A role for 3-O-sulfated heparan sulfate in promoting human cytomegalovirus infection in human iris cells.

Human cytomegalovirus (HCMV) has emerged as a clinically opportunistic pathogen that targets multiple types of ocular cells and tissues, including the iris region of the uveal tract during anterior uveitis. In this report, we used primary cultures of human iris stroma (HIS) cells derived from human eye donors to investigate HCMV entry. The following lines of evidence suggested the role of 3-O-sulfated heparan sulfate (3-OS HS) during HCMV-mediated entry and cell-to-cell fusion in HIS cells. First, 3-O-sulfotransferase-3 (3-OST-3) expression in HIS cells promoted HCMV internalization, while pretreatment of HIS cells with heparinase enzyme or with anti-3-OS HS (G2) peptide significantly reduced the HCMV-mediated formation of plaques/foci. Second, coculture of the HCMV-infected HIS cells with CHO-K1 cells expressing 3-OS HS significantly enhanced cell fusion. Finally, a similar trend of enhanced fusion was observed with cells expressing HCMV glycoproteins (gB, gO, and gH-gL) cocultured with 3-OS HS cells. Taken together, these results highlight the role of 3-OS HS during HCMV plaque formation and cell-to-cell fusion and identify a novel target for future therapeutic interventions.

Through the Looking Glass: Real-Time Video Using 'Smart' Technology Provides Enhanced Intraoperative Logistics.

INTRODUCTION: In the setting of increasingly complex medical therapies and limited physician resources, the recent emergence of 'smart' technology offers tremendous potential for improved logistics, efficiency, and communication between medical team members. In an effort to harness these capabilities, we sought to evaluate the utility of this technology in surgical practice through the employment of a wearable camera device during cardiothoracic organ recovery. METHODS: A single procurement surgeon was trained for use of an Explorer Edition Google Glass (Google Inc., Mountain View, CA) during the recovery process. Live video feed of each procedure was securely broadcast to allow for members of the home transplant team to remotely participate in organ assessment. Primary outcomes involved demonstration of technological feasibility and validation of quality assurance through group assessment. RESULTS: The device was employed for the recovery of four organs: a right single lung, a left single lung, and two bilateral lung harvests. Live video of the visualization process was remotely accessed by the home transplant team, and supplemented final verification of organ quality. In each case, the organs were accepted for transplant without disruption of standard procurement protocols. Media files generated during the procedures were stored in a secure drive for future documentation, evaluation, and education purposes without preservation of patient identifiers. CONCLUSIONS: Live video streaming can improve quality assurance measures by allowing off-site members of the transplant team to participate in the final assessment of donor organ quality. While further studies are needed, this project suggests that the application of mobile 'smart' technology offers not just immediate value, but the potential to transform our approach to the practice of medicine.

Comprehensive analysis of herpes simplex virus 1 (HSV-1) entry mediated by zebrafish 3-O-Sulfotransferase isoforms: implications for the development of a zebrafish model of HSV-1 infection.

Binding of herpes simplex virus 1 (HSV-1) envelope glycoprotein D (gD) to the receptor 3-O-sulfated heparan sulfate (3-OS HS) mediates viral entry. 3-O-Sulfation of HS is catalyzed by the 3-O-sulfotransferase (3-OST) enzyme. Multiple isoforms of 3-OST are differentially expressed in tissues of zebrafish (ZF) embryos. Here, we performed a comprehensive analysis of the role of ZF 3-OST isoforms (3-OST-1, 3-OST-5, 3-OST-6, and 3-OST-7) in HSV-1 entry. We found that a group of 3-OST gene family isoforms (3-OST-2, -3, -4, and -6) with conserved catalytic and substrate-binding residues of the enzyme mediates HSV-1 entry and spread, while the other group (3-OST-1, -5, and -7) lacks these properties. These results demonstrate that HSV-1 entry can be recapitulated by certain ZF 3-OST enzymes, a significant step toward the establishment of a ZF model of HSV-1 infection and tissue-specific tropism.

Is Early Reperfusion a Good Thing? Optimal Timing of CABG Surgery Postacute Myocardial Infarction.

