Ubiquitin-specific protease 7 inhibitors reveal a differentiated mechanism of p53-driven anti-cancer activity.

The USP7 deubiquitinase regulates proteins involved in the cell cycle, DNA repair, and epigenetics and has been implicated in cancer progression. USP7 inhibition has been pursued for the development of anti-cancer therapies. Here, we describe the discovery of potent and specific USP7 inhibitors exemplified by FX1-5303. FX1-5303 was used as a chemical probe to study the USP7-mediated regulation of p53 signaling in cells. It demonstrates mechanistic differences compared to MDM2 antagonists, a related class of anti-tumor agents that act along the same pathway. FX1-5303 synergizes with the clinically approved BCL2 inhibitor venetoclax in acute myeloid leukemia (AML) cell lines and ex vivo patient samples and leads to strong tumor growth inhibition in in vivo mouse xenograft models of multiple myeloma and AML. This work introduces new USP7 inhibitors, differentiates their mechanism of action from MDM2 inhibition, and identifies specific opportunities for their use in the treatment of AML.

Preliminary evaluation of biplane correlation (BCI) stereographic imaging for lung nodule detection.

A biplane correlation (BCI) imaging system obtains images that can be viewed in stereo, thereby minimizing overlapping structures. This study investigated whether using stereoscopic visualization provides superior lung nodule detection compared to standard postero-anterior (PA) image display. Images were acquired at two oblique views of ±3° as well as at a standard PA position from 60 patients. Images were processed using optimal parameters and displayed on a stereoscopic display. The PA image was viewed in the standard format, while the oblique views were paired to provide a stereoscopic view of the subject. A preliminary observer study was performed with four radiologists who viewed and scored the PA image then viewed and scored the BCI stereoscopic image. The BCI stereoscopic viewing of lung nodules resulted in 71 % sensitivity and 0.31 positive predictive value (PPV) index compared to PA results of 86 % sensitivity and 0.26 PPV index. The sensitivity for lung nodule detection with the BCI stereoscopic system was reduced by 15 %; however, the total number of false positives reported was reduced by 35 % resulting in an improved PPV index of 20 %. The preliminary results indicate observer dependency in terms of relative advantage of either system in the detection of lung nodules, but overall equivalency of the two methods with promising potential for BCI as an adjunct diagnostic technique.

A computerized scheme for lung nodule detection in multiprojection chest radiography.

PURPOSE: Our previous study indicated that multiprojection chest radiography could significantly improve radiologists' performance for lung nodule detection in clinical practice. In this study, the authors further verify that multiprojection chest radiography can greatly improve the performance of a computer-aided diagnostic (CAD) scheme. METHODS: Our database consisted of 59 subjects, including 43 subjects with 45 nodules and 16 subjects without nodules. The 45 nodules included 7 real and 38 simulated ones. The authors developed a conventional CAD scheme and a new fusion CAD scheme to detect lung nodules. The conventional CAD scheme consisted of four steps for (1) identification of initial nodule candidates inside lungs, (2) nodule candidate segmentation based on dynamic programming, (3) extraction of 33 features from nodule candidates, and (4) false positive reduction using a piecewise linear classifier. The conventional CAD scheme processed each of the three projection images of a subject independently and discarded the correlation information between the three images. The fusion CAD scheme included the four steps in the conventional CAD scheme and two additional steps for (5) registration of all candidates in the three images of a subject, and (6) integration of correlation information between the registered candidates in the three images. The integration step retained all candidates detected at least twice in the three images of a subject and removed those detected only once in the three images as false positives. A leave-one-subject-out testing method was used for evaluation of the performance levels of the two CAD schemes. RESULTS: At the sensitivities of 70%, 65%, and 60%, our conventional CAD scheme reported 14.7, 11.3, and 8.6 false positives per image, respectively, whereas our fusion CAD scheme reported 3.9, 1.9, and 1.2 false positives per image, and 5.5, 2.8, and 1.7 false positives per patient, respectively. The low performance of the conventional CAD scheme may be attributed to the high noise level in chest radiography, and the small size and low contrast of most nodules. CONCLUSIONS: This study indicated that the fusion of correlation information in multiprojection chest radiography can markedly improve the performance of CAD scheme for lung nodule detection.

