Small RNA identification in Enterobacteriaceae using synteny and genomic backbone retention

Genomic screens for small RNA candidates in Enterobacteriacae genomes were carried out with existing small RNA sequences, conserved flanking genes, and genomic backbone information. The small RNA sequences and contexts from E. coli K12 formed the basis of the search. Sequence identity identified 117 additional small RNA homologs in related genomes. Motifs of continuous sequence stretches added another 48 sRNA regions, termed partial homologs. However, this study is unique in identifying 160 nonhomologous sRNA loci in related genomes based on the conserved flanking gene synteny and the backbone retention information obtained from KEGG-SSDB. Gene synteny and genomic backbone continuity were observed to be correlated with all of the sRNAs in related genomes. This search is the first of its kind toward identification of functionally important regions using gene order and back-bone information. A disruption in flanking gene order or genomic backbone indicates a possible hotspot for alien gene pool integration. This study reports both occurrence of multiple copies of a sRNA and co-occurrence of different sRNAs between a pair of conserved flanking genes. In general, synteny and genomic backbone retention information can be added as additional search criteria toward the design of precise bioinformatics tools for sRNA, gene identification, and gene functional annotations in related genomes.     

How do antigenically varying pathogens avoid cross-reactive responses to invariant antigens?

Pathogens such as trypanosomes and malaria use antigenic variation to evade immune responses and prolong the duration of infections. As pathogens typically express more than one antigen, even relatively rare conserved antigens might be expected to trigger cross-reactive immune responses capable of clearing the infection. We use simple mathematical models that explicitly consider the dynamic interplay between the replicating pathogen, immune responses to different antigens and immune exhaustion to explore how pathogens can escape the responses to both variable and invariant (conserved) antigens. Our results suggest two hypotheses. In the first, limited quantities of invariant antigens on each pathogen may lead to saturation in killing by cross-reactive responses. In the second, antigenic variation of the dominant antigens prolongs the duration of infection sufficiently to allow for exhaustion of the cross-reactive responses to subdominant, invariant epitopes prior to their being able to control the infection. These hypotheses make distinct predictions: the former predicts that cross-reactive responses will always be ineffective while the latter predicts that appropriately timed treatment could, by preventing exhaustion, lead to the generation of long-lasting protective cross-reactive immunity and thus act similarly to a vaccine.     

Stimulation history dictates memory CD8 T cell phenotype: implications for prime-boost vaccination

Heterologous prime-boost vaccination results in increased frequencies of memory T cells. Although these quantitative effects of reexposure to Ag are well documented, little is known about the impact of boosting on the functional qualities of memory T cells. To address this critical issue, we have used three different types of immunization regimens and examined how boosting effects the function and anatomic location of memory CD8 T cells. We found that memory T cell phenotype differed substantially depending on the number of immunizations and that secondary and tertiary responses resulted in the generation of memory CD8 T cells that retained effector-like properties and showed preferential accumulation in nonlymphoid tissues. These results show that memory differentiation is coupled to the history of Ag experience and that prime-boost vaccination strategies have important consequences on memory CD8 T cell quality and surveillance within mucosal tissues.     

Rapid construction and characterization of synthetic antibody libraries without DNA amplification

We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V_H) and light (V_κ) libraries. Four high quality, chemically synthesized polynucleotides (90–140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10⁹ transformants could be synthesized within 1 day. Fusion to β‐lactamase and selection on ampicillin resulted in 3.7 × 10⁸ V_H and 6.9 × 10⁸ V_κ clones highly enriched for full‐length, in‐frame genes. High‐throughput 454 DNA sequencing of >250,000 V_H and V_κ genes from the pre‐ and post‐selection libraries revealed that, in addition to the expected reduction in reading‐frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V_H/V_κ‐β‐lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions. Biotechnol. Bioeng. 2010; 106: 347–357. © 2010 Wiley Periodicals, Inc.     

