Endo-Porter-mediated delivery of phosphorodiamidate morpholino oligos (PMOs) in erythrocyte suspension cultures from Cope's gray treefrog Hyla chrysoscelis

Cope's gray treefrog, Hyla chrysoscelis, is a freeze-tolerant anuran that accumulates cryoprotective glycerol during cold acclimation. H. chrysoscelis erythrocytes express the aquaglyceroporin HC-3, which facilitates transmembrane glycerol and water movement. Aquaglyceroporins have no pharmacological inhibitors, and no genetic knockout tools currently exist for H. chrysoscelis. A phosphorodiamidate morpholino oligo (PMO)-mediated expression knockdown approach was therefore pursued to provide a model for testing the role of HC-3. We describe a novel procedure optimized for specific, efficient knockdown of HC-3 expression in amphibian erythrocyte suspensions cultured at nonmammalian physiological temperatures using Endo-Porter. Our protocol includes three critical components: pre-incubation at 37°C, two rounds of Endo-Porter and HC-3 PMO administration at ~23°C, and continuous shaking at 190 rpm. This combination of steps resulted in 94% reduction in HC-3 protein expression (Western blot), substantial decrease in HC-3 expression in >65% of erythrocytes, and no detectable expression in an additional 30% of cells (immunocytochemistry).     

Predicting the epidemic sizes of influenza A/H1N1, A/H3N2, and B: a statistical method

Background

The epidemic sizes of influenza A/H3N2, A/H1N1, and B infections vary from year to year in the United States. We use publicly available US Centers for Disease Control (CDC) influenza surveillance data between 1997 and 2009 to study the temporal dynamics of influenza over this period.

Methods and Findings

Regional outpatient surveillance data on influenza-like illness (ILI) and virologic surveillance data were combined to define a weekly proxy for the incidence of each strain in the United States. All strains exhibited a negative association between their cumulative incidence proxy (CIP) for the whole season (from calendar week 40 of each year to calendar week 20 of the next year) and the CIP of the other two strains (the complementary CIP) from the start of the season up to calendar week 2 (or 3, 4, or 5) of the next year. We introduce a method to predict a particular strain''s CIP for the whole season by following the incidence of each strain from the start of the season until either the CIP of the chosen strain or its complementary CIP exceed certain thresholds. The method yielded accurate predictions, which generally occurred within a few weeks of the peak of incidence of the chosen strain, sometimes after that peak. For the largest seasons in the data, which were dominated by A/H3N2, prediction of A/H3N2 incidence always occurred at least several weeks in advance of the peak.

Conclusion

Early circulation of one influenza strain is associated with a reduced total incidence of the other strains, consistent with the presence of interference between subtypes. Routine ILI and virologic surveillance data can be combined using this new method to predict the relative size of each influenza strain''s epidemic by following the change in incidence of a given strain in the context of the incidence of cocirculating strains. Please see later in the article for the Editors'' Summary     

Infection with a novel gammaherpesvirus in northern elephant seals (Mirounga angustirostris)

Twenty juvenile northern elephant seals (Mirounga angustirostris) that died between 1998 and 2004 had ulcers on the tongue, palatine mucosa, and/or tonsils. Histologic examination of the lesions revealed cytoplasmic swelling, nuclear pyknosis, and eosinophilic to amphophilic intranuclear inclusions bodies suggestive of herpesviral infection. Electron microscopic examination and polymerase chain reaction analysis confirmed the presence of a herpesvirus. Subsequent DNA sequencing identified this to be a new gammaherpesvirus that was similar to Porcine lymphotropic virus 2, Alcephaline herpesvirus 1 (malignant catarrhal fever virus from wildebeest), and Chlorocebus rhadinovirus 1 from African green monkeys. Identical herpesviral DNA was also detected in blood and mucosal swabs collected from five healthy elephant seal pups.     

Population genomics: linkage disequilibrium holds the key

Current efforts to find disease-causing genes depend on patterns of linkage disequilibrium in human populations. Recent work has shown that linkage disequilibrium can extend over much larger genomic regions than expected, and that the patterns of linkage disequilibrium can differ markedly among populations.     

Molecular characterization of Ascaris suum DNA and of chromatin diminution

A technique for the extraction of pure somatic (post-diminution) and germ line (pre-diminution) DNA from the parasitic nematode Ascaris is described. Uncontaminated post- and pre-diminution DNAs were sheared and reassociated to different C₀t values. Computer analysis of the complete reassociation kinetics determined that 33% of the germ line genome is eliminated during the process of chromatin diminution. The eliminated DNA is comprised of repetitive and unique sequences in an approx. 1:1 ratio.     

