Pathway crosstalk effects: shrinkage and disentanglement using a Bayesian hierarchical model

Identifying the biological pathways that are related to various clinical phenotypes is an important concern in biomedical research. Based on estimated expression levels and/or p values, overrepresentation analysis (ORA) methods provide rankings of pathways, but they are tainted because pathways overlap. This crosstalk phenomenon has not been rigorously studied and classical ORA does not take into consideration: (1) that crosstalk effects in cases of overlapping pathways can cause incorrect rankings of pathways, (2) that crosstalk effects can cause both excess type I errors and type II errors, (3) that rankings of small pathways are unreliable, and (4) that type I error rates can be inflated due to multiple comparisons of pathways. We develop a Bayesian hierarchical model that addresses these problems, providing sensible estimates and rankings, and reducing error rates. We show, on both real and simulated data, that the results of our method are more accurate than the results produced by the classical overrepresentation analysis, providing a better understanding of the underlying biological phenomena involved in the phenotypes under study. The R code and the binary datasets for implementing the analyses described in this article are available online at: http://www.eng.wayne.edu/page.php?id=6402.     

A comparison of anti-HER2 IgA and IgG1 in vivo efficacy is facilitated by high N-glycan sialylation of the IgA

Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece - coinciding with the loss of high-mannose type N-glycan species - as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated anti-proliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels.     

In vitro test systems supporting the development of improved pest control methods: a case study with chemical mixtures and bivalve biofoulers

Using the bivalve macrofouler Corbicula fluminea, the suitability of in vitro testing as a stepping stone towards the improvement of control methods based on chemical mixtures was addressed in this study. In vitro cholinesterase (ChE) activity inhibition following single exposure of C. fluminea tissue to four model chemicals (the organophosphates dimethoate and dichlorvos, copper and sodium dodecyl phosphate [SDS]) was first assessed. Consequently, mixtures of dimethoate with copper and dichlorvos with SDS were tested and modelled; mixtures with ChE revealed synergistic interactions for both chemical pairs. These synergic combinations were subsequently validated in vivo and the increased control potential of these selected combinations was verified, with gains of up to 50% in C. fluminea mortality relative to corresponding single chemical treatments. Such consistency supports the suitability of using time- and cost-effective surrogate testing platforms to assist the development of biofouling control strategies incorporating mixtures.     

An African Ancestry-Specific Allele of CTLA4 Confers Protection against Rheumatoid Arthritis in African Americans

Cytotoxic T-lymphocyte associated protein 4 (CTLA4) is a negative regulator of T-cell proliferation. Polymorphisms in CTLA4 have been inconsistently associated with susceptibility to rheumatoid arthritis (RA) in populations of European ancestry but have not been examined in African Americans. The prevalence of RA in most populations of European and Asian ancestry is ~1.0%; RA is purportedly less common in black Africans, with little known about its prevalence in African Americans. We sought to determine if CTLA4 polymorphisms are associated with RA in African Americans. We performed a 2-stage analysis of 12 haplotype tagging single nucleotide polymorphisms (SNPs) across CTLA4 in a total of 505 African American RA patients and 712 African American controls using Illumina and TaqMan platforms. The minor allele (G) of the rs231778 SNP was 0.054 in RA patients, compared to 0.209 in controls (4.462×10⁻²⁶, Fisher's exact). The presence of the G allele was associated with a substantially reduced odds ratio (OR) of having RA (AG+GG genotypes vs. AA genotype, OR 0.19, 95% CI: 0.13–0.26, p=2.4×10⁻²⁸, Fisher's exact), suggesting a protective effect. This SNP is polymorphic in the African population (minor allele frequency [MAF] 0.09 in the Yoruba population), but is very rare in other groups (MAF=0.002 in 530 Caucasians genotyped for this study). Markers associated with RA in populations of European ancestry (rs3087243 [+60C/T] and rs231775 [+49A/G]) were not replicated in African Americans. We found no confounding of association for rs231778 after stratifying for the HLA-DRB1 shared epitope, presence of anti-cyclic citrullinated peptide antibody, or degree of admixture from the European population. An African ancestry-specific genetic variant of CTLA4 appears to be associated with protection from RA in African Americans. This finding may explain, in part, the relatively low prevalence of RA in black African populations.     

Substrate utilization by Legionella cells after cryopreservation in phosphate buffer.

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.     

Substrate utilization by Campylobacter jejuni and Campylobacter coli.

An attempt was made to elucidate in Campylobacter spp. some of the physiologic characteristics that are reflected in the kinetics of CO2 formation from four 14C-labeled substrates. Campylobacter jejuni and C. coli were grown in a biphasic medium, and highly motile spiral cells were harvested at 12 h. Of the media evaluated for use in the metabolic tests, minimal essential medium without glutamine, diluted with an equal volume of potassium sodium phosphate buffer (pH 7.2), provided the greatest stability and least competition with the substrates to be tested. The cells were incubated with 0.02 M glutamate, glutamine, alpha-ketoglutarate, or formate, or with concentrations of these substrates ranging from 0.0032 to 0.125 M. All four substrates were metabolized very rapidly by both species. A feature of many of these reactions, particularly obvious with alpha-ketoglutarate, was an immediate burst of CO2 production followed by CO2 evolution at a more moderate rate. These diphasic kinetics of substrate utilization were not seen in comparable experiments with Escherichia coli grown and tested under identical conditions. With C. jejuni, CO2 production from formate proceeded rapidly for the entire period of incubation. The rate of metabolism of glutamate, glutamine, and alpha-ketoglutarate by both species was greatly enhanced by increased substrate concentration. The approach to the study of the metabolism of campylobacters here described may be useful in detecting subtle changes in the physiology of cells as they are maintained past their logarithmic growth phase.     

Scanning electron microscopy of neurons isolated from the pedal disk and body column of Hydra

Segments of pedal disk and body column were cut from specimens of Hydra littoralis and separated into epidermis and gastrodermis, then macerated to isolate neurons for scanning electron microscopy. Bipolar and multipolar ganglion cells were present in both tissue layers, whereas sensory cells were found only in the gastrodermis. A single cilium projected from the perikaryon of some bipolar and multipolar ganglion cells; the cilium was long in the pedal disk ganglion cells and short in those from the body column. Ganglion cells from the pedal disk had short, thick processes, whereas those from the body column had long, thin neurites. Gastrodermal sensory cells were characterized as unipolar by the presence of an apical cilium near the perikaryon or as asymmetrical bipolar by the presence of a narrow neck region between the perikaryon and cilium. The axon was short in pedal disk sensory cells and long in those from the body column.     

Multiple opioid receptors: An examination of the dissociation of [3H]leucine enkephalin from rat brain membranes

The experiments reported in this paper address the hypothesis that [3H]leucine enkephalin labels both mu and delta receptors. As reported by other workers, this peptide dissociates from rat brain membranes in a biphasic manner. This is consistent with a two site binding model which hypothesizes that the peptide labels both opioid mu and delta receptors from which it dissociates at different rates. To test this hypothesis, we determined the dissociation of bound ligand from rat brain membranes incubated to equilibrium with [3H]leucine enkephalin in the absence and presence of 100 nM morphine. The data were not significantly different. We conclude that the biphasic off-kinetics of [3H]leucine enkephalin is not evidence for a two-site binding model.