Signal-difference-to-noise comparison of temporal subtraction, kV-switching dual-energy and photon-counting dual-energy x-ray angiography.

BACKGROUND: Dual-energy (DE) x-ray angiography with photon-counting detectors (PCDs) may enable single-exposure DE imaging of coronary vasculature. PURPOSE: To compare the iodine signal-difference-to-noise ratio (SDNR) of single-exposure DE angiography with digital subtraction angiography (DSA) and kV-switching DE angiography for matched patient x-ray exposure. METHODS: In a phantom study, we determined the technique parameters that maximized the iodine SDNR per root entrance air kerma for DSA, kV-switching DE angiography and single-exposure DE angiography. We measured SDNR from images of a phantom consisting of an iodine step-wedge immersed in a water tank of either 20  or 30 cm in thickness. We also imaged a phantom with simulated vessels embedded in background clutter and measured vessel SDNR. For this second phantom, we also applied anti-correlated noise reduction (ACNR) and calculated the resulting iodine SDNR. All images were acquired using a cadmium telluride PCD with two energy bins and analog charge summing for charge sharing suppression. The energy-discrimination capabilities were only used for the single-exposure DE approach. Optimized techniques were compared in terms of SDNR per root air kerma for two levels of x-ray scatter. RESULTS: For the same patient x-ray exposure, the SDNR of single-exposure DE imaging without ACNR was 75% to 85% of that of kV-switching DE imaging (also without ACNR) and DSA, the latter two of which had nearly equal SDNR. The single-exposure DE approach required ∼50% of the tube load of the kV-switching approach to achieve the same SDNR. For matched patient air kermas, the single exposure approach required only ∼25% of the tube load of the kV-switching approach. ACNR increased SDNR by 2.4 and 3.0 for kV-switching and single-exposure DE imaging, respectively. CONCLUSIONS: Photon-counting, single-exposure DE angiography can suppress soft tissues and provide iodine SDNR levels comparable to DSA and kV-switching DE angiography for matched patient radiation exposures. When ACNR is used to reduce DE image noise, the SDNR of single-exposure DE imaging and kV-switching DE imaging exceed that of DSA by more than a factor of two. Compared to kV-switching DE imaging, single-exposure DE imaging requires substantially lower tube loading to achieve the same SDNR.

Experimental optimization of single-exposure dual-energy angiography with photon-counting x-ray detectors.

BACKGROUND: Photon-counting x-ray detectors may enable single-exposure dual-energy (DE) x-ray angiography. PURPOSE: The purpose of this paper is to experimentally optimize the energy thresholds and tube voltage for single-exposure DE x-ray angiography. METHODS: We optimized single-exposure DE x-ray angiography using the iodine signal-difference-to-noise ratio (SDNR) per root patient air kerma (κ) as a figure of merit. We measured the iodine SDNR by imaging an iodine stepwedge immersed in a water tank with a depth of 30 cm in the direction of x-ray propagation. The stepwedge was imaged using tube voltages ranging from 90 to 150 kV and a cadmium telluride (CdTe) x-ray detector with two energy bins and analog charge summing for charge sharing suppression. The energy threshold that separates the two energy bins was varied from approximately 35 keV to approximately 75% of the maximum energy of the x-ray beam. Curve fitting was used to determine the threshold that maximized SDNR / κ $\mathrm{SDNR}/\sqrt {\kappa }$ . The effect of scatter was determined from measurements of the scatter-to-primary ratios (SPRs) of the low-energy and high-energy images and a semi-empirical model of the relationship between SDNR and SPR. Using the optimal parameters, we imaged a phantom with vessel-simulating structures and background clutter. RESULTS: The optimal energy thresholds increased monotonically from ∼50 to ∼85 keV over the range of tube voltages considered. For tube voltages greater than 90 kV, the optimal energy thresholds consistently allocated approximately two thirds of all detected primary photons to the low energy bin; this ratio was preserved without scatter. Consistent with prior modeling studies, SDNR / κ $\mathrm{SDNR}/\sqrt {\kappa }$ increased monotonically with tube voltage from 90 to 150 kV; SDNR / κ $\mathrm{SDNR}/\sqrt {\kappa }$ at 150 kV was approximately 38% higher than that at 90 kV for an iodine area density of ∼50 mg/cm2 . Scatter reduced SDNR by approximately 25% for SPRs of ∼1 and 0.4 in low-energy and high-energy images, respectively. CONCLUSIONS: Achieving optimal image quality in single-exposure DE angiography with photon-counting x-ray detectors will require high tube voltages (i.e., >130 kV) and, for thick patients, energy thresholds that allocate approximately two thirds of all primary photons to the low-energy image. Future work will compare the image quality of singe-exposure photon-counting and kV-switching approaches to DE x-ray angiography.

Theoretical comparison of energy-resolved and digital-subtraction angiography.

