Isolation of Highly Active Monoclonal Antibodies against Multiresistant Gram-Positive Bacteria

Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.     

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues ¹. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light ². Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode ^3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.     

Multistage Background Field Removal (MUBAFIRE)—Compensating for B 0 Distortions at Ultra-High Field

The investigation of tissue magnetic susceptibility and the resultant magnetic field offers a new avenue for quantitative tissue characterisation by MRI. One crucial step in mining the phase and field data for relevant tissue information is the correction of externally induced field shifts. This article outlines a multistep approach comprising several methodologies for background field removal. The virtues of B ₀ long-range variation detection and compensation of more localised external disturbances are unified in a sequential filter chain. The algorithm is tested by means of a numerical Monte Carlo simulation model and applied to in vivo measurements at 3T and 9.4T as well as to a fixed brain tissue measurement at 9.4T. Further, a comparison to conventional filter types has been undertaken.     

Unbalanced 2 x 2 Factorial Designs and the Interaction Effect: A Troublesome Combination

In this power study, ANOVAs of unbalanced and balanced 2 x 2 datasets are compared (N = 120). Datasets are created under the assumption that H1 of the effects is true. The effects are constructed in two ways, assuming: 1. contributions to the effects solely in the treatment groups; 2. contrasting contributions in treatment and control groups. The main question is whether the two ANOVA correction methods for imbalance (applying Sums of Squares Type II or III; SS II or SS III) offer satisfactory power in the presence of an interaction. Overall, SS II showed higher power, but results varied strongly. When compared to a balanced dataset, for some unbalanced datasets the rejection rate of H0 of main effects was undesirably higher. SS III showed consistently somewhat lower power. When the effects were constructed with equal contributions from control and treatment groups, the interaction could be re-estimated satisfactorily. When an interaction was present, SS III led consistently to somewhat lower rejection rates of H0 of main effects, compared to the rejection rates found in equivalent balanced datasets, while SS II produced strongly varying results. In data constructed with only effects in the treatment groups and no effects in the control groups, the H0 of moderate and strong interaction effects was often not rejected and SS II seemed applicable. Even then, SS III provided slightly better results when a true interaction was present. ANOVA allowed not always for a satisfactory re-estimation of the unique interaction effect. Yet, SS II worked better only when an interaction effect could be excluded, whereas SS III results were just marginally worse in that case. Overall, SS III provided consistently 1 to 5% lower rejection rates of H0 in comparison with analyses of balanced datasets, while results of SS II varied too widely for general application.     

Reliability of Intra-Retinal Layer Thickness Estimates

Purpose

Measurement of intra-retinal layer thickness using optical coherence tomography (OCT) has become increasingly prominent in multiple sclerosis (MS) research. Nevertheless, the approaches used for determining the mean layer thicknesses vary greatly. Insufficient data exist on the reliability of different thickness estimates, which is crucial for their application in clinical studies. This study addresses this lack by evaluating the repeatability of different thickness estimates.

Methods

Studies that used intra-retinal layer segmentation of macular OCT scans in patients with MS were retrieved from PubMed. To investigate the repeatability of previously applied layer estimation approaches, we generated datasets of repeating measurements of 15 healthy subjects and 13 multiple sclerosis patients using two OCT devices (Cirrus HD-OCT and Spectralis SD-OCT). We calculated each thickness estimate in each repeated session and analyzed repeatability using intra-class correlation coefficients and coefficients of repeatability.

Results

We identified 27 articles, eleven of them used the Spectralis SD-OCT, nine Cirrus HD-OCT, two studies used both devices and two studies applied RTVue-100. Topcon OCT-1000, Stratus OCT and a research device were used in one study each. In the studies that used the Spectralis, ten different thickness estimates were identified, while thickness estimates of the Cirrus OCT were based on two different scan settings. In the simulation dataset, thickness estimates averaging larger areas showed an excellent repeatability for all retinal layers except the outer plexiform layer (OPL).

Conclusions

Given the good reliability, the thickness estimate of the 6mm-diameter area around the fovea should be favored when OCT is used in clinical research. Assessment of the OPL was weak in general and needs further investigation before OPL thickness can be used as a reliable parameter.     

Structural and functional studies of the first tripartite protein complex at the Trypanosoma brucei flagellar pocket collar

The flagellar pocket (FP) is the only endo- and exocytic organelle in most trypanosomes and, as such, is essential throughout the life cycle of the parasite. The neck of the FP is maintained enclosed around the flagellum via the flagellar pocket collar (FPC). The FPC is a macromolecular cytoskeletal structure and is essential for the formation of the FP and cytokinesis. FPC biogenesis and structure are poorly understood, mainly due to the lack of information on FPC composition. To date, only two FPC proteins, BILBO1 and FPC4, have been characterized. BILBO1 forms a molecular skeleton upon which other FPC proteins can, theoretically, dock onto. We previously identified FPC4 as the first BILBO1 interacting partner and demonstrated that its C-terminal domain interacts with the BILBO1 N-terminal domain (NTD). Here, we report by yeast two-hybrid, bioinformatics, functional and structural studies the characterization of a new FPC component and BILBO1 partner protein, BILBO2 (Tb927.6.3240). Further, we demonstrate that BILBO1 and BILBO2 share a homologous NTD and that both domains interact with FPC4. We have determined a 1.9 Å resolution crystal structure of the BILBO2 NTD in complex with the FPC4 BILBO1-binding domain. Together with mutational analyses, our studies reveal key residues for the function of the BILBO2 NTD and its interaction with FPC4 and evidenced a tripartite interaction between BILBO1, BILBO2, and FPC4. Our work sheds light on the first atomic structure of an FPC protein complex and represents a significant step in deciphering the FPC function in Trypanosoma brucei and other pathogenic kinetoplastids.     

Arabidopsis RETINOBLASTOMA-RELATED Is Required for Stem Cell Maintenance, Cell Differentiation, and Lateral Organ Production

Several genes involved in the regulation of postembryonic organ initiation and growth have been identified. However, it remains largely unclear how developmental cues connect to the cell cycle. RETINOBLASTOMA RELATED (RBR) is a plant homolog of the tumor suppressor Retinoblastoma (pRb), which is a key regulator of the cell cycle. Using inducible RNA interference (RNAi) against Arabidopsis thaliana RBR (RBRi), we reduced RBR expression levels at different stages of plant development. Conditional reduction or loss of RBR function disrupted cell division patterns, promoted context-dependent cell proliferation, and negatively influenced establishment of cell differentiation. Several lineages of toti- and pluripotent cells, including shoot apical meristem stem cells, meristemoid mother cells, and procambial cells, failed to produce appropriately differentiated cells. Meristem activity was altered, leading to a disruption of the CLAVATA-WUSCHEL feedback loop and inhibition of lateral organ formation. Release of RBR from RNAi downregulation restored meristem activity. Gene profiling analyses soon after RBRi induction revealed that a change in RBR homeostasis is perceived as a stress, even before genes regulated by RBR-E2F become deregulated. The results establish RBR as a key cell cycle regulator required for coordination of cell division, differentiation, and cell homeostasis.