Structure-Based Lead Optimization of Enterovirus D68 2A Protease Inhibitors.

Enterovirus D68 (EV-D68) virus is a nonpolio enterovirus that typically causes respiratory illness and, in severe cases, can lead to paralysis and death in children. There is currently no vaccine or antiviral for EV-D68. We previously discovered the viral 2A protease (2Apro) as a viable antiviral drug target and identified telaprevir as a 2Apro inhibitor. 2Apro is a viral cysteine protease that cleaves the viral VP1-2A polyprotein junction. In this study, we report the X-ray crystal structures of EV-D68 2Apro, wild-type, and the C107A mutant and the structure-based lead optimization of telaprevir. Guided by the X-ray crystal structure, we predicted the binding pose of telaprevir in 2Apro using molecular dynamics simulations. We then utilized this model to inform structure-based optimization of the telaprevir's reactive warhead and P1-P4 substitutions. These efforts led to the discovery of 2Apro inhibitors with improved antiviral activity than telaprevir. These compounds represent promising lead compounds for further development as EV-D68 antivirals.

Evaluation of Incise Drape Lift Using 2% Chlorhexidine Gluconate/70% Isopropyl Alcohol Preoperative Skin Preparations in a Human Volunteer Knee Model.

Background: Before surgery, skin is prepped with antiseptics to reduce risk of surgical site infections. An incise drape can be used as an additional modality to immobilize any remaining bacteria. Good adhesion of this drape is critical for infection prevention. Methods: This is a randomized controlled study using a human volunteer knee model (n = 30) to evaluate the adhesion performance of an incise drape comparing 2 skin preparations. A new investigational 2% chlorhexidine gluconate/70% isopropyl alcohol skin prep (prep A) was compared with an existing skin prep containing the same active agents (prep B). Two samples of an iodine-impregnated incise drape were placed on each knee after prepping. Knees were flexed in dry conditions, under a saline-soaked gauze, and after saline lavage. The frequency of drape lift was recorded after each challenge. Results: After dry flex, 4 of 60 samples (6.7%) had lifted on prep A and 0 on prep B (P = .125). After wet flex, 20 of 60 samples (33%) had lifted on prep A, whereas 42 of 60 samples (70%) had lifted on prep B (P < .0001). After lavage, 23 of 60 samples (38%) had lifted on prep A, whereas 48 of 60 samples (80%) had lifted on prep B (P < .0001). Both preps were well tolerated with minimal erythema and no edema, rash, dryness, or denudation observed. No adverse events were reported. Conclusions: Prep A resulted in reduced frequency of incise drape lift from skin under wet conditions in this model compared with prep B.

Inhibitors of tri- and tetra- polyamine oxidation, but not diamine oxidation, impair the initial stages of wound-induced suberization.

The mechanisms regulating, and modulating potato wound-healing processes are of great importance in reducing tuber infections, reducing shrinkage and maintaining quality and nutritional value for growers and consumers. Wound-induced changes in tuber polyamine metabolism have been linked to the modulation of wound healing (WH) and in possibly providing the crucial amount of H2O2 required for suberization processes. In this investigation we determined the effect of inhibition of specific steps within the pathway of polyamine metabolism on polyamine content and the initial accumulation of suberin polyphenolics (SPP) during WH. The accumulation of SPP represents a critical part of the beginning or inchoate phase of tuber WH during closing-layer formation because it serves as a barrier to bacterial infection and is a requisite for the accumulation of suberin polyaliphatics which provide the barrier to fungal infection. Results showed that the inhibitor treatments that caused changes in polyamine content generally did not influence wound-induced accumulation of SPP. Such lack of correlation was found for inhibitors involved in metabolism and oxidation of putrescine (arginine decarboxylase, ornithine decarboxylase, and diamine oxidase). However, accumulation of SPP was dramatically reduced by treatment with guazatine, a potent inhibitor of polyamine oxidase (PAO), and methylglyoxal-bis(guanylhydrazone), a putative inhibitor of S-adenosylmethione decarboxylase which may also cross-react to inhibit PAO. The mode of action of these inhibitors is presumed to be blockage of essential H2O2 production within the WH cell wall. These results are of great importance in understanding the mechanisms modulating WH and ultimately controlling related infections and associated postharvest losses.

Breast mass classification in sonography with transfer learning using a deep convolutional neural network and color conversion.