BACKGROUND: X-ray coronary angiography is a sub-optimal vascular imaging technique because it cannot suppress un-enhanced anatomy that may obscure the visualization of coronary artery disease. PURPOSE: The purpose of this paper is to evaluate the theoretical image quality of energy-resolved x-ray angiography (ERA) implemented with spectroscopic x-ray detectors (SXDs), which may overcome limitations of x-ray coronary angiography. METHODS: We modeled the large-area signal-difference-to-noise (SDNR) of contrast-enhanced vasculature in ERA images and compared with that of digital-subtraction angiography (DSA), which served as a gold standard vascular imaging technique. To this end, we used calibrated numerical models of the response of cadmium telluride SXDs including the effects of charge sharing, electronic noise, and energy thresholding. Our models assumed zero scatter, no pulse pile up and small signals such that image contrast is approximately linear in the area density of contrast agents. For DSA, we similarly modeled x-ray detection by cesium iodide energy-integrating detectors using validated numerical models. For ERA, we investigated iodine and gadolinium (Gd) contrast agents, two-material and three-material decompositions, analog charge summing for charge sharing correction, and optimized image quality with respect to the tube voltage and energy thresholds assuming cadmium telluride SXDs with three energy bins. RESULTS: Our analysis reveals that a three-material decomposition using iodine contrast agents will require x-ray exposures that are approximately 400 times those of DSA to achieve the same SDNR as DSA in coronary applications, and is therefore not feasible in a clinical setting. However, three-material decompositions with Gd contrast agents have the potential to provide SDNR that is ∼45% of that of DSA for matched patient air kerma. For two-material decompositions that suppress soft-tissue, ERA has the potential to produce images with SDNR that is 50%-75% of that of DSA for matched patient air kermas but lower levels of tube loading. Achieving these SDNR levels will require the use of analog charge summing for charge sharing correction, which increased SDNR by up to a factor of 1.7 depending on the contrast agent and whether or not a two-material or three-material decomposition was assumed. CONCLUSIONS: We conclude that three-material ERA implemented with Gd contrast agents and two-material ERA implemented with either iodine or Gd contrast agents, should be investigated as alternatives to DSA in situations where motion artifacts preclude the use of DSA, such as in coronary imaging.

Using bispecific antibodies in forced degradation studies to analyze the structure-function relationships of symmetrically and asymmetrically modified antibodies.

Forced degradation experiments of monoclonal antibodies (mAbs) aid in the identification of critical quality attributes (CQAs) by studying the impact of post-translational modifications (PTMs), such as oxidation, deamidation, glycation, and isomerization, on biological functions. Structure-function characterization of mAbs can be used to identify the PTM CQAs and develop appropriate analytical and process controls. However, the interpretation of forced degradation results can be complicated because samples may contain mixtures of asymmetrically and symmetrically modified mAbs with one or two modified chains. We present a process to selectively create symmetrically and asymmetrically modified antibodies for structure-function characterization using the bispecific DuoBody® platform. Parental molecules mAb1 and mAb2 were first stressed with peracetic acid to induce methionine oxidation. Bispecific antibodies were then prepared from a mixture of oxidized or unoxidized parental mAbs by a controlled Fab-arm exchange process. This process was used to systematically prepare four bispecific mAb products: symmetrically unoxidized, symmetrically oxidized, and both combinations of asymmetrically oxidized bispecific mAbs. Results of this study demonstrated chain-independent, 1:2 stoichiometric binding of the mAb Fc region to both FcRn receptor and to Protein A. The approach was also applied to create asymmetrically deamidated mAbs at the asparagine 330 residue. Results of this study support the proposed 1:1 stoichiometric binding relationship between the FcγRIIIa receptor and the mAb Fc. This approach should be generally applicable to study the potential impact of any modification on biological function.

A genomewide screen for suppressors of Alu-mediated rearrangements reveals a role for PIF1.

Alu-mediated rearrangement of tumor suppressor genes occurs frequently during carcinogenesis. In breast cancer, this mechanism contributes to loss of the wild-type BRCA1 allele in inherited disease and to loss of heterozygosity in sporadic cancer. To identify genes required for suppression of Alu-mediated recombination we performed a genomewide screen of a collection of 4672 yeast gene deletion mutants using a direct repeat recombination assay. The primary screen and subsequent analysis identified 12 candidate genes including TSA, ELG1, and RRM3, which are known to play a significant role in maintaining genomic stability. Genetic analysis of the corresponding human homologs was performed in sporadic breast tumors and in inherited BRCA1-associated carcinomas. Sequencing of these genes in high risk breast cancer families revealed a potential role for the helicase PIF1 in cancer predisposition. PIF1 variant L319P was identified in three breast cancer families; importantly, this variant, which is predicted to be functionally damaging, was not identified in a large series of controls nor has it been reported in either dbSNP or the 1000 Genomes Project. In Schizosaccharomyces pombe, Pfh1 is required to maintain both mitochondrial and nuclear genomic integrity. Functional studies in yeast of human PIF1 L319P revealed that this variant cannot complement the essential functions of Pfh1 in either the nucleus or mitochondria. Our results provide a global view of nonessential genes involved in suppressing Alu-mediated recombination and implicate variation in PIF1 in breast cancer predisposition.

