Automation of clip localization in Digital Tomosynthesis for setup of breast cancer patients

The objective of this study is to develop an automatic clip localization procedure for breast cancer patient setup based on Digital Tomosynthesis (DTS) and to characterize its performance with respect to the overall registration accuracy and robustness. The study was performed under an IRB-approved protocol for 12 breast cancer patients with surgical clips implanted around the tumor cavity. The registration of DTS images to planning CTs was performed using an automatic algorithm developed to overcome specific challenges of localization and registration of clips in the breast setup images. The automatic method consisted of auto-segmentation (intensity-based thresholding with a priori knowledge about clip size and location to distinguish clips from bony features) and auto-registration of the segmented clip clusters. To determine the inherent accuracy and robustness of the registration algorithm, additional simulated DTS data was analyzed. The developed algorithm is efficient in removing false positives and negatives and provides an accuracy of better than 2.3 mm for 60° and 3.3 mm for 40° DTS. When incorporated in clinical software, this algorithm helps to facilitate fast and accurate setup evaluation with minimal dose delivered to patients.     

Serum albumins have five sites for binding of cationic dendrimers

The detailed analysis of the interaction between PAMAM G4 dendrimer and serum albumins was performed using circular dichroism, isothermal titration calorimetry, capillary electrophoresis, zeta-potential and fluorescence polarization. It was shown that serum albumins and PAMAM G4 dendrimer form the complex with stoichiometry of 4-6:1 for G4:HSA and 4-5:1 for G4:BSA molar ratio. The possible sites of PAMAM G4 dendrimers binding to protein surface were discussed. Also, it has been proposed that dendrimer does not significantly affect the protein secondary structure studied by circular dichroism.     

High-Throughput Screening for Novel Inhibitors of Neisseria gonorrhoeae Penicillin-Binding Protein 2

The increasing prevalence of N. gonorrhoeae strains exhibiting decreased susceptibility to third-generation cephalosporins and the recent isolation of two distinct strains with high-level resistance to cefixime or ceftriaxone heralds the possible demise of β-lactam antibiotics as effective treatments for gonorrhea. To identify new compounds that inhibit penicillin-binding proteins (PBPs), which are proven targets for β-lactam antibiotics, we developed a high-throughput assay that uses fluorescence polarization (FP) to distinguish the fluorescent penicillin, Bocillin-FL, in free or PBP-bound form. This assay was used to screen a 50,000 compound library for potential inhibitors of N. gonorrhoeae PBP 2, and 32 compounds were identified that exhibited >50% inhibition of Bocillin-FL binding to PBP 2. These included a cephalosporin that provided validation of the assay. After elimination of compounds that failed to exhibit concentration-dependent inhibition, the antimicrobial activity of the remaining 24 was tested. Of these, 7 showed antimicrobial activity against susceptible and penicillin- or cephalosporin-resistant strains of N. gonorrhoeae. In molecular docking simulations using the crystal structure of PBP 2, two of these inhibitors docked into the active site of the enzyme and each mediate interactions with the active site serine nucleophile. This study demonstrates the validity of a FP-based assay to find novel inhibitors of PBPs and paves the way for more comprehensive high-throughput screening against highly resistant strains of N. gonorrhoeae. It also provides a set of lead compounds for optimization of anti-gonococcal agents.     

The role of the β5-α11 loop in the active-site dynamics of acylated penicillin-binding protein A from Mycobacterium tuberculosis

Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constants for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5-α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5-α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.     

Influence of Light on Carbon Utilization in Aerobic Anoxygenic Phototrophs

Aerobic anoxygenic phototrophs contain photosynthetic reaction centers composed of bacteriochlorophyll. These organisms are photoheterotrophs, as they require organic carbon substrates for their growth whereas light-derived energy has only an auxiliary function. To establish the contribution of light energy to their metabolism, we grew the phototrophic strain Erythrobacter sp. NAP1 in a carbon-limited chemostat regimen on defined carbon sources (glutamate, pyruvate, acetate, and glucose) under conditions of different light intensities. When grown in a light-dark cycle, these bacteria accumulated 25% to 110% more biomass in terms of carbon than cultures grown in the dark. Cultures grown on glutamate accumulated the most biomass at moderate light intensities of 50 to 150 μmol m⁻² s⁻¹ but were inhibited at higher light intensities. In the case of pyruvate, we did not find any inhibition of growth by high irradiance. The extent of anaplerotic carbon fixation was detemined by radioactive bicarbonate incorporation assays. While the carboxylation activity provided 4% to 11% of the cellular carbon in the pyruvate-grown culture, in the glutamate-grown cells it provided only approximately 1% of the carbon. Additionally, we tested the effect of light on respiration and photosynthetic electron flow. With increasing light intensity, respiration decreased to approximately 25% of its dark value and was replaced by photophosphorylation. The additional energy from light allows the aerobic anoxygenic phototrophs to accumulate the supplied organic carbon which would otherwise be respired. The higher efficiency of organic carbon utilization may provide an important competitive advantage during growth under carbon-limited conditions.     

Dendrimer-protein interactions studied by tryptophan room temperature phosphorescence

Dendrimers are a relatively new class of materials with unique molecular architectures, which provide promising opportunities for biological applications as DNA carriers and drug delivery systems. Progress in these fields, however, requires knowledge of their potential interactions with biological components at cellular and molecular level. This study utilizes Trp phosphorescence spectroscopy to examine possible perturbations of the protein native fold in solution by neutral, positively and negatively charged fifth generation polyamidoamine (PAMAM) dendrimers. Phosphorescence lifetime measurements, conducted on model proteins varying in the degree of burial of the triplet probe and in quaternary structure, show that dendrimers interact with proteins in solutions forming stable complexes in which the protein structure may be significantly altered, particularly in superficial, flexible regions of the polypeptide. Both electrostatic and non-electrostatic interactions can give rise to stable complexes, whose affinity and limited number of binding sites distinguish them from mere aspecific molecular associations. Of direct relevance for the application of these polymers in the medical field, structural alterations have also been detected in human plasma proteins such as serum albumin and immunoglobulins. The above results suggest that Trp phosphorescence may provide a useful monitor for working out experimental conditions and protocols that help preserve the structural integrity of proteins in the presence of these polymers.     

Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle

Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal‐fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse‐chase LC‐MS‐based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine‐derived fluxes: Oxidation of glucose‐derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation.     

A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal

Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N∶2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53^−/− cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.