Design and Development of a New Multi-Projection X-Ray System for Chest Imaging.

Overlapping anatomical structures may confound the detection of abnormal pathology, including lung nodules, in conventional single-projection chest radiography. To minimize this fundamental limiting factor, a dedicated digital multi-projection system for chest imaging was recently developed at the Radiology Department of Duke University. We are reporting the design of the multi-projection imaging system and its initial performance in an ongoing clinical trial. The system is capable of acquiring multiple full-field projections of the same patient along both the horizontal and vertical axes at variable speeds and acquisition frame rates. These images acquired in rapid succession from slightly different angles about the posterior-anterior (PA) orientation can be correlated to minimize the influence of overlying anatomy. The developed system has been tested for repeatability and motion blur artifacts to investigate its robustness for clinical trials. Excellent geometrical consistency was found in the tube motion, with positional errors for clinical settings within 1%. The effect of tube-motion on the image quality measured in terms of impact on the Modulation Transfer Function (MTF) was found to be minimal. The system was deemed clinic-ready and a clinical trial was subsequently launched. The flexibility of image acquisition built into the system provides a unique opportunity to easily modify it for different clinical applications, including tomosynthesis, correlation imaging (CI), and stereoscopic imaging.

Probing molecular docking in a charged model binding site.

A model binding site was used to investigate charge-charge interactions in molecular docking. This simple site, a small (180A(3)) engineered cavity in cyctochrome c peroxidase (CCP), is negatively charged and completely buried from solvent, allowing us to explore the balance between electrostatic energy and ligand desolvation energy in a system where many of the common approximations in docking do not apply. A database with about 5300 molecules was docked into this cavity. Retrospective testing with known ligands and decoys showed that overall the balance between electrostatic interaction and desolvation energy was captured. More interesting were prospective docking scre"ens that looked for novel ligands, especially those that might reveal problems with the docking and energy methods. Based on screens of the 5300 compound database, both high-scoring and low-scoring molecules were acquired and tested for binding. Out of 16 new, high-scoring compounds tested, 15 were observed to bind. All of these were small heterocyclic cations. Binding constants were measured for a few of these, they ranged between 20microM and 60microM. Crystal structures were determined for ten of these ligands in complex with the protein. The observed ligand geometry corresponded closely to that predicted by docking. Several low-scoring alkyl amino cations were also tested and found to bind. The low docking score of these molecules owed to the relatively high charge density of the charged amino group and the corresponding high desolvation penalty. When the complex structures of those ligands were determined, a bound water molecule was observed interacting with the amino group and a backbone carbonyl group of the cavity. This water molecule mitigates the desolvation penalty and improves the interaction energy relative to that of the "naked" site used in the docking screen. Finally, six low-scoring neutral molecules were also tested, with a view to looking for false negative predictions. Whereas most of these did not bind, two did (phenol and 3-fluorocatechol). Crystal structures for these two ligands in complex with the cavity site suggest reasons for their binding. That these neutral molecules do, in fact bind, contradicts previous results in this site and, along with the alkyl amines, provides instructive false negatives that help identify weaknesses in our scoring functions. Several improvements of these are considered.

Estimating diversity of Indo-Pacific coral reef stomatopods through DNA barcoding of stomatopod larvae.