PURPOSE: We propose a deep learning-based approach to breast mass classification in sonography and compare it with the assessment of four experienced radiologists employing breast imaging reporting and data system 4th edition lexicon and assessment protocol. METHODS: Several transfer learning techniques are employed to develop classifiers based on a set of 882 ultrasound images of breast masses. Additionally, we introduce the concept of a matching layer. The aim of this layer is to rescale pixel intensities of the grayscale ultrasound images and convert those images to red, green, blue (RGB) to more efficiently utilize the discriminative power of the convolutional neural network pretrained on the ImageNet dataset. We present how this conversion can be determined during fine-tuning using back-propagation. Next, we compare the performance of the transfer learning techniques with and without the color conversion. To show the usefulness of our approach, we additionally evaluate it using two publicly available datasets. RESULTS: Color conversion increased the areas under the receiver operating curve for each transfer learning method. For the better-performing approach utilizing the fine-tuning and the matching layer, the area under the curve was equal to 0.936 on a test set of 150 cases. The areas under the curves for the radiologists reading the same set of cases ranged from 0.806 to 0.882. In the case of the two separate datasets, utilizing the proposed approach we achieved areas under the curve of around 0.890. CONCLUSIONS: The concept of the matching layer is generalizable and can be used to improve the overall performance of the transfer learning techniques using deep convolutional neural networks. When fully developed as a clinical tool, the methods proposed in this paper have the potential to help radiologists with breast mass classification in ultrasound.

Evaluation of activity and potential for development of antimicrobial resistance to a new tinted 2% chlorhexidine gluconate/70% isopropyl alcohol film-forming sterile preoperative skin preparation.

OBJECTIVES: Chlorhexidine gluconate (CHG) is routinely used for skin antisepsis before surgery. Its activity may be affected by formulation ingredients and the presence of organic matter such as blood and proteins. This in vitro study was designed to evaluate the antimicrobial activity of a new CHG skin prep containing a film-forming copolymer, and detect its potential for developing resistance and the potential for cross-resistance to antibiotics after CHG exposure. METHODS: Antimicrobial activity was evaluated in the presence and absence of serum in an in vitro time-kill study. Emergence of resistance to CHG and cross-resistance with antibiotic procedures were performed in vitro using 10 repository isolates from eight species and eight clinical isolate strains equal to the repository isolate strains (four isolates, two resistant and two non-resistant per species). RESULTS: A 5 log10 reduction (99.999%) for all organisms was observed using the copolymer formulation. The activity remained unchanged in the presence of serum. The minimum inhibitory concentration (MIC) did not increase for any of the strains evaluated for emergence of resistance. In addition, there was no change in MIC related to cross-resistance observed for any of the organism/antibiotic combinations tested. CONCLUSIONS: These results suggest that the film-forming copolymer and the tint in the new CHG skin prep did not interfere with antimicrobial efficacy, even in the presence of an organic soil load, and that the tested formulations showed no potential for developing resistance or cross-resistance with antibiotics.

Bacterial expression of the phosphodiester-binding site of the cation-independent mannose 6-phosphate receptor for crystallographic and NMR studies.

The cation-independent mannose 6-phosphate receptor (CI-MPR) is a multifunctional protein that interacts with diverse ligands and plays central roles in autophagy, development, and tumor suppression. By delivering newly synthesized phosphomannosyl-containing acid hydrolases from the Golgi to endosomal compartments, CI-MPR is an essential component in the generation of lysosomes that are critical for the maintenance of cellular homeostasis. The ability of CI-MPR to interact with ∼60 different acid hydrolases is facilitated by its large extracellular region, with four out of its 15 domains binding phosphomannosyl residues. Although the glycan specificity of CI-MPR has been elucidated, the molecular basis of carbohydrate binding has not been determined for two out of these four carbohydrate recognition domains (CRD). Here we report expression of CI-MPR's CRD located in domain 5 that preferentially binds phosphodiester-containing glycans. Domain 5 of CI-MPR was expressed in Escherichia coli BL21 (DE3) cells as a fusion protein containing an N-terminal histidine tag and the small ubiquitin-like modifier (SUMO) protein. The His6-SUMO-CRD construct was recovered from inclusion bodies, refolded in buffer to facilitate disulfide bond formation, and subjected to Ni-NTA affinity chromatography and size exclusion chromatography. Surface plasmon resonance analyses demonstrated that the purified protein was active and bound phosphorylated glycans. Characterization by NMR spectroscopy revealed high quality (1)H-(15)N HSQC spectra. Additionally, crystallization conditions were identified and a crystallographic data set of the CRD was collected to 1.8Šresolution. Together, these studies demonstrate the feasibility of producing CI-MPR's CRD suitable for three-dimensional structure determination by NMR spectroscopic and X-ray crystallographic approaches.