OBJECTIVES: The optimal timing of coronary artery bypass grafting (CABG) following an acute myocardial infarction (AMI) is a topic of debate. The present study was designed to evaluate patients undergoing CABG both early (<5 days) and late (>5 days) after AMI in the era of percutaneous coronary intervention. METHODS: The medical records at our institution from 2008 through 2012 were reviewed. A total of 128 patients underwent CABG after AMI during this time period and fulfilled criteria for the study. Death, stroke, renal failure, need for intraaortic balloon pump, postoperative ventilator days, and length of stay were examined. RESULTS: Patients undergoing early CABG had an increased need for an intraaortic balloon pump. There were no other correlations that we could discern between early and late CABG. CONCLUSIONS: Our data demonstrate no statistical difference in mortality or in the factors of morbidity studied between either early or late CABG after AMI.

Susceptibility of human iris stromal cells to herpes simplex virus 1 entry.

Ocular herpes simplex virus 1 (HSV-1) infection can lead to multiple complications, including iritis, an inflammation of the iris. Here, we use human iris stroma cells as a novel in vitro model to demonstrate HSV-1 entry and the inflammatory mediators that can damage the iris. The upregulated cytokines observed in this study provide a new understanding of the intrinsic immune mechanisms that can contribute to the onset of iritis.

Cofractionation of HMGB proteins with histone dimers.

An effective and flexible method is presented that can be used to investigate cofractionation of groups of nuclear proteins. The method was used to analyze chromatin-related proteins, of which high-mobility group B (HMGB) proteins consistently cofractionated by cation-exchange chromatography with the histone dimer (H2A-H2B). This led to the hypothesis that the two form a complex, further suggested by gel filtration, in which the HMGBs with core histones eluted as a defined high-molecular-weight peak. A necessary requirement for further studying protein interactions is that the constituents are of the highest possible purity and the pure histone dimers and tetramers used in this study were derived from pure histone octamers with their native marks. There is a growing interest in protein-protein interactions and an increasing focus on protein-interaction domains: most frequently, pull-down assays are used to examine these. The technology presented here can provide an effective system that complements pull-down assays.

Gas phase thermal reactions of exo-8-cyclopropyl-bicyclo[4.2.0]oct-2-ene (1-exo).

The title compound 1-exo (with minor amounts of its C8 epimer 1-endo) was prepared by Wolff-Kishner reduction of the cycloadduct of 1,3-cyclohexadiene and cyclopropylketene. The [1,3]-migration product 2-endo was synthesized by efficient selective cyclopropanation of endo-5-vinylbicyclo[2.2.2]oct-2-ene at the exocyclic π-bond. Gas phase thermal reactions of 1-exo afforded C8 epimerization to 1-endo, [1,3]- migrations to 2-exo and 2-endo, direct fragmentation to cyclohexadiene and vinylcyclopropane, and CPC rearrangement in the following relative kinetic order: kep > k13 > kf > kCPC.

Zebrafish encoded 3-O-sulfotransferase-2 generated heparan sulfate serves as a receptor during HSV-1 entry and spread.

Previously we reported the role of zebrafish (ZF) encoded glucosaminyl 3-O-sulfotransferase-3 (3-OST-3) isoform in assisting herpes simplex virus type-1 (HSV-1) entry and spread by generating an entry receptor to HSV-1 envelope glycoprotein D (gD). However, the ability of ZF encoded 3-OST-2 isoform to participate in HSV-1 entry has not been determined although it is predominantly expressed in ZF brain, a prime target for HSV-1 to infect and establish lifelong latency. Here we report the expression cloning of ZF encoded 3-OST-2 isoform and demonstrate HSV-1 entry into resistant Chinese hamster ovary (CHO-K1) cells expressing the clone. Additional significance of ZF encoded 3-OST-2 receptor was demonstrated using medically important isolates of HSV-1. In addition, interference to HSV-1 entry was observed upon co-expression of HSV-1 gD and ZF 3-OST-2. Similarly HSV-1 entry was significantly inhibited by the pre-treatment of cells with enzyme HS lyases (heparinase II/III). Finally, ZF-3-OST-2 expressing CHO-K1 was able to fuse with HSV-1 glycoprotein expressing cells suggesting their role in HSV-1 spread. Taken together our result demonstrates a role for ZF 3-OST-2 in HSV-1 pathogenesis.

Over-prescribing of antibiotics and imaging in the management of uncomplicated URIs in emergency departments.