The Schizosaccharomyces pombe Pfh1p DNA helicase is essential for the maintenance of nuclear and mitochondrial DNA.

Schizosaccharomyces pombe Pfh1p is an essential member of the Pif family of 5'-3' DNA helicases. The two Saccharomyces cerevisiae homologs, Pif1p and Rrm3p, function in nuclear DNA replication, telomere length regulation, and mitochondrial genome integrity. We demonstrate here the existence of multiple Pfh1p isoforms that localized to either nuclei or mitochondria. The catalytic activity of Pfh1p was essential in both cellular compartments. The absence of nuclear Pfh1p resulted in G(2) arrest and accumulation of DNA damage foci, a finding suggestive of an essential role in DNA replication. Exogenous DNA damage resulted in localization of Pfh1p to DNA damage foci, suggesting that nuclear Pfh1p also functions in DNA repair. The absence of mitochondrial Pfh1p caused rapid depletion of mitochondrial DNA. Despite localization to nuclei and mitochondria in S. pombe, neither of the S. cerevisiae homologs, nor human PIF1, suppressed the lethality of pfh1Delta cells. However, the essential nuclear function of Pfh1p could be supplied by Rrm3p. Expression of Rrm3p suppressed the accumulation of DNA damage foci but not the hydroxyurea sensitivity of cells depleted of nuclear Pfh1p. Together, these data demonstrate that Pfh1p has essential roles in the replication of both nuclear and mitochondrial DNA.

Enzymatic resolution of chiral phosphinate esters.

The bacterial phosphotriesterase has been shown to catalyze the stereoselective hydrolysis of phosphinate esters. The wild-type enzyme preferentially hydrolyzes the SP-enantiomers of methyl phenyl p-X-phenylphosphinate esters by 3 orders of magnitude. The mutant enzyme, I106T/F132A/H254G/H257W, exhibits the opposite stereoselectivity and hydrolyzes the RP-enantiomer up to 30 times faster than the corresponding SP-enantiomer. The enantiomerically pure phosphinate esters, prepared from the kinetic resolution of racemic mixtures, can serve as the entry point for the chemoenzymatic preparation of P-chiral phosphines and phosphine oxides.

Mechanism for the hydrolysis of organophosphates by the bacterial phosphotriesterase.

Phosphotriesterase (PTE) from Pseudomonas diminuta is a zinc metalloenzyme that hydrolyzes a variety of organophosphorus compounds. The kinetic parameters of Zn/Zn PTE, Cd/Cd PTE, and a mixed-metal Zn/Cd hybrid PTE were obtained with a variety of substrates to determine the role of each metal ion in binding and catalysis. pH-rate profiles for the hydrolysis of diethyl p-nitrophenyl phosphate (I) and diethyl p-chlorophenyl phosphate (II) demonstrated that the ionization of a single group in the pH range of 5-10 was critical for substrate turnover. The pK(a) values determined from the kinetic assays were dependent on the identity of the metal ion that occupied the alpha site within the binuclear metal center. These results suggest that the hydrolytic nucleophile is activated as a hydroxide via the ionization of a water molecule attached to the alpha-metal ion. The kinetic constants for the hydrolysis of II and diethyl p-chlorophenyl thiophosphate (IV) were determined for the metal substituted forms of PTE. The kinetic constants for IV were greater than those for II. The inverse thio effect is consistent with the polarization of the phosphoryl oxygen/sulfur bond via a direct ligation to the metal center. The rate enhancement is greater when Cd(2+) occupies the beta-metal-ion position. A series of alanine and asparagine mutations were used to characterize the catalytic roles of Asp233, His254, and Asp301. Mutations to either Asp233 or His254 resulted in an enhanced rate of hydrolysis for the sluggish substrate, diethyl p-chlorophenyl phosphate, and a decrease in the kinetic constants for paraoxon (I). These results are consistent with the existence of a proton relay from Asp301 to His254 to Asp233 that is used to ferry protons away from the active site with substrates that do not require activation of the leaving group phenol. A mechanism for the hydrolysis of organophosphates by the bacterial PTE has been proposed.

Operational control of stereoselectivity during the enzymatic hydrolysis of racemic organophosphorus compounds.

The wild-type bacterial phosphotriesterase catalyzes the stereoselective hydrolysis of racemic pairs of organophosphorus compounds. The enzymatic stereoselectivity can be substantially enhanced via systematic alteration of the pKa for the leaving group phenol in the target substrates. These changes alter the rate-limiting step for substrate turnover from a diffusional event to phosphorus-oxygen bond cleavage. Turnover ratios in excess of 5000:1 were achieved using phenols with pKa values greater than 8.5. This method has enabled the isolation of the RP-enantiomer of 4-acetylphenyl methyl phenylphosphonate with an enantiomeric excess of >99% via a kinetic resolution of the racemate.