Identification of a fourth mannose 6-phosphate binding site in the cation-independent mannose 6-phosphate receptor.

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays an essential role in lysosome biogenesis by targeting ∼ 60 different phosphomannosyl-containing acid hydrolases to the lysosome. This type I membrane glycoprotein has a large extracellular region comprised of 15 homologous domains. Two mannose 6-phosphate (M6P) binding sites have been mapped to domains 3 and 9, whereas domain 5 binds preferentially to the phosphodiester, M6P-N-acetylglucosamine (GlcNAc). A structure-based sequence alignment predicts that the C-terminal domain 15 contains three out of the four conserved residues identified as essential for carbohydrate recognition by domains 3, 5 and 9 of the CI-MPR, but lacks two cysteine residues that are predicted to form a disulfide bond. To determine whether domain 15 of the CI-MPR has lectin activity and to probe its carbohydrate-binding specificity, truncated forms of the CI-MPR were tested for binding to acid hydrolases with defined N-glycans in surface plasmon resonance analyses, and used to interrogate a phosphorylated glycan microarray. The results show that a construct encoding domains 14-15 binds both M6P and M6P-GlcNAc with similar affinity (Kd = 13 and 17 µM, respectively). Site-directed mutagenesis studies demonstrate the essential role of the conserved Tyr residue in domain 15 for phosphomannosyl binding. A structural model of domain 15 was generated that predicted an Arg residue to be in the binding pocket and mutagenesis studies confirmed its important role in carbohydrate binding. Together, these results show that the CI-MPR contains a fourth carbohydrate-recognition site capable of binding both phosphomonoesters and phosphodiesters.

Wounding induces changes in tuber polyamine content, polyamine metabolic gene expression, and enzyme activity during closing layer formation and initiation of wound periderm formation.

Tuber wound-healing processes are complex, and the associated regulation and modulation of these processes are poorly understood. Polyamines (PA) are involved in modulating a variety of responses to biotic and abiotic plant stresses and have been suggested to be involved in tuber wound responses. However, the time course of wound-induced changes in tuber PA content, activity of key biosynthetic enzymes and associated gene expression has not been determined and coordinated with major wound-healing processes. The objective of this study was to determine these wound-induced changes and their coordination with wound-healing processes. Wounding induced increases in putrescine (Put) and spermidine (Spd), but had only minor effects on spermine (Spm) content during the 168 h time course which encompassed the initiation and completion of the closing layer formation, and the initiation of cell division and wound periderm formation. As determinants of the first committed step in PA biosynthesis, arginine and ornithine decarboxylase (ADC and ODC, respectively) activities were below levels of detectability in resting tubers and expression of genes encoding these two enzymes was low. Within 6h of wounding, increases in the in vitro activities of ADC and ODC and expression of their cognate genes were observed. Expression of a gene encoding S-adenosylmethionine decarboxylase, required for Spd and Spm biosynthesis, was also increased 6h after wounding and remained elevated throughout the time course. Expression of a polyamine catabolic gene, encoding polyamine oxidase, was down-regulated after wounding. Results indicated a rapid wound-induced increase in PA biosynthesis during closing layer formation and the time of nuclei entry and exit from S-phase. PA content remained elevated as wound-induced cells became meristematic and initiated formation of the wound periderm suggesting sustained involvement in wound-healing.

Quantitative volumetric breast imaging with 3D inverse scatter computed tomography.

A method was developed to map tissue properties of the entire breast including sound speed and attenuation using fully 3D nonlinear inverse-scattering tomography. Clinical measurements suggest that in breast tissue benign and cancerous lesions may be identified in part by these inherent acoustic parameters. Sound speed accuracy and linearity are very high over a wide range (1325-1700 m/sec) with ~1.5 mm resolution at 2 MHz in transmission mode. Attenuation tomograms provide image contrast over a wide range (0-4 dB/cm/MHz) and assist classification of masses. High resolution 0.6 mm volumetric reflection tomograms are acquired with bandwidth 2-8 MHz, are refraction-corrected with the transmission tissue data and are precisely registered in 3D with the transmission volumes. USCT promises an automated whole-breast scan providing a global view of the entire breast in 3D, facilitating comparison to prior exams in a reproducible geometry. Scanner design, automated operation and results of our trial with over 125 subjects with confirmed breast masses will be presented with detailed comparison to conventional sonography and MRI.

