Mechanics of the IL2RA Gene Activation Revealed by Modeling and Atomic Force Microscopy

Transcription implies recruitment of RNA polymerase II and transcription factors (TFs) by DNA melting near transcription start site (TSS). Combining atomic force microscopy and computer modeling, we investigate the structural and dynamical properties of the IL2RA promoter and identify an intrinsically negative supercoil in the PRRII region (containing Elf-1 and HMGA1 binding sites), located upstream of a curved DNA region encompassing TSS. Conformational changes, evidenced by time-lapse studies, result in the progressive positioning of curvature apex towards the TSS, likely facilitating local DNA melting. In vitro assays confirm specific binding of the General Transcription Factors (GTFs) TBP and TFIIB over TATA-TSS position, where an inhibitory nucleosome prevented preinitiation complex (PIC) formation and uncontrolled DNA melting. These findings represent a substantial advance showing, first, that the structural properties of the IL2RA promoter are encoded in the DNA sequence and second, that during the initiation process DNA conformation is dynamic and not static

Molecular Detection and Genotyping of Noroviruses

Noroviruses (NoVs) are a major cause of gastroenteritis worldwide in humans and animals and are known as very infectious viral agents. They are spread through feces and vomit via several transmission routes involving person-to-person contact, food, and water. Investigation of these transmission routes requires sensitive methods for detection of NoVs. As NoVs cannot be cultivated to date, detection of these viruses relies on the use of molecular methods such as (real-time) reverse transcriptase polymerase chain reaction (RT-PCR). Regardless of the matrix, detection of NoVs generally requires three subsequent steps: a virus extraction step, RNA purification, and molecular detection of the purified RNA, occasionally followed by molecular genotyping. The current review mainly focused on the molecular detection and genotyping of NoVs. The most conserved region in the genome of human infective NoVs is the ORF1/ORF2 junction and has been used as a preferred target region for molecular detection of NoVs by methods such as (real-time) RT-PCR, NASBA, and LAMP. In case of animal NoVs, broad range molecular assays have most frequently been applied for molecular detection. Regarding genotyping of NoVs, five regions situated in the polymerase and capsid genes have been used for conventional RT-PCR amplification and sequencing. As the expected levels of NoVs on food and in water are very low and inhibition of molecular methods can occur in these matrices, quality control including adequate positive and negative controls is an essential part of NoV detection. Although the development of molecular methods for NoV detection has certainly aided in the understanding of NoV transmission, it has also led to new problems such as the question whether low levels of human NoV detected on fresh produce and shellfish could pose a threat to public health. © 2012 Springer Science+Business Media New York

A Review of Known and Hypothetical Transmission Routes for Noroviruses

Human noroviruses (NoVs) are considered a worldwide leading cause of acute non-bacterial gastroenteritis. Due to a combination of prolonged shedding of high virus levels in feces, virus particle shedding during asymptomatic infections, and a high environmental persistence, NoVs are easily transmitted pathogens. Norovirus (NoV) outbreaks have often been reported and tend to affect a lot of people. NoV is spread via feces and vomit, but this NoV spread can occur through several transmission routes. While person-to-person transmission is without a doubt the dominant transmission route, human infective NoV outbreaks are often initiated by contaminated food or water. Zoonotic transmission of NoV has been investigated, but has thus far not been demonstrated. The presented review aims to give an overview of these NoV transmission routes. Regarding NoV person-to-person transmission, the NoV GII. 4 genotype is discussed in the current review as it has been very successful for several decades but reasons for its success have only recently been suggested. Both pre-harvest and post-harvest contamination of food products can lead to NoV food borne illness. Pre-harvest contamination of food products mainly occurs via contact with polluted irrigation water in case of fresh produce or with contaminated harvesting water in case of bivalve molluscan shellfish. On the other hand, an infected food handler is considered as a major cause of post-harvest contamination of food products. Both transmission routes are reviewed by a summary of described NoV food borne outbreaks between 2000 and 2010. A third NoV transmission route occurs via water and the spread of NoV via river water, ground water, and surface water is reviewed. Finally, although zoonotic transmission remains hypothetical, a summary on the bovine and porcine NoV presence observed in animals is given and the presence of human infective NoV in animals is discussed. © 2012 Springer Science+Business Media New York