bluuebunny/crossref_metadata_2025 huggingface dataset:
Divine Goodness, Predestination, and the Hypostatic Union: St. Thomas on the Temporal Realization of the Father's Eternal Plan in the Incarnate Son
Authors: Nutt Roger W.
Published: July 2016

This article considers Aquinas' doctrine of predestination as an eternal reality in God in light of its temporal realization in time by the incarnation of the eternal Son. In particular, Aquinas' repeated recourse to the ratio of the divine goodness as the motive of predestination is documented in conjunction with his teaching on the fittingness of the incarnation. In this light, the relation of the natural sonship of Christ to the grace of adoption is developed by Aquinas as the temporal realization of the eternal reality of predestination in God.
In vivo transplantation of fetal human gut‐derived enteric neural crest cells
Authors: Cooper J. E. , Natarajan D. , McCann C. J. , Choudhury S. , Godwin H. , Burns A. J. , Thapar N.
Published: July 2016

The prospect of using neural cell replacement for the treatment of severe enteric neuropathies has seen significant progress in the last decade. The ability to harvest and transplant enteric neural crest cells (ENCCs) that functionally integrate within recipient intestine has recently been confirmed by in vivo murine studies. Although similar cells can be harvested from human fetal and postnatal gut, no studies have as yet verified their functional viability upon in vivo transplantation. We sought to determine whether ENCCs harvested from human fetal bowel are capable of engraftment and functional integration within recipient intestine following in vivo transplantation into postnatal murine colon. Enteric neural crest cells selected and harvested from fetal human gut using the neurotrophin receptor p75NTR were lentivirally labeled with either GFP or calcium‐sensitive GCaMP and transplanted into the hindgut of Rag2−/γc−/C5−‐immunodeficient mice at postnatal day 21. Transplanted intestines were assessed immunohistochemically for engraftment and differentiation of donor cells. Functional viability and integration with host neuromusculature was assessed using calcium imaging. Transplanted human fetal gut‐derived ENCC showed engraftment within the recipient postnatal colon in 8/15 mice (53.3%). At 4 weeks posttransplantation, donor cells had spread from the site of transplantation and extended projections over distances of 1.2 ± 0.6 mm (n = 5), and differentiated into enteric nervous system (ENS) appropriate neurons and glia. These cells formed branching networks located with the myenteric plexus. Calcium transients (change in intensity F/F0 = 1.25 ± 0.03; 15 cells) were recorded in transplanted cells upon stimulation of the recipient endogenous ENS demonstrating their viability and establishment of functional connections.
Pancreatic polypeptide stimulates mouse gastric motor activity through peripheral neural mechanisms
Authors: Amato A. , Baldassano S. , Caldara G. F. , Mulè F.
Published: July 2016

Background

Pancreatic polypeptide (PP) is supposed to be one of the major endogenous agonists of the neuropeptide Y4 receptor. Pancreatic polypeptide can influence gastrointestinal motility, acting mainly through vagal mechanisms, but whether PP acts directly on the stomach has not been explored yet. The aims of this study were to investigate the effects of PP on mouse gastric emptying, on spontaneous tone of whole stomach in vitro and to examine the mechanism of action.

Methods

Gastric emptying was measured by red phenol method after i.p. PP administration (1–3 nmol per mouse). Responses induced by PP (1–300 mmol L−1) on gastric endoluminal pressure were analyzed in vitro in the presence of different drugs. Gastric genic expression of Y4 receptor was verified by RT‐PCR.

Key Results

Pancreatic polypeptide dose‐dependently increased non‐nutrient liquid gastric emptying rate. In vitro, PP produced a concentration‐dependent contraction that was abolished by tetrodotoxin, a neural blocker of Na+ voltage‐dependent channels. The contractile response was significantly reduced by atropine, a muscarinic receptor antagonist, and by SR48968, an NK2 receptor antagonist, while it was potentiated by neostigmine, an inhibitor of acetylcholinesterase. The joint application of atropine and SR48968 fully abolished PP contractile effect. Reverse transcriptase‐polymerase chain reaction analysis revealed the presence of Y4 receptor mRNA in mouse stomach with a greater expression in antrum than in fundus.