There is a push to fully document the biodiversity of the world within 25 years. However, the magnitude of this challenge, particularly in marine environments, is not well known. In this study, we apply DNA barcoding to explore the biodiversity of gonodactylid stomatopods (mantis shrimp) in both the Coral Triangle and the Red Sea. Comparison of sequences from 189 unknown stomatopod larvae to 327 known adults representing 67 taxa in the superfamily Gonodactyloidea revealed 22 distinct larval operational taxonomic units (OTUs). In the Western Pacific, 10 larval OTUs were members of the Gonodactylidae and Protosquillidae where success of positive identification was expected to be 96.5%. However, only five OTUs could be identified to species and at least three OTUs represent new species unknown in their adult form. In the Red Sea where the identification rate was expected to be 75% in the Gonodactylidae, none of four larval OTUs could be identified to species; at least two represent new species unknown in their adult forms. Results indicate that the biodiversity in this well-studied group in the Coral Triangle and Red Sea may be underestimated by a minimum of 50% to more than 150%, suggesting a much greater challenge in lesser-studied groups. Although the DNA barcoding methodology was effective, its overall success was limited due to the newly discovered taxonomic limitations of the reference sequence database, highlighting the importance of synergy between molecular geneticists and taxonomists in understanding and documenting our world's biodiversity, both in marine and terrestrial environments.

Imaging properties of digital magnification radiography.

Flat panel detectors exhibit improved signal-to-noise ratio (SNR) and display capabilities compared to film. This improvement necessitates a new evaluation of optimal geometry for conventional projection imaging applications such as digital projection mammography as well as for advanced x-ray imaging applications including cone-beam computed tomography (CT), tomosynthesis, and mammotomography. Such an evaluation was undertaken in this study to examine the effects of x-ray source distribution, inherent detector resolution, magnification, scatter rejection, and noise characteristics including noise aliasing. A model for x-ray image acquisition was used to develop generic results applicable to flat panel detectors with similar x-ray absorption characteristics. The model assumed a Gaussian distribution for the focal spot and a rectangular distribution for a pixel. A generic model for the modulated transfer function (MTF) of indirect flat panel detectors was derived by a nonlinear fit of empirical receptor data to the Burgess model for phosphor MTFs. Noise characteristics were investigated using a generic noise power spectrum (NPS) model for indirect phosphor-based detectors. The detective quantum efficiency (DQE) was then calculated from the MTF and NPS models. The results were examined as a function of focal spot size (0.1, 0.3, and 0.6 mm) and pixel size (50, 100, 150, and 200 microm) for magnification ranges 1 to 3. Mammography, general radiography (also applicable to mammotomography), and chest radiography applications were explored using x-ray energies of 28, 74, and 120 kVp, respectively. Nodule detection was examined using the effective point source scatter model, effective DQE, and the Hotelling SNR2 efficiency. Results indicate that magnification can potentially improve the signal and noise performance of digital images. Results also show that a cross over point occurs in the spatial frequency above and below which the effects of magnification differ indicating that there are task dependent tradeoffs associated with magnification. The cross over point varies depending upon focal spot size, pixel size, x-ray energy, and source-to-image-distance (SID). For mammography, the cross over point occurs for a 0.3 mm focal spot while a 0.6 mm focal spot indicates that magnification does not improve image quality due to focal spot blurring. Thus, the benefit of magnification may be limited. For general radiography (as well as mammotomography), and chest radiography, the cross over point changes with SID. For a system with a 0.3 mm focal spot, 100 microm pixel size, a 2 m SID, and the applicable tissue thickness and scatter components, optimal magnification improved SNR2 by approximately 1.2 times for mammography and 1.5 times for general radiography (and mammotomography). These results indicate that the optimal geometry can improve image quality without changing patient dose or otherwise reduce dose without compromising image quality.

Randomized, controlled surgical trial of preoperative tumor curettage of basal cell carcinoma in Mohs micrographic surgery.

