Structure and function of primase RepB′ encoded by broad-host-range plasmid RSF1010 that replicates exclusively in leading-strand mode

For the initiation of DNA replication, dsDNA is unwound by helicases. Primases then recognize specific sequences on the template DNA strands and synthesize complementary oligonucleotide primers that are elongated by DNA polymerases in leading- and lagging-strand mode. The bacterial plasmid RSF1010 provides a model for the initiation of DNA replication, because it encodes the smallest known primase RepB′ (35.9 kDa), features only 1 single-stranded primase initiation site on each strand (ssiA and ssiB, each 40 nt long with 5′- and 3′-terminal 6 and 13 single-stranded nucleotides, respectively, and nucleotides 7–27 forming a hairpin), and is replicated exclusively in leading strand mode. We present the crystal structure of full-length dumbbell-shaped RepB′ consisting of an N-terminal catalytic domain separated by a long α-helix and tether from the C-terminal helix-bundle domain and the structure of the catalytic domain in a specific complex with the 6 5′-terminal single-stranded nucleotides and the C7–G27 base pair of ssiA, its single-stranded 3′-terminus being deleted. The catalytic domains of RepB′ and the archaeal/eukaryotic family of Pri-type primases share a common fold with conserved catalytic amino acids, but RepB′ lacks the zinc-binding motif typical of the Pri-type primases. According to complementation studies the catalytic domain shows primase activity only in the presence of the helix-bundle domain. Primases that are highly homologous to RepB′ are encoded by broad-host-range IncQ and IncQ-like plasmids that share primase initiation sites ssiA and ssiB and high sequence identity with RSF1010.     

In vitro assembly of relaxosomes at the transfer origin of plasmid RP4.

During initiation of conjugative transfer of DNA containing the transfer origin (oriT) of the promiscuous plasmid RP4, the proteins TraI, TraJ, and TraH interact and assemble a specialized nucleoprotein complex (the relaxosome) at oriT. The structure can be visualized on electron micrographs. Site- and strand-specific nicking at the transfer origin in vitro is dependent on the proteins TraI and TraJ and on Mg2+ ions. Substrate specificity is directed exclusively towards the cognate transfer origin: the RP4-specified TraJ protein cannot recognize the closely related oriT of plasmid R751. After nicking, TraI protein remains attached to the 5'-terminal 2'-deoxycytidyl residue at the nic site [Pansegrau, W., Ziegelin, G. & Lanka, E. (1990) J. Biol. Chem. 265, 10637-10644]. Nicking and relaxosome formation require supercoiled DNA. Thus, a complicated structure involving multiple plasmid-specified proteins and a defined region of DNA must be formed at the transfer origin to prepare the plasmid for generating the single strand to be transferred.     

Relaxase (TraI) of IncP alpha plasmid RP4 catalyzes a site-specific cleaving-joining reaction of single-stranded DNA.

Conjugative DNA transfer of the self-transmissible broad-host-range plasmid RP4 is initiated by strand- and site-specific cleavage at the nick site (nic) of the transfer origin (oriT). Cleavage results in covalent attachment of the plasmid-encoded relaxase (TraI) to the 5'-terminal 2'-deoxycytidine residue at nic. We demonstrate that Tyr22 is the center of the catalytic site of TraI, mediating cleavage via formation of a phosphodiester between the DNA 5' phosphoryl and the aromatic hydroxyl group. The specificity of cleavage seen with form I oriT DNA was verified with short oligodeoxy-ribonucleotides embracing the nick region. The reaction requires TraI and Mg2+ but is independent of the relaxosome component TraJ. Cleavage produces one oligonucleotide fragment with a free 3' hydroxyl, the other part forms a covalent TraI-oligonucleotide adduct. Like nicking of form I oriT DNA, TraI-catalyzed oligonucleotide cleavage reaches an equilibrium when about 30% of the input TraI exists as a covalent protein-DNA complex. In the presence of two differently sized oligonucleotides, defined hybrid oligonucleotides are produced, demonstrating that TraI catalyzes recombination of two single strands at nic. This finding shows that TraI possesses cleaving-joining activity resembling that of a type I topoisomerase. Reactions are dependent on the sequence of the 3'-terminal 6 nucleotides adjacent to nic. Only certain base changes in a few positions are tolerated, whereas the sequence of the 5' terminal nucleotides apparently is irrelevant for recognition by TraI. The reactions described here further support the hypothesis that DNA transfer via conjugation involves a rolling circle-like mechanism which generates the immigrant single strand while DNA-bound TraI protein scans for the occurrence of a second cleavage site at the donor-recipient interface.     

Compare Display Schemes for Lung Nodule CT Screening

This study investigated the relative efficiencies of a stereographic display and two monoscopic display schemes for detecting lung nodules in chest computed tomography (CT). The ultimate goal was to determine whether stereoscopic display provides advantages for visualization and interpretation of three-dimensional (3D) medical image datasets. A retrospective study that compared lung nodule detection performances achieved using three different schemes for displaying 3D CT data was conducted. The display modes included slice-by-slice, orthogonal maximum intensity projection (MIP), and stereoscopic display. One hundred lung-cancer screening CT examinations containing 647 nodules were interpreted by eight radiologists, in each of the display modes. Reading times and displayed slab thickness versus time were recorded, as well as the probability, location, and size for each detected nodule. Nodule detection performance was analyzed using the receiver operating characteristic method. The stereo display mode provided higher detection performance with a shorter interpretation time, as compared to the other display modes tested in the study, although the difference was not statistically significant. The analysis also showed that there was no difference in the patterns of displayed slab thickness versus time between the stereo and MIP display modes. Most radiologists preferred reading the 3D data at a slab thickness that corresponded to five CT slices. Our results indicate that stereo display has the potential to improve radiologists' performance for detecting lung nodules in CT datasets. The experience gained in conducting the study also strongly suggests that further benefits can be achieved through providing readers with additional functionality.     

Supervised machine learning for diagnostic classification from large-scale neuroimaging datasets

Brain Imaging and Behavior - There are growing concerns about the generalizability of machine learning classifiers in neuroimaging. In order to evaluate this aspect across relatively large...     

Concise review on short bowel syndrome: Etiology,pathophysiology, and management

Adults have approximately 20 feet of small intestine, which is the primary site for absorbing essential nutrients and water. Resection of the intestine for any medical reason may result in short bowel syndrome (SBS), leading to loss of major absorptive surface area and resulting in various malabsorption and motility disorders. The mainstay of treatment is personalized close dietary management. Here we present SBS with its pathophysiology and different nutritional management options available. The central perspective of this paper is to provide a concise review of SBS and the treatment options available, along with how proper nutrition can solve major dietary issues in SBS and help patients recover faster.