Predicting Odor Pleasantness with an Electronic Nose

A primary goal for artificial nose (eNose) technology is to report perceptual qualities of novel odors. Currently, however, eNoses primarily detect and discriminate between odorants they previously “learned”. We tuned an eNose to human odor pleasantness estimates. We then used the eNose to predict the pleasantness of novel odorants, and tested these predictions in naïve subjects who had not participated in the tuning procedure. We found that our apparatus generated odorant pleasantness ratings with above 80% similarity to average human ratings, and with above 90% accuracy at discriminating between categorically pleasant or unpleasant odorants. Similar results were obtained in two cultures, native Israeli and native Ethiopian, without retuning of the apparatus. These findings suggest that unlike in vision and audition, in olfaction there is a systematic predictable link between stimulus structure and stimulus pleasantness. This goes in contrast to the popular notion that odorant pleasantness is completely subjective, and may provide a new method for odor screening and environmental monitoring, as well as a critical building block for digital transmission of smell.     

Electroendocytosis: stimulation of adsorptive and fluid-phase uptake by pulsed low electric fields

We present a novel approach for stimulating uptake via endocytic pathways by exposing cells to a train of pulsed low electric fields (LEF) in the range of 2.5-20 V/cm. Electric field treatment of COS 5-7 and HaCaT cells in the presence of BSA-FITC augments the adsorption of the probe to plasma membranes with subsequent enhanced internalization. The uptake of BSA-FITC is maximal when the cells are exposed to LEF in the presence of the probe while uptake of a fluid-phase marker, propidium iodide (PI), is more effective when the probe is added immediately after termination of a 1-min exposure. LEF-stimulated uptake decays with a half-life of about 3 and 1 min for and BSA-FITC and PI, respectively. The uptake is inefficient at 4 degrees C but increases with temperature. The uptake proceeds via cell membrane vesiculation, showing a high extent of colocalization of BSA-FITC with plasma membrane vesicles labeled with a phospholipid fluorescent analogue. Unlike constitutive endocytosis where the BSA-FITC is exposed to acidic pH, in LEF-induced uptake the probe is exposed to the more alkaline pH of the cytosol. The staining kinetics of nuclear targets by PI reflects the release of the probe from the LEF-induced vesicles into the cytosol 1-3 h after exposure. The LEF-induced adsorptive pathway was approximately 2.5 more effective than the LEF-induced fluid-phase one. The observed 5- to 6-fold increase of BSA-FITC uptake induced by LEF may be partially attributed to a clathrin-dependent route (up to 25%), whereas the rest of the uptake may be assigned to macropinocytotic and clathrin/caveolin independent pathways or to a novel, yet unidentified, route driven by LEF. This study provides a basis for a general approach towards the efficient incorporation of a variety of molecules such as antibodies, enzymes or genes into cells.     

Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment

Chronic viral infections often result in ineffective CD8 T-cell responses due to functional exhaustion or physical deletion of virus-specific T cells. However, how persisting virus impacts various CD8 T-cell effector functions and influences other aspects of CD8 T-cell dynamics, such as immunodominance and tissue distribution, remains largely unknown. Using different strains of lymphocytic choriomeningitis virus (LCMV), we compared responses to the same CD8 T-cell epitopes during acute or chronic infection. Persistent infection led to a disruption of the normal immunodominance hierarchy of CD8 T-cell responses seen following acute infection and dramatically altered the tissue distribution of LCMV-specific CD8 T cells in lymphoid and nonlymphoid tissues. Most importantly, CD8 T-cell functional impairment occurred in a hierarchical fashion in chronically infected mice. Production of interleukin 2 and the ability to lyse target cells in vitro were the first functions compromised, followed by the ability to make tumor necrosis factor alpha, while gamma interferon production was most resistant to functional exhaustion. Antigen appeared to be the driving force for this loss of function, since a strong correlation existed between the viral load and the level of exhaustion. Further, epitopes presented at higher levels in vivo resulted in physical deletion, while those presented at lower levels induced functional exhaustion. A model is proposed in which antigen levels drive the hierarchical loss of different CD8 T-cell effector functions during chronic infection, leading to distinct stages of functional impairment and eventually to physical deletion of virus-specific T cells. These results have implications for the study of human chronic infections, where similar T-cell deletion and functional dysregulation has been observed.