One Group of Genetically Similar Listeria monocytogenes Strains Frequently Dominates and Persists in Several Fish Slaughter- and Smokehouses

Contamination of foods with the human pathogen Listeria monocytogenes may occur during processing, and the purpose of this study was to determine whether genetically similar strains colonize different processing plants or whether specific persistent strains are unique to each processing plant. We hypothesized that specific L. monocytogenes strains may be better adapted to specific environmental niches in the processing environment. L. monocytogenes contamination patterns were identified by the collection of 686 and 267 samples from the processing environments: raw fish and products of four fish smokehouses and four fish slaughterhouses, respectively. Samples were collected both during production and after cleaning and disinfection. Typically, these samplings were separated by 1 to 3 months. Sampling sites were targeted toward areas likely to harbor the bacterium. L. monocytogenes was isolated from 213 samples, and one strain from each positive sample was typed by RAPD (random amplified polymorphic DNA) analysis with four different primers. The 213 strains were divided into 37 RAPD types. One RAPD type was predominant; 86 of 213 strains belonged to this type. This type was found in three smokehouses and two slaughterhouses and was predominant in three of these plants. A subset of 35 strains was also analyzed by amplified fragment length polymorphism typing, which confirmed the genetic similarity of the groups. Moreover, strains of the dominant RAPD type were indistinguishable from strains isolated frequently from smoked fish products 10 years ago. One smokehouse was surveyed for a year and a half, and the dominant RAPD type persisted throughout the survey period and accounted for 94 of 118 isolates. Our study indicates that strains of L. monocytogenes that are genetically very closely related may be especially adapted to colonizing the processing equipment or especially resistant to cleaning and disinfection.     

Phylogeny of the Alismatales (Monocotyledons) and the relationship of Acorus (Acorales?)

A phylogenetic analysis of the early branching lineages of the monocotyledons is performed using data from two plastid genes (rbcL and matK), five mitochondrial genes (atp1, ccmB, cob, mttB and nad5) and morphology. The complete matrix includes 93 terminals representing Acorus, the 14 families currently recognized within Alismatales, and numerous lineages of monocotyledons and other angiosperms. Total evidence analysis results in an almost completely resolved strict consensus tree, but all data partitions, genomic as well as morphological, are incongruent. The effects of RNA editing and potentially processed paralogous sequences are explored and discussed. Despite a decrease in incongruence length differences after exclusion of edited sites, the major data partitions remain significantly incongruent. The 14 families of Alismatales are all found to be monophyletic, but Acorus is found to be included in Alismatales rather than being the sister group to all other monocotyledons. The placement is strongly supported by the mitochondrial data, atp1 in particular, but it cannot be explained as an artifact caused by patterns of editing or by sampling of processed paralogues.     

Collagen concentration and biomechanical properties of samples from the lower uterine cervix in relation to age and parity in non-pregnant women

Background  

During normal pregnancy the cervix has a load bearing function. The cervical tissue consists mainly of an extracellular matrix (ECM) rich in collagen; important for the biomechanical properties. The aim of the present study was to evaluate how the biomechanical strength of samples from the distal cervix is associated with collagen content in relation to age and parity. This study demonstrates a method to investigate cervical tissue from women who still have their uterus in situ.     

Papaya glutamine cyclotransferase shows a singular five-fold beta-propeller architecture that suggests a novel reaction mechanism

Cyclisation of N-terminal glutamine and/or glutamate to yield pyroglutamate is an essential posttranslational event affecting a plethora of bioactive peptides and proteins. It is directly linked with pathologies ranging from neurodegenerative diseases to inflammation and several types of cancers. The reaction is catalysed by ubiquitous glutaminyl cyclotransferases (QCs), which present two distinct prototypes. Mammalian QCs are zinc-dependent enzymes with an alpha/beta-hydrolase fold. Here we present the 1.6-A-resolution structure of the other prototype, the plant analogue from Carica papaya (PQC). The hatbox-shaped molecule consists of an unusual five-fold beta-propeller traversed by a central channel, a topology that has hitherto been described only for some sugar-binding proteins and an extracellular nucleotidase. The high resistance of the enzyme to denaturation and proteolytic degradation is explained by its architecture, which is uniquely stabilised by a series of tethering elements that confer rigidity. Strikingly, the N-terminus of PQC specifically interacts with residues around the entrance to the central channel of a symmetry-related molecule, suggesting that this location is the putative active site. Cyclisation would follow a novel general-acid/base working mechanism, pivoting around a strictly conserved glutamate. This study provides a lead structure not only for plant QC orthologues, but also for bacteria, including potential human pathogens causing diphtheria, plague and malaria.     

Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins

Affinity tags are highly efficient tools for protein purification. They allow the purification of virtually any protein without any prior knowledge of its biochemical properties. The use of affinity tags has therefore become widespread in several areas of research e.g., high throughput expression studies aimed at finding a biological function to large numbers of yet uncharacterized proteins. In some cases, the presence of the affinity tag in the recombinant protein is unwanted or may represent a disadvantage for the projected application of the protein, like for clinical use. Therefore, an increasing number of approaches are available at present that are designed for the removal of the affinity tag from the recombinant protein. Most of these methods employ recombinant endoproteases that recognize a specific sequence. These process enzymes can subsequently be removed from the process by affinity purification, since they also include a tag. Here, a survey of the most common affinity tags and the current methods for tag removal is presented, with special emphasis on the removal of N-terminal histidine tags using TAGZyme, a system based on exopeptidase cleavage. In the quest to reduce the significant costs associated with protein purification at large scale, relevant aspects involved in the development of downstream processes for pharmaceutical protein production that incorporate a tag removal step are also discussed. A comparison of the yield of standard vs. affinity purification together with an example of tag removal using TAGZyme is also included.     

Survival of Bactericidal Antibiotic Treatment by a Persister Subpopulation of Listeria monocytogenes

Listeria monocytogenes can cause the serious infection listeriosis, which despite antibiotic treatment has a high mortality. Understanding the response of L. monocytogenes to antibiotic exposure is therefore important to ensure treatment success. Some bacteria survive antibiotic treatment by formation of persisters, which are a dormant antibiotic-tolerant subpopulation. The purpose of this study was to determine whether L. monocytogenes can form persisters and how bacterial physiology affects the number of persisters in the population. A stationary-phase culture of L. monocytogenes was adjusted to 10⁸ CFU ml⁻¹, and 10³ to 10⁴ CFU ml⁻¹ survived 72-h treatment with 100 μg of norfloxacin ml⁻¹, indicating a persister subpopulation. This survival was not caused by antibiotic resistance as regrown persisters were as sensitive to norfloxacin as the parental strain. Higher numbers of persisters (10⁵ to 10⁶) were surviving when older stationary phase or surface-associated cells were treated with 100 μg of norfloxacin ml⁻¹. The number of persisters was similar when a ΔsigB mutant and the wild type were treated with norfloxacin, but the killing rate was higher in the ΔsigB mutant. Dormant norfloxacin persisters could be activated by the addition of fermentable carbohydrates and subsequently killed by gentamicin; however, a stable surviving subpopulation of 10³ CFU ml⁻¹ remained. Nitrofurantoin that has a growth-independent mode of action was effective against both growing and dormant cells, suggesting that eradication of persisters is possible. Our study adds L. monocytogenes to the list of bacterial species capable of surviving bactericidal antibiotics in a dormant stage, and this persister phenomenon should be borne in mind when developing treatment regimens.     

Phylogeny of the Liliales (Monocotyledons) with special emphasis on data partition congruence and RNA editing

A phylogenetic analysis of the monocot order Liliales was performed using sequence data from three mitochondrial (atp1, cob, nad5) and two plastid genes (rbcL, ndhF). The complete data matrix includes 46 terminals representing all 10 families currently included in Liliales. The two major partitions, mitochondrial and plastid data, were congruent, and parsimony analysis resulted in 50 equally parsimonious trees and a well resolved consensus tree confirming monophyly of all families. Mitochondrial genes are known to include RNA edited sites, and in some cases unprocessed genes are replaced by retro‐processed gene copies, that is processed paralogs. To test the effects on phylogeny reconstruction of predicted edited sites and potentially unintentionally sampled processed paralogs, a number of analyses were performed using subsets of the complete data matrix. In general, predicted edited sites were more homoplasious than the other characters and increased incongruence among most data partitions. The predicted edited sites have a non‐random phylogenetic signal in conflict with the signal of the non‐edited sites. The potentially misleading signal was caused partially by the apparent presence of processed paralogs in Galanthus (Amaryllidaceae), part of the outgroup, but also by a deviating evolutionary pattern of predicted edited sites in Liliaceae compared with the remainder of the Liliales. Despite the problems that processed paralogs may cause, we argue that they should not a priori be excluded from phylogenetic analysis.     

Efficacy switching SAR of mGluR5 allosteric modulators: highly potent positive and negative modulators from one chemotype

A series of metabotropic glutamate 5 receptor (mGluR5) allosteric ligands with positive, negative or no modulatory efficacy is described. The ability of this series to yield both mGluR5 PAMs and NAMs with single-digit nanomolar potency is unusual, and the underlying SAR is detailed.