Proteomic Profiling of Burkholderia cenocepacia Clonal Isolates with Different Virulence Potential Retrieved from a Cystic Fibrosis Patient during Chronic Lung Infection

Respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. In this work, we assessed the virulence potential of three B. cenocepacia clonal isolates obtained from a CF patient between the onset of infection (isolate IST439) and before death with cepacia syndrome 3.5 years later (isolate IST4113 followed by IST4134), based on their ability to invade epithelial cells and compromise epithelial monolayer integrity. The two clonal isolates retrieved during late-stage disease were significantly more virulent than IST439. Proteomic profiling by 2-D DIGE of the last isolate recovered before the patient’s death, IST4134, and clonal isolate IST439, was performed and compared with a prior analysis of IST4113 vs. IST439. The cytoplasmic and membrane-associated enriched fractions were examined and 52 proteins were found to be similarly altered in the two last isolates compared with IST439. These proteins are involved in metabolic functions, nucleotide synthesis, translation and protein folding, cell envelope biogenesis and iron homeostasis. Results are suggestive of the important role played by metabolic reprogramming in the virulence potential and persistence of B. cenocepacia, in particular regarding bacterial adaptation to microaerophilic conditions. Also, the content of the virulence determinant AidA was higher in the last 2 isolates. Significant levels of siderophores were found to be secreted by the three clonal isolates in an iron-depleted environment, but the two late isolates were more tolerant to low iron concentrations than IST439, consistent with the relative abundance of proteins involved in iron uptake.     

Site-Specific Phosphorylation of the DNA Damage Response Mediator Rad9 by Cyclin-Dependent Kinases Regulates Activation of Checkpoint Kinase 1

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases.     

Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.     

Application of 2D BN/SDS-PAGE coupled with mass spectrometry for identification of VDAC-associated protein complexes related to mitochondrial binding sites for type I brain hexokinase

Two types of binding sites for hexokinase, designated as Type A or Type B sites, have been shown to coexist on brain mitochondria. The ratio of these sites varies between species.HK1 attaches by reversibly binding to the voltage dependent anion channel (VDAC). Regarding the nature of hexokinase binding sites, we investigated if it was linked to distinct VDAC interactomes. We approached this question by 2D BN/SDS-PAGE of mitochondria, followed by mass spectrometry.Our results are consistent with the possibility that the ratio of Type A/Type B sites is due to differential VDAC interactions in bovine and rat neuronal cells.     

Decision-Making in Multiple Sclerosis Consultations in Italy: Third Observer and Patient Assessments

Objective

To assess decision-making in multiple sclerosis (MS) from third observer and patient perspectives.

Method

Audio recordings of first-ever consultations with a participating physician (88 outpatients, 10 physicians) at four tertiary MS care clinics in Italy, were rated by a third observer using the Observing Patient Involvement in Shared Decision Making (OPTION) and by patients using the Perceived Involvement in Care Scale (PICS).

Results

Mean patient age was 37.5, 66% were women, 72% had MS, and 28% had possible MS or other disease. Mean PICS subscale scores (range 0 poor, 100 best possible) were 71.9 (SD 24.3) for "physician facilitation" (PICS-F); 74.6 (SD 22.9) for "patient information exchange" (PICS-I); and only 22.5 (SD 16.2) for "patient decision making" (PICS-DM). Mean OPTION total score (0 poor, 100 best possible) was 29.6 (SD 10.3). Poorest OPTION scores were found for items assessing “preferred patient approach to receiving information” and “preferred patient level of involvement.” Highest scores were for “clinician drawing attention to identified problem”, “indicating need for decision making,” and “need to review the decision.” Consultation time, woman physician, patient-physician gender concordance and PICS-F were associated with higher OPTION total score; older physician and second opinion consultation were associated with lower OPTION score.

Conclusions

In line with findings in other settings, our third observer findings indicated limited patient involvement abilities of MS physicians during first consultations. Patient perceptions of physician skills were better than third observers’, although they correlated. Consultations with women physicians, and younger physicians, were associated with higher third observer and patient-based scores. Our findings reveal a need to empower Italian MS physicians with better communication and shared decision-making skills, and show in particular that attention to MS patient preferences for reception of information and involvement in health decisions, need to be improved.     