BACKGROUND: The use of preoperative tumor curettage in Mohs micrographic surgery has never been prospectively systematically assessed. OBJECTIVE: To assess the utility of preoperative tumor curettage in Mohs micrographic surgery for primary or recurrent, well-defined basal cell carcinoma less than 2 cm in diameter located on the head or neck. METHODS: Patients were randomized to either preoperative tumor curettage or control group and were compared in terms of percent surface area increase from tumor surface area to wound surface area, absolute surface area increase, number of tissue layers removed, types of repairs performed, and postoperative complications. Multivariate analysis was performed to see if tumor location, appearance (exophytic or flat), or histology affected any of the above. RESULTS: The preoperative tumor curettage group had a 399% (95% confidence interval [CI] 346-452) mean surface area increase from tumor to wound surface area versus 263% (95% CI 216-311) for control group (P=.0002). The preoperative tumor curettage group had a mean absolute surface area increase of 1.78 cm2 (95% CI 1.57-1.99) versus 1.40 cm2 (95% CI 1.15-1.65) for control group (P=.02). The preoperative tumor curettage group had fewer tissue layers removed (P=.3). Preoperative tumor curettage had no effect on types of repairs performed or number or type of postoperative complications. Tumor appearance and histology had no effect on any of the above end points. CONCLUSION: Preoperative tumor curettage was associated with significantly greater percent surface area increase and absolute surface area increase from tumor surface area to wound surface area. This difference did not affect type of repair performed or postoperative complications.

Paraneoplastic dermatoses.

Skin manifestations of systemic disease and malignancy are protean. The recognition of a potentially paraneoplastic dermatosis as such must prompt an investigation for occult malignancy. Lack of familiarity with cutaneous clues of internal malignancy may delay diagnosis and treatment of cancer. It is important to consider a paraneoplastic process in the differential diagnosis of a number of eruptive and treatment-resistant dermatoses. These dermatoses may be the first sign of an occult neoplasm. Their recognition may assist in cancer detection and the swift induction of appropriate therapy.

A single-center study of aminolevulinic acid and 417 NM photodynamic therapy in the treatment of moderate to severe acne vulgaris.

This article describes a single-center trial involving twenty-two patients with acne vulgaris. Blu-U light was used in all patients, while half were pre-treated with aminolevulinic acid. Preliminary results show promise for this treatment in mild to moderate inflammatory acne vulgaris.

Surgical margins of excision for basal cell carcinoma and squamous cell carcinoma.

In excising basal and squamous cell carcinomata, the surgical margin that is wide enough to completely remove the tumor an acceptable percentage of the time and narrow enough to minimize removal of excessive normal tissue must be selected. This task can be reliably accomplished with comprehensive knowledge of factors that affect subclinical tumor extension such as tumor appearance, diameter, histology, location, treatment status, and, in the case of squamous cell carcinoma, vertical invasion depth and involvement of subcutaneous fat. Information regarding these factors along with specific recommendations about excisional margins for basal cell and squamous cell carcinomata is presented.

Structural and regulatory elements of HCV NS5B polymerase--ß-loop and C-terminal tail--are required for activity of allosteric thumb site II inhibitors.

Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B--the C-terminal tail and ß-loop--in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the ß-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor-NS5B complex are absent in the inhibitor-bound constructs in which interactions between C-terminal tail and ß-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and ß-loop.

Pre-linguistic children with cleft palate: growth of gesture, vocalization, and word use.

Children with cleft lip and/or palate show early delays in speech and vocabulary development that may have an impact on later communication and social development. While delays in the complexity of babbling may put children at risk for later delays in speech and language development, there is considerable variability in development. This study focused on the rate of children's communication acts, canonical vocalizations, and word use as they made the transition from the pre-linguistic to linguistic development. The study included 15 children with non-syndromic cleft lip and/or palate who were seen at three time points between 17-34 months age. Communication rates were calculated from parent-child language samples collected during play activities. Assignment to linguistic stages was based on the children's expressive vocabulary, as reported on the MacArthur Communicative Development Inventory: Words and Sentences. From the pre-linguistic to linguistic level, the children's average rate per minute of: communicative acts overall increased significantly from 1.49 to 3.07 per minute; canonical vocalizations from 0.21 to 0.90 per minute; and word use from 0.16 to 3.61 per minute. Rates of communicative acts were associated with later word use. It appears that children with clefts rely on non-verbal communicative acts when verbal development is delayed.

