Pediatric evaluations for deep learning CT denoising.

BACKGROUND: Deep learning (DL) CT denoising models have the potential to improve image quality for lower radiation dose exams. These models are generally trained with large quantities of adult patient image data. However, CT, and increasingly DL denoising methods, are used in both adult and pediatric populations. Pediatric body habitus and size can differ significantly from adults and vary dramatically from newborns to adolescents. Ensuring that pediatric subgroups of different body sizes are not disadvantaged by DL methods requires evaluations capable of assessing performance in each subgroup. PURPOSE: To assess DL CT denoising in pediatric and adult-sized patients, we built a framework of computer simulated image quality (IQ) control phantoms and evaluation methodology. METHODS: The computer simulated IQ phantoms in the framework featured pediatric-sized versions of standard CatPhan 600 and MITA-LCD phantoms with a range of diameters matching the mean effective diameters of pediatric patients ranging from newborns to 18 years old. These phantoms were used in simulating CT images that were then inputs for a DL denoiser to evaluate performance in different sized patients. Adult CT test images were simulated using standard-sized phantoms scanned with adult scan protocols. Pediatric CT test images were simulated with pediatric-sized phantoms and adjusted pediatric protocols. The framework's evaluation methodology consisted of denoising both adult and pediatric test images then assessing changes in image quality, including noise, image sharpness, CT number accuracy, and low contrast detectability. To demonstrate the use of the framework, a REDCNN denoising model trained on adult patient images was evaluated. To validate that the DL model performance measured with the proposed pediatric IQ phantoms was representative of performance in more realistic patient anatomy, anthropomorphic pediatric XCAT phantoms of the same age range were also used to compare noise reduction performance. RESULTS: Using the proposed pediatric-sized IQ phantom framework, size differences between adult and pediatric-sized phantoms were observed to substantially influence the adult trained DL denoising model's performance. When applied to adult images, the DL model achieved a 60% reduction in noise standard deviation without substantial loss in sharpness in mid or high spatial frequencies. However, in smaller phantoms the denoising performance dropped due to different image noise textures resulting from the smaller field of view (FOV) between adult and pediatric protocols. In the validation study, noise reduction trends in the pediatric-sized IQ phantoms were found to be consistent with those found in anthropomorphic phantoms. CONCLUSION: We developed a framework of using pediatric-sized IQ phantoms for pediatric subgroup evaluation of DL denoising models. Using the framework, we found the performance of an adult trained DL denoiser did not generalize well in the smaller diameter phantoms corresponding to younger pediatric patient sizes. Our work suggests noise texture differences from FOV changes between adult and pediatric protocols can contribute to poor generalizability in DL denoising and that the proposed framework is an effective means to identify these performance disparities for a given model.

Emerging technologies and their impact on regulatory science.

There is an evolution and increasing need for the utilization of emerging cellular, molecular and in silico technologies and novel approaches for safety assessment of food, drugs, and personal care products. Convergence of these emerging technologies is also enabling rapid advances and approaches that may impact regulatory decisions and approvals. Although the development of emerging technologies may allow rapid advances in regulatory decision making, there is concern that these new technologies have not been thoroughly evaluated to determine if they are ready for regulatory application, singularly or in combinations. The magnitude of these combined technical advances may outpace the ability to assess fit for purpose and to allow routine application of these new methods for regulatory purposes. There is a need to develop strategies to evaluate the new technologies to determine which ones are ready for regulatory use. The opportunity to apply these potentially faster, more accurate, and cost-effective approaches remains an important goal to facilitate their incorporation into regulatory use. However, without a clear strategy to evaluate emerging technologies rapidly and appropriately, the value of these efforts may go unrecognized or may take longer. It is important for the regulatory science field to keep up with the research in these technically advanced areas and to understand the science behind these new approaches. The regulatory field must understand the critical quality attributes of these novel approaches and learn from each other's experience so that workforces can be trained to prepare for emerging global regulatory challenges. Moreover, it is essential that the regulatory community must work with the technology developers to harness collective capabilities towards developing a strategy for evaluation of these new and novel assessment tools.

Quality Improvement Initiatives to Assess and Improve PET/CT Injection Infiltration Rates at Multiple Centers.

PET/CT radiotracer infiltration is not uncommon and is often outside the imaging field of view. Infiltration can negatively affect image quality, image quantification, and patient management. Until recently, there has not been a simple way to routinely practice PET radiopharmaceutical administration quality control and quality assurance. Our objectives were to quantify infiltration rates, determine associative factors for infiltration, and assess whether rates could be reduced at multiple centers and then sustained. Methods: A "design, measure, analyze, improve, and control" quality improvement methodology requiring novel technology was used to try to improve PET/CT injection quality. Teams were educated on the importance of quality injections. Baseline infiltration rates were measured, center-specific associative factors were analyzed, team meetings were held, improvement plans were established and executed, and rates remeasured. To ensure that injection-quality gains were retained, real-time feedback and ongoing monitoring were used. Sustainability was assessed. Results: Seven centers and 56 technologists provided data on 5,541 injections. The centers' aggregated baseline infiltration rate was 6.2% (range, 2%-16%). On the basis of their specific associative factors, 4 centers developed improvement plans and reduced their aggregated infiltration rate from 8.9% to 4.6% (P < 0.0001). Ongoing injection monitoring showed sustainability. Significant variation was found in center- and technologist-level infiltration rates (P < 0.0001 and P = 0.0020, respectively). Conclusion: A quality improvement approach with new technology can help centers measure infiltration rates, determine associative factors, implement interventions, and improve and sustain injection quality. Because PET/CT images help guide patient management, the monitoring and improvement of radiotracer injection quality are important.

Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption.

The mammalian small heat shock protein (sHSPs) family is comprised of 10 members and includes HSPB1, which is proposed to play an essential role in cellular physiology, acting as a molecular chaperone to regulate diverse cellular processes. Whilst differential roles for sHSPs are suggested for specific tissues, the relative contribution of individual sHSP family members in cellular and organ physiology remains unclear. To address the function of HSPB1 in vivo and determine its tissue-specific expression during development and in the adult, we generated knock-in mice where the coding sequence of hspb1 is replaced by a lacZ reporter gene. Hspb1 expression marks myogenic differentiation with specific expression first confined to developing cardiac muscles and the vascular system, and later in skeletal muscles with specific expression at advanced stages of myoblast differentiation. In the adult, hspb1 expression was observed in other tissues, such as stratified squamous epithelium of skin, oronasal cavity, tongue, esophagus, and uterine cervix but its expression was most prominent in the musculature. Interestingly, in cardiac muscle hsbp1 expression was down-regulated during the neonatal period and maintained to a relatively low steady-level throughout adulthood. Despite this widespread expression, hspb1-/- mice were viable and fertile with no apparent morphological abnormalities in tissues under physiological conditions. However, at the cellular level and under stress conditions (heat challenge), HSPB1 act synergistically with the stress-induced HSPA1 (HSP70) in thermotolerance development, protecting cells from apoptosis. Our data thus indicate a nonessential role for HSPB1 in embryonic development and for maintenance of tissues under physiological conditions, but also shows that it plays an important role by acting synergistically with other HSPs during stress conditions to exert cytoprotection and anti-apoptotic effects.

Monitoring 14-3-3 protein interactions with a homogeneous fluorescence polarization assay.

The 14-3-3 proteins mediate phosphorylation-dependent protein-protein interactions. Through binding to numerous client proteins, 14-3-3 controls a wide range of physiological processes and has been implicated in a variety of diseases, including cancer and neurodegenerative disorders. To better understand the structure and function of 14-3-3 proteins and to develop small-molecule modulators of 14-3-3 proteins for physiological studies and potential therapeutic interventions, the authors have designed and optimized a highly sensitive fluorescence polarization (FP)-based 14-3-3 assay. Using the interaction of 14-3-3 with a fluorescently labeled phosphopeptide from Raf-1 as a model system, they have achieved a simple 1-step "mix-and-measure" method for analyzing 14-3-3 proteins. This is a solution-based, versatile method that can be used to monitor the binding of 14-3-3 with a variety of client proteins. The 14-3-3 FP assay is highly stable and has achieved a robust performance in a 384-well format with a demonstrated signal-to-noise ratio greater than 10 and a Z' factor greater than 0.7. Because of its simplicity and high sensitivity, this assay is generally applicable to studying 14-3-3/client-protein interactions and especially valuable for high-throughput screening of 14-3-3 modulators.

Positive and negative regulatory roles of the WW-like domain in TEL-PDGFbetaR transformation.

TEL-platelet-derived growth factor-beta receptor (TEL-PDGFbetaR) is expressed in chronic myelomonocytic leukemias associated with t(5;12)(q33;p13), and the fusion tyrosine kinase retains a conserved WW-like domain in the PDGFbetaR autoinhibitory juxtamembrane region. Here we report that mutation of the 2 conserved tryptophan residues of the WW-like domain has opposing effects on TELPDGFbetaR kinase activation. Alanine substitution of W593, essential for protein-protein interaction in the context of other WW domains, impaired TEL-PDGFbetaR-mediated transformation of hematopoietic cells due to inhibition of TEL-PDGFbetaR kinase activity. In contrast, alanine substitution of W566, essential for structural integrity of WW domain in other contexts, had no effect on TEL-PDGFbetaR activation and oncogenic activity. Surprisingly, however, the W566A mutation suppressed the W593A phenotype. Double mutant W566A/W593A was indistinguishable from the wild-type fusion protein with regard to kinase activity, ability to confer factor-independent growth to Ba/F3 cells, or ability to induce a myeloproliferative disease in mice. Additional mutational analysis identified other substitutions within the WW-like domain in addition to W566A that could also suppress the W593A phenotype, including mutations predicted to diminish the autoinhibitory function of the juxtamembrane region. Therefore, the WW-like domain in the context of TELPDGFbetaR may have both positive and negative regulatory roles in kinase activation.