MicroRNA Regulation of Adipose Derived Stem Cells in Aging Rats

Background

Perturbations in abdominal fat secreted adipokines play a key role in metabolic syndrome. This process is further altered during the aging process, probably due to alterations in the preadipocytes (aka. stromal vascular fraction cells-SVF cells or adipose derived stem cells-ASCs) composition and/or function. Since microRNAs regulate genes involved both in development and aging processes, we hypothesized that the impaired adipose function with aging is due to altered microRNA regulation of adipogenic pathways in SVF cells.

Methodology and Principal Findings

Alterations in mRNA and proteins associated with adipogenic differentiation (ERK5 and PPARg) but not osteogenic (RUNX2) pathways were observed in SVF cells isolated from visceral adipose tissue with aging (6 to 30 mo) in female Fischer 344 x Brown Norway Hybrid (FBN) rats. The impaired differentiation capacity with aging correlated with altered levels of miRNAs involved in adipocyte differentiation (miRNA-143) and osteogenic pathways (miRNA-204). Gain and loss of function studies using premir or antagomir-143 validated the age associated adipocyte dysfunction.

Conclusions and Significance

Our studies for the first time indicate a role for miRNA mediated regulation of SVF cells with aging. This discovery is important in the light of the findings that dysfunctional adipose derived stem cells contribute to age related chronic diseases.     

A Shift towards Pro-Inflammatory CD16+ Monocyte Subsets with Preserved Cytokine Production Potential after Kidney Transplantation

Background

The presence of monocyte-macrophage lineage cells in rejecting kidney transplants is associated with worse graft outcome. At present, it is still unclear how the monocyte-macrophage related responses develop after transplantation. Here, we studied the dynamics, phenotypic and functional characteristics of circulating monocytes during the first 6 months after transplantation and aimed to establish the differences between kidney transplant recipients and healthy individuals.

Methods

Phenotype, activation status and cytokine production capacity of classical (CD14++CD16−), intermediate (CD14++CD16+) and non-classical (CD14+CD16++), monocytes were determined by flow cytometry in a cohort of 33 healthy individuals, 30 renal transplant recipients at transplantation, 19 recipients at 3 months and 16 recipients at 6 months after transplantation using a cross-sectional approach.

Results

The percentage of both CD16+ monocyte subsets was significantly increased in transplant recipients compared to healthy individuals, indicative of triggered innate immunity (p≤0.039). Enhanced production capacity of tumor necrosis factor-α, interferon-γ and interleukin-1β was observed by monocytes at transplantation compared to healthy individuals. Remarkably, three months post-transplant, in presence of potent immunosuppressive drugs and despite improved kidney function, interferon-γ, tumor necrosis factor-α and interleukin-10 production capacity still remained significantly increased.

Conclusion

Our data demonstrate a skewed balance towards pro-inflammatory CD16+ monocytes that is present at the time of transplantation and retained for at least 6 months after transplantation. This shift could be one of the important drivers of early post-transplant cellular immunity.     

Equilibrium-Phase High Spatial Resolution Contrast-Enhanced MR Angiography at 1.5T in Preoperative Imaging for Perforator Flap Breast Reconstruction

Objectives

The aim was (i) to evaluate the accuracy of equilibrium-phase high spatial resolution (EP) contrast-enhanced magnetic resonance angiography (CE-MRA) at 1.5T using a blood pool contrast agent for the preoperative evaluation of deep inferior epigastric artery perforator branches (DIEP), and (ii) to compare image quality with conventional first-pass CE-MRA.

Methods

Twenty-three consecutive patients were included. All patients underwent preoperative CE-MRA to determine quality and location of DIEP. First-pass imaging after a single bolus injection of 10 mL gadofosveset trisodium was followed by EP imaging. MRA data were compared to intra-operative findings, which served as the reference standard.

Results

There was 100% agreement between EP CE-MRA and surgical findings in identifying the single best perforator branch. All EP acquisitions were of diagnostic quality, whereas in 10 patients the quality of the first-pass acquisition was qualified as non-diagnostic. Both signal- and contrast-to-noise ratios were significantly higher for EP imaging in comparison with first-pass acquisitions (p<0.01).

Conclusions

EP CE-MRA of DIEP in the preoperative evaluation of patients undergoing a breast reconstruction procedure is highly accurate in identifying the single best perforator branch at 1.5Tesla (T). Besides accuracy, image quality of EP imaging proved superior to conventional first-pass CE-MRA.