Protein Microarray On-Demand: A Novel Protein Microarray System

We describe a novel, simple and low-cost protein microarray strategy wherein the microarrays are generated by printing expression ready plasmid DNAs onto slides that can be converted into protein arrays on-demand. The printed expression plasmids serve dual purposes as they not only direct the synthesis of the protein of interest; they also serve to capture the newly synthesized proteins through a high affinity DNA-protein interaction. To accomplish this we have exploited the high-affinity binding (∼3–7×10 −13 M) of E. coli Tus protein to Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication. In our system, each protein of interest is synthesized as a Tus fusion protein and each expression construct directing the protein synthesis contains embedded Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for the newly synthesized Tus fusion protein. This “all DNA” microarray can be converted to a protein microarray on-demand without need for any additional capture reagent.

Long-Range Chromosome Organization in E. coli: A Site-Specific System Isolates the Ter Macrodomain

The organization of the Escherichia coli chromosome into a ring composed of four macrodomains and two less-structured regions influences the segregation of sister chromatids and the mobility of chromosomal DNA. The structuring of the terminus region (Ter) into a macrodomain relies on the interaction of the protein MatP with a 13-bp target called matS repeated 23 times in the 800-kb-long domain. Here, by using a new method that allows the transposition of any chromosomal segment at a defined position on the genetic map, we reveal a site-specific system that restricts to the Ter region a constraining process that reduces DNA mobility and delays loci segregation. Remarkably, the constraining process is regulated during the cell cycle and occurs only when the Ter MD is associated with the division machinery at mid-cell. The change of DNA properties does not rely on the presence of a trans-acting mechanism but rather involves a cis-effect acting at a long distance from the Ter region. Two specific 12-bp sequences located in the flanking Left and Right macrodomains and a newly identified protein designated YfbV conserved with MatP through evolution are required to impede the spreading of the constraining process to the rest of the chromosome. Our results unravel a site-specific system required to restrict to the Ter region the consequences of anchoring the Ter MD to the division machinery

Replisome speed determines the efficiency of the Tus-Ter replication termination barrier

In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites1. Escherichia coli sets a \u27replication fork trap\u27 that allows the first arriving fork to enter but not to leave the terminus region2, 3, 4, 5. The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction3, 4, 5, 6, 7. However, the efficiency of fork blockage by Tus-Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro8, 9. Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus-Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first example where intrinsic differences in rates of individual replisomes have different biological outcomes

Spondylodiscitis: standards of current treatment

Background: Spinal infections are an important clinical problem that often require aggressive medical therapy, and sometimes even surgery. Known risk factors are advanced age, diabetes mellitus, rheumatoid arthritis, immunosuppression, alcoholism, long-term steroid use, concomitant infections, poly-trauma, malignant tumor, and previous surgery or invasive procedures (discography, chemonucleolysis, and surgical procedures involving or adjacent to the intervertebral disc space). The most common level of involvement is at the lumbar spine, followed by the thoracic, cervical and sacral levels: lesions at the thoracic spine tend to lead more frequently to neurological symptoms. Objective: The aim of the current paper is to describe current evidence-based standards of therapy in the management of SD by emphasizing pharmacological therapy and principles and indications for bracing and surgery. Methods: A PubMed and Google Scholar search using various forms and combinations of the key words: spondylodiscitis, spine, infection, therapy, surgery, radiology, treatment. Reference citations from publications identified in the literature search were reviewed. Publications highlighted in this article were extracted based on relevancy to established, putative, and emerging diagnostic and therapeutic standards, either conservative (antibiotic therapy and bracing) or surgical. Findings: To date, conservative therapy, based on targeted antibiotic therapy plus bracing, represents the mainstay in the management of SD. Proper diagnosis and tailored therapy can improve clinical results and decrease the chance of failure. Surgery should be an option only for patients with complications of this disease, namely deformity, neural compression and neurological compromise. Current standards in the setting of SD are continuously evolving, as can be seen in the recent advances in the field of radiological diagnostics, and the use of growth factors and cell-therapy strategies to promote infection eradication and bone healing after surgery. © 2012 Informa UK Ltd All rights reserve