Accelerated partial breast irradiation and posttreatment imaging evaluation.

Accelerated partial breast irradiation (APBI) is a technique that allows irradiation of only that part of the breast that is at greatest risk for recurrence of breast cancer. Because only a portion of the breast is irradiated, APBI can be performed in a relatively short period of time, usually in 5 days rather than the traditional 6 weeks. When used in carefully selected patients, APBI also allows normal portions of the breast parenchyma and regional vital organs to be spared from unnecessary irradiation. Common post-APBI imaging findings include focal skin thickening, seroma, scar, and skin retraction. Studies are underway that will compare a cohort of patients who underwent whole-breast irradiation with a cohort who underwent APBI to help determine whether the two techniques lead to significantly different imaging findings. Additional multicenter studies will be needed to document and analyze any such differences. In the future, APBI may play a significant role in selected patients, with pretherapy dynamic contrast material-enhanced magnetic resonance imaging of the breast possibly aiding in the selection process.

Mannose 6-phosphate receptor homology (MRH) domain-containing lectins in the secretory pathway.

BACKGROUND: The mannose 6-phosphate receptor homology (MRH) domain-containing family of proteins, which include recycling receptors (mannose 6-phosphate receptors, MPRs), resident endoplasmic reticulum (ER) proteins (glucosidase II ß-subunit, XTP3-B, OS-9), and a Golgi glycosyltransferase (GlcNAc-phosphotransferase γ-subunit), are characterized by the presence of one or more MRH domains. Many MRH domains act as lectins and bind specific phosphorylated (MPRs) or non-phosphorylated (glucosidase II ß-subunit, XTP3-B and OS-9) high mannose-type N-glycans. The MPRs are the only proteins known to bind mannose 6-phosphate (Man-6-P) residues via their MRH domains. SCOPE OF REVIEW: Recent biochemical and structural studies that have provided valuable insight into the glycan specificity and mechanisms of carbohydrate recognition by this diverse group of MRH domain-containing proteins are highlighted. MAJOR CONCLUSIONS: Currently, three-dimensional structures are known for ten MRH domains, revealing the conservation of a similar fold. OS-9 and the MPRs use the same four residues (Gln, Arg, Glu, and Tyr) to bind mannose. GENERAL SIGNIFICANCE: The MRH domain-containing proteins play key roles in the secretory pathway: glucosidase II, XTP3-B, and OS-9 are involved in the recognition of nascent glycoproteins, whereas the MPRs play an essential role in lysosome biogenesis by targeting Man-6-P-containing lysosomal enzymes to the lysosome.

Twists and turns of the cation-dependent mannose 6-phosphate receptor. Ligand-bound versus ligand-free receptor.

Mannose 6-phosphate receptors (MPRs) participate in the biogenesis of lysosomes in higher eukaryotes by transporting soluble acid hydrolases from the trans-Golgi network to late endosomal compartments. The receptors release their ligands into the acidic environment of the late endosome and then return to the trans-Golgi network to repeat the process. However, the mechanism that facilitates ligand binding and dissociation upon changes in pH is not known. We report the crystal structure of the extracytoplasmic domain of the homodimeric cation-dependent MPR in a ligand-free form at pH 6.5. A comparison of the ligand-bound and ligand-free structures reveals a significant change in quaternary structure as well as a reorganization of the binding pocket, with the most prominent change being the relocation of a loop (residues Glu(134)-Cys(141)). The movements involved in the bound-to-free transition of the cation-dependent MPR are reminiscent of those of the oxy-to-deoxy hemoglobin transition. These results allow us to propose a mechanism by which the receptor regulates its ligand binding upon changes in pH; the pK(a) of Glu(133) appears to be responsible for ligand release in the acidic environment of the late endosomal compartment, and the pK(a) values of the sugar phosphate and His(105) are accountable for its inability to bind ligand at the cell surface where the pH is about 7.4.