Conclusions & Inferences

The present findings demonstrate that exogenous PP stimulates mouse gastric motor activity, by acting directly on the stomach. This effect appears due to the activation of enteric excitatory neurons releasing acetylcholine and tachykinins.
Stage‐directed individualized therapy in esophageal cancer
Authors: Goense Lucas , van Rossum Peter S.N. , Kandioler Daniela , Ruurda Jelle P. , Goh Khean‐Lee , Luyer Misha D. , Krasna Mark J. , van Hillegersberg Richard
Published: July 2016

Esophageal cancer is the eighth most common cancer worldwide, and the incidence of esophageal carcinoma is rapidly increasing. With the advent of new staging and treatment techniques, esophageal cancer can now be managed through various strategies. A good understanding of the advances and limitations of new staging techniques and how these can guide in individualizing treatment is important to improve outcomes for esophageal cancer patients. This paper outlines the recent progress in staging and treatment of esophageal cancer, with particularly attention to endoscopic techniques for early‐stage esophageal cancer, multimodality treatment for locally advanced esophageal cancer, assessment of response to neoadjuvant treatment, and the role of cervical lymph node dissection. Furthermore, advances in robot‐assisted surgical techniques and postoperative recovery protocols that may further improve outcomes after esophagectomy are discussed.
Tetramethylenedisulfotetramine: pest control gone awry
Authors: Shakarjian Michael P. , Laukova Marcela , Velíšková Jana , Stanton Patric K. , Heck Diane E. , Velíšek Libor
Published: July 2016

Incidences of pesticide poisonings are a significant cause of morbidity and mortality worldwide. The seizure‐inducing rodenticide tetramethylenedisulfotetramine is one of the most toxic of these agents. Although banned, it has been responsible for thousands of accidental, intentional, and mass poisonings in mainland China and elsewhere. An optimal regimen for treatment of poisoning has not been established. Its facile synthesis from easily obtained starting materials, extreme potency, and lack of odor, color, or taste make it a potential chemical threat agent. This review describes the toxicologic properties of this agent, more recent advances in our understanding of its properties, and recommendations for future research.
The Effects of Shock from Defibrillation Threshold Testing on Cardiac Systolic and Diastolic Function
Authors: KODANI TAKESHI , MINE TAKANAO , KISHIMA HIDEYUKI , MASUYAMA TOHRU
Published: July 2016

Background

Inappropriate implantable cardioverter defibrillator (ICD) shocks are associated with increased overall mortality. However, it remains unclear whether shocks from defibrillation threshold (DFT) testing directly impair cardiac function.

Methods

DFT testing was performed in 34 patients who underwent ICD/cardiac resynchronization therapy with a defibrillator implantation/generator exchange. Heart rate and cardiac function, including left ventricular (LV) systolic pressure, LV end‐diastolic pressure, peak positive and negative dp/dt ( + dp/dt and –dp/dt, respectively) of LV pressure, and the tau index, were assessed with a Mikro‐Cath™ diagnostic pressure catheter (CD Leycom, Zoetermeer, the Netherlands). These parameters were measured before and 1 minute, 3 minutes, 5 minutes, 10 minutes, and 15 minutes after DFT testing.

Results

Peak positive dp/dt increased over baseline at each interval (976 ± 229 mm Hg/s vs 1,039 ± 258, 1,049 ± 245, 1,042 ± 247, 1,037 ± 259, and 1,034 ± 254 mm Hg/s, respectively; P < 0.01). Furthermore, peak negative dp/dt (–1,140 ± 397 mm Hg/s vs –1,185 ± 447, 1,193 ± 435, –1,195 ± 434, –1,189 ± 449, and –1,186 ± 459 mm Hg/s, respectively; P < 0.01) and the tau index (65.1 ± 18.5 vs 62.5 ± 16.8*, 62.4 ± 15.9**, 63.0 ± 16.8*, and 62.8 ± 18.7*, respectively; *P < 0.05, **P < 0.01) decreased compared to those at baseline at each interval.