Co-immunoprecipitation from transfected cells.

One of the most commonly used methods for determining whether two proteins can interact is co-immunoprecipitation. Co-immunoprecipitation relies on the ability of an antibody to stably and specifically bind complexes containing a bait protein. The antibody provides a means of immobilizing these complexes on a solid matrix, which in the protocol presented here is accomplished through interaction with Protein A, so that irrelevant proteins can be washed away. The presence of target proteins in the bait complexes is determined by Western blot. Because of the biochemical diversity of protein-protein interactions, it is not possible to describe a single set of conditions that will work for every immunoprecipitation experiment. Instead, the goal of this chapter is to provide practical starting conditions for co-immunoprecipitation assays and to describe potential modifications to the procedure so that conditions can be optimized.

Internet resources for studying protein-protein interactions.

The Internet is now an essential tool for scientists. This chapter presents a beginner's guide to some of the valuable resources freely available on the Internet that are relevant to the study of protein-protein interactions. The format is designed to parallel a typical experimental project, indicating web sites to visit at each step. Although detailed instructions for using each site are not provided, the goal of each visit and what kind of information you can expect to obtain are indicated. A final section lists some directory sites that can point to additional web tools.

A sphingosine-dependent protein kinase that specifically phosphorylates 14-3-3 (SDK1) is identified as the kinase domain of PKCdelta: a preliminary note.

A specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, eta, or zeta, respectively, only in the presence of sphingosine (Sph) or N,N-dimethyl-Sph (DMS), was termed "sphingosine-dependent protein kinase-1" (SDK1) [J. Biol. Chem. 273(34) (1998) 21834]. We have now identified SDK1 as a protein having the same amino acid sequence as in the C-terminal-half kinase domain of PKCdelta, with approximately 40 kDa molecular mass, based on large-scale purification of a protein from rat liver, and partial sequence using three different combinations of LC-MS or LC-MS/MS with respective search engine. PKCdelta did not display any SDK1 activity and PKCdelta activity was inhibited by Sph and DMS. However, strong SDK1 activity, only in the presence of Sph or DMS, became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40 kDa kinase domain.

Sphingosine-dependent protein kinase-1, directed to 14-3-3, is identified as the kinase domain of protein kinase C delta.

Some protein kinases are known to be activated by d-erythro-sphingosine (Sph) or N,N-dimethyl-d-erythro-sphingosine (DMS), but not by ceramide, Sph-1-P, other sphingolipids, or phospholipids. Among these, a specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, 14-3-3eta, or 14-3-3zeta, respectively, was termed "sphingosine-dependent protein kinase-1" (SDK1) (Megidish, T., Cooper, J., Zhang, L., Fu, H., and Hakomori, S. (1998) J. Biol. Chem. 273, 21834-21845). We have now identified SDK1 as a protein having the C-terminal half kinase domain of protein kinase Cdelta (PKCdelta) based on the following observations. (i). Large-scale preparation and purification of proteins showing SDK1 activity from rat liver (by six steps of chromatography) gave a final fraction with an enhanced level of an approximately 40-kDa protein band. This fraction had SDK1 activity approximately 50000-fold higher than that in the initial extract. (ii). This protein had approximately 53% sequence identity to the Ser/Thr kinase domain of PKCdelta based on peptide mapping using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry data. (iii). A search for amino acid homology based on the BLAST algorithm indicated that the only protein with high homology to the approximately 40-kDa band is the kinase domain of PKCdelta. The kinase activity of PKCdelta did not depend on Sph or DMS; rather, it was inhibited by these sphingoid bases, i.e. PKCdelta did not display any SDK1 activity. However, strong SDK1 activity became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40-kDa kinase domain. PKCdelta and SDK1 showed different lipid requirements and substrate specificity, although both kinase activities were inhibited by common PKC inhibitors. The high susceptibility of SDK1 to Sph and DMS accounts for their important modulatory role in signal transduction.

Role of the 14-3-3 C-terminal loop in ligand interaction.

14-3-3 proteins are a family of conserved dimeric molecules that interact with a broad range of target proteins, most of which contain phosphoserine/threonine. The amphipathic groove of 14-3-3 is the main structural feature involved in mediating its associations. We have studied another domain of 14-3-3, the C-terminal loop, to determine what role it plays in ligand interaction. A truncated form of 14-3-3zeta lacking this C-terminal loop was generated and found to bind with higher affinity than the wild-type 14-3-3zeta protein to the ligands Raf-1 and Bad. Interestingly, the truncated 14-3-3zeta also showed increased association with the 14-3-3 binding-deficient Bad/S136A mutant. Taken together, these data support a role for the C-terminal loop as a general inhibitor of 14-3-3/ligand interactions. This may provide a mechanism by which inappropriate associations with 14-3-3 are prevented.