Obesity and the Role of Short Duration Submaximal Work on Cardiovascular and Cerebral Hemodynamics

The objective of this study was to compare gas exchange, cardiac and cerebral hemodynamic responses between 10 non-obese and 10 obese men during submaximal work. With the increasing prevalence of obesity, there is a need to understand the impact of obesity on work-induced responses. Participants completed a step-wise incremental cycling until they reached 60% of their age-predicted maximum heart rate. Gas exchange, cardiac and pre-frontal cortex hemodynamic responses were simultaneously measured during rest, work, and recovery. The non-obese group reached ~43% of their predicted maximal aerobic capacity as compared to ~34% in the obese group, with the non-obese working at a relatively higher workload and for more duration than the obese. The obese had elevated baseline heart rate and reduced whole-body oxygen uptake per body weight at baseline and task termination. Other cardiac and cerebral responses, although increased from baseline, were similar between groups during submaximal effort. In the obese, during recovery oxygen uptake and heart-rate recovery were slowest; cardiac output and rate pressure product were greatest, and left ventricle ejection time was shortest. However, both groups exhibited similar cerebral hemodynamics during recovery. These finding imply that, irrespective of their low aerobic fitness, obesity does not impair myocardial performance and cerebrovascular function during graded submaximal work, however, recovery from a short duration of work was influenced by their fitness level. Since a majority of activities of daily living are performed at individual’s submaximal level, understanding influence of obesity on submaximal work is critical.     

Misfolded proteins, endoplasmic reticulum stress and neurodegeneration

The accumulation of misfolded proteins (e.g. mutant or damaged proteins) triggers cellular stress responses that protect cells against the toxic buildup of such proteins. However, prolonged stress due to the buildup of these toxic proteins induces specific death pathways. Dissecting these pathways should be valuable in understanding the pathogenesis of, and ultimately in designing therapy for, neurodegenerative diseases that feature misfolded proteins.     

Inhibition of hepatitis delta virus RNA editing by short inhibitory RNA-mediated knockdown of ADAR1 but not ADAR2 expression

Hepatitis delta virus (HDV) requires host RNA editing at the viral RNA amber/W site. Of the two host genes responsible for RNA editing via deamination of adenosines in double-stranded RNAs, short inhibitory RNA-mediated knockdown of host ADAR1 expression but not that of ADAR2 led to decreased HDV amber/W editing and virus production. Despite substantial sequence and structural variation among the amber/W sites of the three HDV genotypes, ADAR1a was primarily responsible for editing all three. We conclude that ADAR1 is primarily responsible for editing HDV RNA at the amber/W site during HDV infection.     

Increased RNA editing and inhibition of hepatitis delta virus replication by high-level expression of ADAR1 and ADAR2

Hepatitis delta virus (HDV) is a subviral human pathogen that uses specific RNA editing activity of the host to produce two essential forms of the sole viral protein, hepatitis delta antigen (HDAg). Editing at the amber/W site of HDV antigenomic RNA leads to the production of the longer form (HDAg-L), which is required for RNA packaging but which is a potent trans-dominant inhibitor of HDV RNA replication. Editing in infected cells is thought to be catalyzed by one or more of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). We examined the effects of increased ADAR1 and ADAR2 expression on HDV RNA editing and replication in transfected Huh7 cells. We found that both ADARs dramatically increased RNA editing, which was correlated with strong inhibition of HDV RNA replication. While increased HDAg-L production was the primary mechanism of inhibition, we observed at least two additional means by which ADARs can suppress HDV replication. High-level expression of both ADAR1 and ADAR2 led to extensive hyperediting at non-amber/W sites and subsequent production of HDAg variants that acted as trans-dominant inhibitors of HDV RNA replication. Moreover, we also observed weak inhibition of HDV RNA replication by mutated forms of ADARs defective for deaminase activity. Our results indicate that HDV requires highly regulated and selective editing and that the level of ADAR expression can play an important role: overexpression of ADARs inhibits HDV RNA replication and compromises virus viability.     

siRNA-based inhibition specific for mutant SOD1 with single nucleotide alternation in familial ALS, compared with ribozyme and DNA enzyme

In many of autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with SOD1 mutation, a missense point mutation may induce the disease by its gain of adverse property. Reduction of such a mutant protein expression is expected to improve the disease phenotype. Duplex of 21-nt RNA, known as siRNA, has recently emerged as a powerful tool to silence gene, but the sequence specificity and efficacies have not been fully studied in comparison with ribozyme and DNA enzyme. We could make the siRNA which recognized even a single nucleotide alternation and selectively suppress G93A SOD1 expression leaving wild-type SOD1 intact. In mammalian cells, the siRNA much more efficiently suppressed the expression of mutant SOD1 than ribozyme or DNA enzyme. Furthermore, these siRNAs could suppress cell death of Neuro2a induced by over-expression of mutant SOD1s with stress of proteasome inhibition. Our results support the feasibility of utilizing siRNA-based gene therapy of familial ALS with mutant SOD1.     

Comparative protein interactomics of neuroglobin and myoglobin

Neuroglobin is a hypoxia-inducible O(2) -binding protein with neuroprotective effects in cell and animal models of stroke and Alzheimer's disease. The mechanism underlying neuroglobin's cytoprotective action is unknown, although several possibilities have been proposed, including anti-oxidative and anti-apoptotic effects. We used affinity purification-mass spectrometry methods to identify neuroglobin-interacting proteins in normoxic and hypoxic murine neuronal (HN33) cell lysates, and to compare these interactions with those of a structurally and functionally related protein, myoglobin. We report that the protein interactomes of neuroglobin and myoglobin overlap substantially and are modified by hypoxia. In addition, neuroglobin-interacting proteins include partners consistent with both anti-oxidative and anti-apoptotic functions, as well as with a relationship to several neurodegenerative diseases.     