Conclusion

Shock after DFT testing improved LV systolic and diastolic function immediately, especially in patients with preserved LV ejection fraction.
Towards a better understanding of the dynamics of value creation in R&D intensive small firms
Authors: Casault Sébastien , Groen Aard J. , Linton Jonathan D.
Published: July 2016

Standard financial and economic theories suggest that the stock value of R&D intensive High Technology Small Firms (HTSF) undergo a geometric random walk. Such a model neglects to account for observed periods where firms experience large fluctuations due to uncertainty related to its R&D activities, external competitive or regulatory environments. Empirical evidence also shows that the behavior of these firms is difficult to describe – let alone predict – using this Gaussian process. With ambidexterity as a theoretical basis, we show that the value of HTSF can be statistically described as the result of a combination of two distinct random walks: an exploitative steady state component characterized by Neo‐Marshallian equilibrium and low volatility; and a more dynamic component with high volatility reflecting bursts of large and rapid changes associated with Schumpeterian outcomes of explorative processes. A mixture of two normal distributions provides an overall function that is more reflective of the empirical evidence and provides a quantitative measure for the theory that firms engage in concurrent exploration/exploitation activities. A linear relationship between the two components of the mixture distribution that describe the stock value of these firms also emerges. By understanding this dual nature and its impact on stock value, firms can better manage resources and prepare for the increase in variability that are associated with exploration activities. A more accurate financial description of HTSF that reduces or that anticipates uncertainty may lead to financial tools and option pricing methods that put a premium on the value of HTSF markets, incentivizing investors to invest more in such firms.
Airway remodelling in COPD: It's not asthma!
Authors: Jones Robyn L. , Noble Peter B. , Elliot John G. , James Alan L.
Published: July 2016

COPD is defined as airflow limitation that is not reversed by treatment. In asthma, airflow limitation is not only reversible, but also inducible. This is called ‘airway hyperresponsiveness’ (AHR) and is associated with thickening of the airway wall, predominantly the layer of airway smooth muscle, due to more cells, bigger cells and more extracellular matrix (ECM) in proportion to the increase in smooth muscle. AHR is also observed in COPD if the changes in airflow are expressed as a percent of the baseline lung function. However, the absolute change in baseline lung function that can be induced in COPD is actually less than that seen in normal subjects, suggesting that the airways in COPD are resistant not only to opening, but also to closing. This observation agrees with physiological measures showing increased airway wall stiffness in COPD. Like asthma, airway wall thickness is increased in COPD, including the layer of smooth muscle. Unlike asthma, however, fixed airflow obstruction appears to be characterized by a disproportionate increase in the ECM within the smooth muscle layer. In this review, we summarize the studies of airway matrix deposition in COPD and put forward the proposal that the airway remodelling in COPD is different from that in asthma and call for a systematic analysis of airway matrix deposition in COPD.
Arterial stiffness in obstructive sleep apnoea: Is there a difference between daytime and night‐time?
Authors: Sarinc Ulasli Sevinc , Sariaydin Muzaffer , Ozkececi Gulay , Gunay Ersin , Halici Bilal , Unlu Mehmet
Published: July 2016

Background and objective

Obstructive sleep apnoea syndrome (OSAS) is a common chronic systemic disease in the general population, with known associated cardiovascular outcomes. We aimed to investigate arterial stiffness in OSAS patients and compare daytime and night‐time values with control subjects.

Methods

A total of 104 patients undergoing investigation for OSAS with polysomnography also underwent pulse wave velocity (PWV) and augmentation index (AIx) measurements with Mobil‐O‐Graph for 24 h. Eighty‐two patients were found to have OSAS and 22 did not have OSAS and acted as controls.

Results

PWV values of the 82 OSAS patients during the 24‐h period, both daytime and night‐time, were significantly higher than that of the control subjects. Moreover, night‐time levels of AIx were significantly higher in OSAS patients than control subjects (P = 0.025). PWV during night‐time was higher than daytime measurements in OSAS patients (P = 0.012). Apnoea hypopnoea index (AHI) was significantly correlated with PWV and AIx over 24 h (P = 0.0001, r = 0.412; P = 0.002, r = 0.333, respectively). Positive correlations were also found between oxygen desaturation index (ODI) with PWV and AIx during the night (P = 0.0001, r = 0.480; P = 0.002, r = 0.325, respectively). However, daytime AIx was not significantly correlated with ODI (P = 0.052, r = 0.205).

