a 50% to 100% higher reproduction number

The Interaction of the Antitoxin DM43 with a Snake Venom Metalloproteinase Analyzed by Mass Spectrometry and Surface Plasmon Resonance

DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non-covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry (MS) and surface plasmon resonance (SPR) to improve the molecular characterization of this heterocomplex. The stoichiometry of the interaction was confirmed by nanoelectrospray ionization-quadrupole-time-of-flight MS; from native solution conditions, the complex showed a molecular mass of ~94 kDa, indicating that one molecule of jararhagin (50 kDa) interacts with one monomer of DM43 (43 kDa). Although readily observed in solution, the dimeric structure of the inhibitor was barely preserved in the gas phase. This result suggests that, in contrast to the toxin-antitoxin complex, hydrophobic interactions are the primary driving force for the inhibitor dimerization. For the real-time interaction analysis, the toxin was captured on a sensor chip derivatized with the anti-jararhagin monoclonal antibody MAJar 2. The sensorgrams obtained after successive injections of DM43 in a concentration series were globally fitted to a simple bimolecular interaction, yielding the following kinetic rates for the DM43/jararhagin interaction: k(a) = 3.54 ± 0.03 × 10(4) M(-1) s(-1) and k(d) = 1.16 ± 0.07 × 10(-5) s(-1), resulting in an equilibrium dissociation constant (K(D) ) of 0.33 ± 0.06 nM. Taken together, MS and SPR results show that DM43 binds to its target toxin with high affinity and constitute the first accurate quantitative study on the extent of the interaction between a natural inhibitor and a metalloproteinase toxin, with unequivocal implications for the use of this kind of molecule as template for the rational development of novel antivenom therapies