Sequence-specific biosensors report drug-induced changes in epigenetic silencing in living cells

Treatment with demethylating drugs can induce demethylation and reactivation of abnormally silenced tumor suppressor genes in cancer cells, but it can also induce potentially deleterious loss of methylation of repetitive elements. To enable the observation of unwanted drug effects related to loss of methylation of repetitive DNA, we have developed a novel biosensor capable of reporting changes in DNA accessibility via luminescence, in living cells. The biosensor design comprises two independent modules, each with a polydactyl zinc finger domain fused to a half intein and to a split-luciferase domain that can be joined by conditional protein splicing after binding to adjacent DNA targets. We show that an artificial zinc finger design specifically targeting DNA sequences near the promoter region of the L1PA2 subfamily of Line-1 retroelements is able to generate luminescent signals, reporting loss of epigenetic silencing and increased DNA accessibility of retroelements in human cells treated with the demethylating drugs decitabine or 5-azacytidine.     

Selection of a reference gene for studies on lipid‐related aquatic adaptations of toothed whales (Grampus griseus)

Toothed whales are one group of marine mammals that has developed special adaptations, such as echolocation for predation, to successfully live in a dynamic aquatic environment. Their fat metabolism may differ from that of other mammals because toothed whales have acoustic fats. Gene expression in the metabolic pathways of animals can change with respect to their evolution and environment. A real‐time quantitative polymerase chain reaction (RT‐qPCR) is a reliable technique for studying the relative expressions of genes. However, since the accuracy of RT‐qPCR data is totally dependent on the reference gene, the selection of the reference gene is an essential step. In this study, 10 candidate reference genes (ZC3H10, FTL, LGALS1, RPL27, GAPDH, FTH1, DCN, TCTP, NDUS5, and UBIM) were initially tested for amplification efficiency using RT‐qPCR. After excluding DCN, the remaining nine genes, which are nearly 100% efficient, were selected for the gene stability analysis. Stable reference genes across eight different fat tissue, liver, and muscle samples from Grampus griseus were identified by four algorithms, which were provided in Genorm, NormFinder, BestKeeper, and Delta CT. Finally, a RefFinder comprehensive ranking was performed based on the stability values, and the nine genes were ranked as follows: LGALS1 > FTL > GAPDH > ZC3H10 > FTH1 > NDUS5 > TCTP > RPL27 > UBIM. The LGALS1 and FTL genes were identified as the most stable novel reference genes. The third‐ranked gene, GAPDH, is a well‐known housekeeping gene for mammals. Ultimately, we suggest the use of LGALS1 as a reliable novel reference gene for genomics studies on the lipid‐related aquatic adaptations of toothed whales.     

Cholecystokinin-Induced Desensitization, Receptor Phosphorylation, and Internalization in the CHP212 Neuroblastoma Cell Line

Abstract: Agonist stimulation of cells often results in desensitization of the response, to protect the cell from overstimulation. We have previously shown that the type A cholecystokinin (CCK) receptor on the pancreatic acinar cell and on the model CHO-CCKR cell line undergoes desensitization in response to CCK, with receptor phosphorylation and internalization playing key roles. Although these mechanisms contribute in a cell-specific manner, no analogous information exists for the CCK receptor expressed on neuronal cells, where in vivo data demonstrate a particularly sensitive response to CCK. The present study was designed to explore CCK receptor desensitization in the CHP212 neuroblastoma cell line, focusing on inositol 1,4,5-trisphosphate (IP₃) responses to CCK and on recognized molecular and cellular mechanisms of desensitization. CCK promptly stimulated IP₃ responses in these cells, with hormonal responsiveness rapidly and completely desensitized. Both receptor phosphorylation and internalization were observed to occur, with the former occurring most rapidly and likely being responsible for the earliest desensitization observed. Although the time course of receptor phosphorylation and dephosphorylation, and the groups of kinases involved in the neuroblastoma cell line, were most similar to those in the pancreatic cell, the movement of the agonist-bound receptor in these cells was quite different from that in the pancreatic cell and most similar to that in the CHO-CCKR cell line. This hybrid response supports the cell-specific nature of CCK receptor regulation and provides an important system to explore the molecular determinants of these processes.     

Multivalent Binding of a Ligand-Coated Particle: Role of Shape,Size, and Ligand Heterogeneity

We utilize a multiscale modeling framework to study the effect of shape, size, and ligand composition on the efficacy of binding of a ligand-coated particle to a substrate functionalized with the target receptors. First, we show how molecular dynamics along with steered molecular dynamics calculations can be used to accurately parameterize the molecular-binding free energy and the effective spring constant for a receptor-ligand pair. We demonstrate this for two ligands that bind to the ${\alpha }_5{\beta }_1$-domain of integrin. Next, we show how these effective potentials can be used to build computational models at the meso- and continuum-scales. These models incorporate the molecular nature of the receptor-ligand interactions and yet provide an inexpensive route to study the multivalent interaction of receptors and ligands through the construction of Bell potentials customized to the molecular identities. We quantify the binding efficacy of the ligand-coated-particle in terms of its multivalency, binding free-energy landscape, and the losses in the configurational entropies. We show that 1) the binding avidity for particle sizes less than 350 nm is set by the competition between the enthalpic and entropic contributions, whereas that for sizes above 350 nm is dominated by the enthalpy of binding; 2) anisotropic particles display higher levels of multivalent binding compared to those of spherical particles; and 3) variations in ligand composition can alter binding avidity without altering the average multivalency. The methods and results presented here have wide applications in the rational design of functionalized carriers and also in understanding cell adhesion.