BACKGROUND: Unnecessary use of resources for common illnesses has substantial effect on patient care and costs. Evidence-based guidelines do not recommend antibiotics or imaging for uncomplicated upper respiratory infections (URIs). The objective of the current study was to examine medical care providers' compliance with guidelines in treating uncomplicated URIs in emergency departments (EDs) in the US. METHODS: Nationally representative data from the NHAMCS 2007 and 2008 were used. Uncomplicated URIs were identified through ICD-9 codes of nasopharyngitis, laryngitis, bronchitis, URI not otherwise specified and influenza involving upper respiratory tract. Exclusion criteria were concurrent comorbidities, follow-up visits, and age < 18 or >64 years. Most frequently prescribed classes of antibiotics were identified. Multivariate analyses were conducted to identify the factors associated with the prescribing of antibiotics and use of imaging studies. RESULTS: In 2007 and 2008, there were 2.2 million adult uncomplicated URI visits without any other concurrent diagnoses in EDs in the US. Approximately 52% were given antibiotic prescriptions, over one-third of which were macrolides, and nearly half of the visits performed imaging studies. About 51% had a diagnosis of bronchitis, 35% URI NOS, 9% nasopharyngitis, laryngitis or influenza, and 4% multiple URI diagnoses. The diagnosis of bronchitis, fever at presentation, older ages, male gender, longer waiting time, and metropolitan areas were associated with a greater likelihood of prescribing antibiotics or imaging studies, controlling for confounding factors. CONCLUSION: Despite the recommendations and campaign efforts by the CDC and many medical associations, the prescribing of antibiotics in treating uncomplicated URIs in the EDs remains prevalent. Furthermore, overutilization of imaging studies is prevalent. Changes at levels of health care system and hospitals are needed to avoid unnecessary resource utilization. In addition, further patient education about antibiotic use in the community may greatly facilitate the transition out of an antibiotic-dependent consumer culture.

Thermal isomerizations of cis,anti,cis-tricyclo[7.4.0.0(2,8)]tridec-10-ene.

cis,anti,cis-Tricyclo[7.4.0.0(2,8)]tridec-10-ene (13TCT) undergoes [1,3] sigmatropic rearrangements at 315 °C in the gas phase to the si product 1 and to the sr product 2 with si/sr = 2.1. The dominant thermal isomerization process, however, is epimerization at C8 to afford product 3. That stereomutation at C8 occurs 50% faster than the si and sr shifts combined.

Ultrafast visualization of incipient plasticity in dynamically compressed matter.

Plasticity is ubiquitous and plays a critical role in material deformation and damage; it inherently involves the atomistic length scale and picosecond time scale. A fundamental understanding of the elastic-plastic deformation transition, in particular, incipient plasticity, has been a grand challenge in high-pressure and high-strain-rate environments, impeded largely by experimental limitations on spatial and temporal resolution. Here, we report femtosecond MeV electron diffraction measurements visualizing the three-dimensional (3D) response of single-crystal aluminum to the ultrafast laser-induced compression. We capture lattice transitioning from a purely elastic to a plastically relaxed state within 5 ps, after reaching an elastic limit of ~25 GPa. Our results allow the direct determination of dislocation nucleation and transport that constitute the underlying defect kinetics of incipient plasticity. Large-scale molecular dynamics simulations show good agreement with the experiment and provide an atomic-level description of the dislocation-mediated plasticity.

III. Identification of novel CXCR3 chemokine receptor antagonists with a pyrazinyl-piperazinyl-piperidine scaffold.

The SAR of a novel pyrazinyl-piperazinyl-piperidine scaffold with CXCR3 receptor antagonist activity was explored. Optimization of the DMPK profile and reduction of hERG inhibition is described. Compound 16e with single-digit CXCR3 affinity, good rat PK and hERG profiles has been identified as a lead for further study.

Biphenyl/diphenyl ether renin inhibitors: filling the S1 pocket of renin via the S3 pocket.

Structure-based design led to the discovery of a novel class of renin inhibitors in which an unprecedented phenyl ring filling the S1 site is attached to the phenyl ring filling the S3 pocket. Optimization for several parameters including potency in the presence of human plasma, selectivity against CYP3A4 inhibition and improved rat oral bioavailability led to the identification of 8d which demonstrated antihypertensive efficacy in a transgenic rat model of human hypertension.

On the stereochemical characteristic of the thermal reactions of vinylcyclobutane.