Effective DQE (eDQE) for monoscopic and stereoscopic chest radiography imaging systems with the incorporation of anatomical noise.

PURPOSE: Stereoscopic chest biplane correlation imaging (stereo∕BCI) has been proposed as an alternative modality to single view chest x-ray (CXR). The metrics effective modulation transfer function (eMTF), effective normalized noise power spectrum (eNNPS), and effective detective quantum efficiency (eDQE) have been proposed as clinically relevant metrics for assessing clinical system performance taking into consideration the magnification and scatter effects. This study compared the metrics eMTF, eNNPS, eDQE, and detectability index for stereo∕BCI and single view CXR under isodose conditions at two magnifications for two anthropomorphic phantoms of differing sizes. METHODS: Measurements for the eMTF were taken for two phantom sizes with an opaque edge test device using established techniques. The eNNPS was measured at two isodose conditions for two phantoms using established techniques. The scatter was measured for two phantoms using an established beam stop method. All measurements were also taken at two different magnifications with two phantoms. A geometrical phantom was used for comparison with prior results for CXR although the results for an anatomy free phantom are not expected to vary for BCI. RESULTS: Stereo∕BCI resulted in improved metrics compared to single view CXR. Results indicated that magnification can potentially improve the detection performance primarily due to the air gap which reduced scatter by ∼20%. For both phantoms, at isodose, eDQE(0) for stereo∕BCI was ∼100 times higher than that for CXR. Magnification at isodose improved eDQE(0) by ∼10 times for stereo∕BCI. Increasing the dose did not improve eDQE. The detectability index for stereo∕BCI was ∼100 times better than single view CXR for all conditions. The detectability index was also not improved with increased dose. CONCLUSIONS: The findings indicate that stereo∕BCI with magnification may improve detectability of subtle lung nodules compared to single view CXR. Results were improved with magnification for the smaller phantom but not for the larger phantom. The effective DQE and the detectability index did not improve with increasing dose.

Roles for ordered and bulk solvent in ligand recognition and docking in two related cavities.

A key challenge in structure-based discovery is accounting for modulation of protein-ligand interactions by ordered and bulk solvent. To investigate this, we compared ligand binding to a buried cavity in Cytochrome c Peroxidase (CcP), where affinity is dominated by a single ionic interaction, versus a cavity variant partly opened to solvent by loop deletion. This opening had unexpected effects on ligand orientation, affinity, and ordered water structure. Some ligands lost over ten-fold in affinity and reoriented in the cavity, while others retained their geometries, formed new interactions with water networks, and improved affinity. To test our ability to discover new ligands against this opened site prospectively, a 534,000 fragment library was docked against the open cavity using two models of ligand solvation. Using an older solvation model that prioritized many neutral molecules, three such uncharged docking hits were tested, none of which was observed to bind; these molecules were not highly ranked by the new, context-dependent solvation score. Using this new method, another 15 highly-ranked molecules were tested for binding. In contrast to the previous result, 14 of these bound detectably, with affinities ranging from 8 µM to 2 mM. In crystal structures, four of these new ligands superposed well with the docking predictions but two did not, reflecting unanticipated interactions with newly ordered waters molecules. Comparing recognition between this open cavity and its buried analog begins to isolate the roles of ordered solvent in a system that lends itself readily to prospective testing and that may be broadly useful to the community.

Blind prediction of charged ligand binding affinities in a model binding site.