Benign synthesis of 2-ethylhexanoic acid by cytochrome P450cam: enzymatic, crystallographic, and theoretical studies

This study examines the ability of P450cam to catalyze the formation of 2-ethylhexanoic acid from 2-ethylhexanol relative to its activity on the natural substrate camphor. As is the case for camphor, the P450cam exhibits stereoselectivity for binding (R)- and (S)-2-ethylhexanol. Kinetic studies indicate (R)-2-ethylhexanoic acid is produced 3.5 times as fast as the (S)-enantiomer. In a racemic mixture of 2-ethylhexanol, P450cam produces 50% more (R)-2-ethylhexanoic acid than (S)-2-ethylhexanoic acid. The reason for stereoselective 2-ethylhexanoic acid production is seen in regioselectivity assays, where (R)-2-ethylhexanoic acid comprises 50% of total products while (S)-2-ethylhexanoic acid comprises only 13%. (R)- and (S)-2-ethylhexanol exhibit similar characteristics with respect to the amount of oxygen and reducing equivalents consumed, however, with (S)-2-ethylhexanol turnover producing more water than the (R)-enantiomer. Crystallographic studies of P450cam with (R)- or (S)-2-ethylhexanoic acid suggest that the (R)-enantiomer binds in a more ordered state. These results indicate that wild-type P450cam displays stereoselectivity toward 2-ethylhexanoic acid synthesis, providing a platform for rational active site design.     

Characterization of a T4+/Leu-8+ T cell clone that directly helps B cell Ig production by secreting B cell differentiation factor

A human T4+/Leu-8+ T cell clone (YA2) was established by phytohemagglutinin activation and interleukin 2 (IL 2) propagation. Functional characterization of this clone demonstrated that it provided potent help towards Ig production by pokeweed mitogen-stimulated B cells in the presence of small numbers of autologous T cells or by Staphylococcus aureus Cowan I (SAC)-activated B cells in the presence of B cell growth factor (BCGF). YA2 provided no help to resting B cells and minimal help to either unactivated B cells cultured with BCGF or SAC-activated B cells. Supernatant generated from clone YA2 by IL 2 stimulation had significant B cell differentiation activity but no BCGF or IL 2 activity. Thus, YA2 is a T4+/Leu-8+ potent direct helper only to B cells that are activated and proliferating due to its selective secretion of a differentiation factor, and not an activation and growth factor. The availability of phenotypically defined cloned populations of T cells with restricted functional helper activity related to the secretion of selected B cell tropic factors should prove useful in the dissection of the role of individual T cell subsets in the regulation of the human B cell cycle.     

Treatment-Induced Changes in Plasma Adiponectin Do Not Reduce Urinary Albumin Excretion in the Diabetes Prevention Program Cohort

Background and Objectives

Molecular data suggests that adiponectin may directly regulate urinary albumin excretion. In the Diabetes Prevention Program (DPP) we measured adiponectin and albuminuria before and after intervention, and we previously reported increases in adiponectin with interventions. Here we have used the DPP dataset to test the hypothesis that treatment-related increases in adiponectin may reduce albuminuria in obesity.

Design, Setting, Participants and Methods

We evaluated cross-sectional correlations between plasma adiponectin and urinary albumin excretion at baseline, and the relationship of treatment-related changes in adiponectin and albuminuria. Baseline and follow-up urine albumin to creatinine ratios (ACR (albumin to creatinine ratio)) and plasma adiponectin concentration were available in 2553 subjects.

Results

Adjusting for age, sex and race/ethnicity, we observed a statistically significant but weak inverse relationship between adiponectin and ACR at baseline (conditional Spearman’s rho = (-) 0.04, p = 0.04). Although DPP treatments significantly increased plasma adiponectin, there were no treatment effects on ACR and no differences in ACR across treatment groups. There was a weak direct (not inverse) association between change in adiponectin and change in albuminuria (adjusted Spearman’s rho = (+) 0.04, p = 0.03).

Conclusions

In a large, well-characterized cohort of obese dysglycemic subjects we observed a weak inverse association between circulating adiponectin concentrations and urinary albumin excretion at baseline. Contrary to the hypothesized effect, treatment-related increases in plasma adiponectin were not associated with a reduction in ACR. The association of change in adiponectin with change in ACR should be assessed in populations with overt albuminuria before excluding a beneficial effect of increasing adiponectin to reduce ACR in obesity.