This Perspective outlines the stereochemical and mechanistic complexities inherent in the thermal reactions converting vinylcyclobutane to cyclohexene, butadiene, and ethylene. The structural isomerization and the fragmentation processes seem, at first sight, to be obvious and simple. When considered more carefully and investigated with the aid of deuterium-labeled stereochemically well-defined vinylcyclobutane derivatives there emerges a complex kinetic situation traced by 56 structure-to-structure transformations and 12 independent kinetic parameters. Experimental determinations of stereochemical details of stereomutations and [1,3] carbon sigmatropic shifts are now being pursued and will in time contribute to gaining relevant evidence casting light on the reaction dynamics involved as flexible short-lived diradical intermediates trace the paths leading from one d(2)-labeled vinylcyclobutane starting material to a mixture of 16 structures.

Optimization of orally bioavailable alkyl amine renin inhibitors.

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC(50) of 0.83nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10mg/kg resulted in >20h reduction of blood pressure in a double transgenic rat model of hypertension.

In-Vivo Toxicity Studies and In-Vitro Inactivation of SARS-CoV-2 by Povidone-iodine In-situ Gel Forming Formulations.

To curb the spread of SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, we characterize the virucidal activity of long-acting Povidone Iodine (PVP-I) compositions developed using an in-situ gel forming technology. The PVP-I gel forming nasal spray (IVIEW-1503) and PVP-I gel forming ophthalmic eye drop (IVIEW-1201) rapidly inactivated SARS-CoV-2, inhibiting the viral infection of VERO76 cells. No toxicity was observed for the PVP-I formulations. Significant inactivation was noted with preincubation of the virus with these PVP-I formulations at the lowest concentrations tested. It has been demonstrated that both PVP-I formulations can inactivate SARS-CoV-2 virus efficiently in both a dose-dependent and a time-dependent manner. These results suggest IVIEW-1503 and IVIEW-1201 could be potential agents to reduce or prevent the transmission of the virus through the nasal cavity and the eye, respectively. Further studies are needed to clinically evaluate these formulations in early-stage COVID-19 patients.

Quantitative analyses of mixtures of 2-deuterio-1-vinylcyclobutanes.

Making distinctions between two stereoisomers characterized by diastereotopic deuterium atoms can ordinarily be achieved using standard NMR spectroscopic methods. Mixtures of stereoisomers having both diastereotopic and enantiotopic deuterium labels, however, may be difficult to analyze quantitatively. The present work introduces a simple way to gain quantitative analyses of mixtures of the four stereoisomeric 2-deuterio-1-vinylcyclobutanes, an essential prerequisite to establishing the stereochemical characteristics of the thermal stereomutations of vinylcyclobutane and its structural isomerizations to cyclohexene. The unconventional NMR method introduced and validated in this work will likely prove convenient and generally applicable to related stereochemical investigations.

Quantitative analyses of stereoisomeric 3,4-d2-cyclohexenes in the presence of 3,6-d2-cyclohexenes.

The challenging analytical problem posed by mixtures of the four isomeric 3,4-d(2)-cyclohexenes and the three isomeric 3,6-d(2)-cyclohexenes has been solved through a novel one-dimensional NMR spectroscopic method dependent on recording (13)C resonances while broadband decoupling both proton and deuteron nuclei. Upfield deuterium perturbations of (13)C chemical shifts in chair conformationally locked cyclohexane derivatives readily secured from a mixture of the seven deuterium-labeled cyclohexenes allow quantitative analytical assessments of the four possible 3,4-d(2)-cyclohexenes in the mixture. This analytical capability is an essential prerequisite for uncovering the relative participations of the four possible stereochemical paths followed by the thermal structural isomerizations of vinylcyclobutane to cyclohexene. The unconventional NMR method validated in this work will likely prove invaluable in related stereochemical investigations.

Design and optimization of renin inhibitors: Orally bioavailable alkyl amines.

Structure-based drug design led to the identification of a novel class of potent, low MW alkylamine renin inhibitors. Oral administration of lead compound 21l, with MW of 508 and IC(50) of 0.47nM, caused a sustained reduction in mean arterial blood pressure in a double transgenic rat model of hypertension.

Sulfonamido-aryl ethers as bradykinin B1 receptor antagonists.

The synthesis and identification of sulfonamido-aryl ethers as potent bradykinin B1 receptor antagonists from a approximately 60,000 member encoded combinatorial library are reported. Two distinct series of compounds exhibiting different structure-activity relationships were identified in a bradykinin B1 whole-cell receptor-binding assay. Specific examples exhibit K(i) values of approximately 10nM.