Predicting absolute protein-ligand binding affinities remains a frontier challenge in ligand discovery and design. This becomes more difficult when ionic interactions are involved because of the large opposing solvation and electrostatic attraction energies. In a blind test, we examined whether alchemical free-energy calculations could predict binding affinities of 14 charged and 5 neutral compounds previously untested as ligands for a cavity binding site in cytochrome c peroxidase. In this simplified site, polar and cationic ligands compete with solvent to interact with a buried aspartate. Predictions were tested by calorimetry, spectroscopy, and crystallography. Of the 15 compounds predicted to bind, 13 were experimentally confirmed, while 4 compounds were false negative predictions. Predictions had a root-mean-square error of 1.95 kcal/mol to the experimental affinities, and predicted poses had an average RMSD of 1.7Å to the crystallographic poses. This test serves as a benchmark for these thermodynamically rigorous calculations at predicting binding affinities for charged compounds and gives insights into the existing sources of error, which are primarily electrostatic interactions inside proteins. Our experiments also provide a useful set of ionic binding affinities in a simplified system for testing new affinity prediction methods.

Predicting ligand binding affinity with alchemical free energy methods in a polar model binding site.

We present a combined experimental and modeling study of organic ligand molecules binding to a slightly polar engineered cavity site in T4 lysozyme (L99A/M102Q). For modeling, we computed alchemical absolute binding free energies. These were blind tests performed prospectively on 13 diverse, previously untested candidate ligand molecules. We predicted that eight compounds would bind to the cavity and five would not; 11 of 13 predictions were correct at this level. The RMS error to the measurable absolute binding energies was 1.8 kcal/mol. In addition, we computed "relative" binding free energies for six phenol derivatives starting from two known ligands: phenol and catechol. The average RMS error in the relative free energy prediction was 2.5 kcal/mol (phenol) and 1.1 kcal/mol (catechol). To understand these results at atomic resolution, we obtained x-ray co-complex structures for nine of the diverse ligands and for all six phenol analogs. The average RMSD of the predicted pose to the experiment was 2.0 A (diverse set), 1.8 A (phenol-derived predictions), and 1.2 A (catechol-derived predictions). We found that predicting accurate affinities and rank-orderings required near-native starting orientations of the ligand in the binding site. Unanticipated binding modes, multiple ligand binding, and protein conformational change all proved challenging for the free energy methods. We believe that these results can help guide future improvements in physics-based absolute binding free energy methods.

Rescoring docking hit lists for model cavity sites: predictions and experimental testing.

Molecular docking computationally screens thousands to millions of organic molecules against protein structures, looking for those with complementary fits. Many approximations are made, often resulting in low "hit rates." A strategy to overcome these approximations is to rescore top-ranked docked molecules using a better but slower method. One such is afforded by molecular mechanics-generalized Born surface area (MM-GBSA) techniques. These more physically realistic methods have improved models for solvation and electrostatic interactions and conformational change compared to most docking programs. To investigate MM-GBSA rescoring, we re-ranked docking hit lists in three small buried sites: a hydrophobic cavity that binds apolar ligands, a slightly polar cavity that binds aryl and hydrogen-bonding ligands, and an anionic cavity that binds cationic ligands. These sites are simple; consequently, incorrect predictions can be attributed to particular errors in the method, and many likely ligands may actually be tested. In retrospective calculations, MM-GBSA techniques with binding-site minimization better distinguished the known ligands for each cavity from the known decoys compared to the docking calculation alone. This encouraged us to test rescoring prospectively on molecules that ranked poorly by docking but that ranked well when rescored by MM-GBSA. A total of 33 molecules highly ranked by MM-GBSA for the three cavities were tested experimentally. Of these, 23 were observed to bind--these are docking false negatives rescued by rescoring. The 10 remaining molecules are true negatives by docking and false positives by MM-GBSA. X-ray crystal structures were determined for 21 of these 23 molecules. In many cases, the geometry prediction by MM-GBSA improved the initial docking pose and more closely resembled the crystallographic result; yet in several cases, the rescored geometry failed to capture large conformational changes in the protein. Intriguingly, rescoring not only rescued docking false positives, but also introduced several new false positives into the top-ranking molecules. We consider the origins of the successes and failures in MM-GBSA rescoring in these model cavity sites and the prospects for rescoring in biologically relevant targets.