Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.     

Alginate and pectin composite films crosslinked with Ca ions: Effect of the plasticizer concentration

The manufacture of composite biofilms of alginate and LM-pectin crosslinked with calcium ions requires a two-step contact with Ca²⁺: initially a low-structured pre-film is formatted which is further crosslinked in a second contact with a more concentrated Ca²⁺ solution containing plasticizer. This research evaluated the influence of the plasticizer (glycerol) concentration (1–15% w/v) in this finishing reticulation step on final films characteristics. The results indicated that the extent of the simultaneous Ca²⁺ crosslinking and plasticization with glycerol was determined by the level of structural organization obtained in the pre-reticulation. Increasing the glycerol concentration of the crosslinking solution increased film solubility in water, moisture content, volumetric swelling and flexibility and decreased the resistance to tensile stress. Transparent alginate and pectin composite films with acceptable mechanical properties, low solubility and limited degree of swelling were obtained with 10% glycerol in the second contact solution.     

Mass transfer in aqueous two-phases system packed column

SCH 201781 is a direct thrombin inhibitor recently under study in clinical trials to determine its safety and efficacy for the treatment of venous and arterial thrombosis. In aqueous solution, SCH 201781 exists as three forms, a ring-opened hydrated form and two ring-closed diastereomers. An automated solid-phase extraction LC-MS/MS method that chromatographically separates and measures each form was developed and validated from 1 to 1000 ng/mL in human plasma. For calibration curve standards, within- and between-run precision (%CV) ranged from 0.6 to 13.7%, while accuracy (%bias) ranged from -4.8 to 13.1%. For quality control samples, within- and between-run %CV ranged from 1.5 to 9.9% while %bias ranged from -9.1 to 4.9%. The method requires a sample volume of 0.8 ml and utilizes 2H6-labeled SCH 201781 as the internal standard. For sample processing, an Isolute C-8 96-well solid phase extraction plate and a Tomtec Quadra 96 sample processor is employed. Separation of the three forms of SCH 201781 is achieved using a 5 microm, 2 mm x 100 mm Asahipak C8 HPLC column and gradient elution. A Sciex API 365 equipped with a turbo ionspray source is used in the selected reaction monitoring mode for detection. The validated method was used to support clinical studies.     

Xylanase mass transfer studies in aqueous two-phase systems using spray and sieve plate columns

Aqueous two-phase systems (ATPSs) have long been used for biomolecule partitioning; these systems offer the possibility of using continuous or semicontinuous extraction processes. They require relatively simple equipment like spray or sieve plate columns that can be adapted for use in ATPSs. The aim of this work was to study the semicontinuous extraction of a model enzyme, xylanase, in spray and sieve plate columns, since, unlike centrifugal contactors, the cost of construction and maintenance of this equipment is low and it is easy to operate. For the spray column, the dispersed phase hold-up and overall mass transfer coefficients K(D) a were evaluated for different column heights and for different superficial velocities of the dispersed phase (light phase). Results indicated that an increase in superficial velocity in the range of 0-0.18 mm/s of the dispersed phase had a positive effect on K(D) a and on hold-up in all column heights studied, 75, 161 and 246 mm. For the same superficial velocity of the dispersed phase, the larger the hold-up was, the shorter the column. For the sieve plate column, the effects of the superficial velocity of the dispersed phase and the number of plates were also studied. Results showed that the K(D) a and hold-up increased with an increase in both parameters. The selectivity of separation of xylanase and BSA (model contaminant) was very high, since 60% of the enzyme was extracted in the light phase, whereas no significant amount of BSA was extracted. The possibility of using the sieve plate column in continuous operation for enzyme extraction was studied because previous work had only addressed the semicontinuous extraction of enzyme. The residence time distribution of the PEG phase using different superficial velocities of the salt phase was studied in continuous operation. The time required to reach the steady state was 40 min, and 70% of the xylanase was recovered. It was found that the Modified Power Spline software was well adjusted to the experimental results.     

Evolutionary dynamics of methicillin-resistant Staphylococcus aureus within a healthcare system

BackgroundIn the past decade, several countries have seen gradual replacement of endemic multi-resistant healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) with clones that are more susceptible to antibiotic treatment. One example is Singapore, where MRSA ST239, the dominant clone since molecular profiling of MRSA began in the mid-1980s, has been replaced by ST22 isolates belonging to EMRSA-15, a recently emerged pandemic lineage originating from Europe.ResultsWe investigated the population structure of MRSA in Singaporean hospitals spanning three decades, using whole genome sequencing. Applying Bayesian phylogenetic methods we report that prior to the introduction of ST22, the ST239 MRSA population in Singapore originated from multiple introductions from the surrounding region; it was frequently transferred within the healthcare system resulting in a heterogeneous hospital population. Following the introduction of ST22 around the beginning of the millennium, this clone spread rapidly through Singaporean hospitals, supplanting the endemic ST239 population. Coalescent analysis revealed that although the genetic diversity of ST239 initially decreased as ST22 became more dominant, from 2007 onwards the genetic diversity of ST239 began to increase once more, which was not associated with the emergence of a sub-clone of ST239. Comparative genomic analysis of the accessory genome of the extant ST239 population identified that the Arginine Catabolic Mobile Element arose multiple times, thereby introducing genes associated with enhanced skin colonization into this population.ConclusionsOur results clearly demonstrate that, alongside clinical practice and antibiotic usage, competition between clones also has an important role in driving the evolution of nosocomial pathogen populations.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0643-z) contains supplementary material, which is available to authorized users.     

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.

Results

Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage φ-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage.

Conclusions

The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1599-9) contains supplementary material, which is available to authorized users.     

Global remodelling of cellular microenvironment due to loss of collagen VII

The mammalian cellular microenvironment is shaped by soluble factors and structural components, the extracellular matrix, providing physical support, regulating adhesion and signalling. A global, quantitative mass spectrometry strategy, combined with bioinformatics data processing, was developed to assess proteome differences in the microenvironment of primary human fibroblasts. We studied secreted proteins of fibroblasts from normal and pathologically altered skin and their post‐translational modifications. The influence of collagen VII, an important structural component, which is lost in genetic skin fragility, was used as model. Loss of collagen VII had a global impact on the cellular microenvironment and was associated with proteome alterations highly relevant for disease pathogenesis including decrease in basement membrane components, increase in dermal matrix proteins, TGF‐β and metalloproteases, but not higher protease activity. The definition of the proteome of fibroblast microenvironment and its plasticity in health and disease identified novel disease mechanisms and potential targets of intervention.