Conclusion

OSAS patients, without known cardiovascular disease, have increased PWV, indicating an increased arterial stiffness, compared with control subjects and correlations between AHI and arterial stiffness indices suggest increased arterial stiffness with increased disease severity. Therefore, arterial stiffness should be considered as a possible cause for cardiovascular complications in OSAS patients.
Growth, Real Exchange Rates and Trade Protectionism since the Financial Crisis
Authors: Georgiadis Georgios , Gräb Johannes
Published: July 2016

Existing evidence suggests that protectionist activity since the financial crisis has been muted, raising the question whether the historically well‐documented relationship between growth, real exchange rates and trade protectionism has broken down. We use a novel and comprehensive dataset that stretches beyond the traditionally considered tariff and trade defense measures to study the responsiveness of trade policies to business cycles and price competitiveness in the time period since the financial crisis. We find that the specter of protectionism has not been banished. Countries continue to pursue more trade‐restrictive policies when they experience recessions and/or when their competitiveness deteriorates. Our results suggest that the global economy continues to be exposed to the risk of a creeping return of trade protectionism.
Monitoring of adenovirus (ADV)‐specific T cells in a boy with ADV pneumonia and disseminated disease after lung transplantation
Authors: Schultze‐Florey R.E. , Tischer S. , Schwerk N. , Heim A. , Eiz‐Vesper B. , Maecker‐Kolhoff B.
Published: July 2016

Human adenovirus (ADV) infections are the cause of severe morbidity and mortality in transplant recipients. Cidofovir (CDV) is the current standard antiviral treatment. We report the case of a 3‐year‐old boy after lung transplantation with severe ADV sepsis, who was monitored for ADV‐specific T cells during his disease and recovery. A strong increase in ADV‐specific T cells was accompanied by resolution of ADV in blood and bronchoalveolar lavage fluid. Antiviral treatment with CDV was individually adapted according to anti‐ADV immune responses, which provides a new method for tailoring antiviral treatment in lung transplant recipients.
Laparoscopic Cholecystoduodenostomy in Dogs: Canine Cadaver Feasibility Study
Authors: Díaz‐Güemes Martín‐Portugués Idoia , Maria Matos‐Azevedo Ana , Enciso Sanz Silvia , Sánchez‐Margallo Francisco Miguel
Published: July 2016

Objective

To evaluate the feasibility of laparoscopic cholecystoduodenostomy in canine cadavers using barbed self‐locking sutures.

Study Design

In vivo experimental study.

Animals

Fresh male Beagle cadavers (n=5).

Methods

Surgery was performed by a single veterinary surgeon. Dogs were placed in dorsal recumbency and 15° reverse Trendelenburg position. The surgical procedure was performed with four 5 mm entry ports and a 5 mm 30° telescope. The cholecystoduodenostomy technique included dissection, incision of the gallbladder, and lavage, followed by gallbladder transposition over the duodenum, incision of the duodenum, and anastomosis. The latter was performed with a 4‐0 barbed self‐locking suture (V‐Loc® 180). Subsequently, a leak test was performed by submerging the anastomosis in saline and insufflating air into the duodenum through a catheter. Total operative time and completion times for each procedural step were recorded.

Results

The median total operative time was 151 minutes (range, 129–159). One conversion to open surgery occurred because of vascular hemorrhage. The 3 longest intraoperative steps were posterior wall anastomosis, gallbladder dissection, and anterior wall anastomosis. Intraoperative anastomotic leakage sites were identified in 3 of 5 dogs. Leaks were managed by placement of a single reinforcing conventional intracorporeal suture, which was adequate to obtain a watertight anastomosis.

Conclusion

This technique cannot be recommended in clinical practice until further studies are performed and the technique is further refined.
Level variations in speech: Effect on masking release in hearing-impaired listeners
Authors: Reed Charlotte M. , Desloge Joseph G. , Braida Louis D. , Perez Zachary D. , Léger Agnès C.
Published: July 2016

Acoustic speech is marked by time-varying changes in the amplitude envelope that may pose difficulties for hearing-impaired listeners. Removal of these variations (e.g., by the Hilbert transform) could improve speech reception for such listeners, particularly in fluctuating interference. Léger, Reed, Desloge, Swaminathan, and Braida [(2015b). J. Acoust. Soc. Am. 138, 389–403] observed that a normalized measure of masking release obtained for hearing-impaired listeners using speech processed to preserve temporal fine-structure (TFS) cues was larger than that for unprocessed or envelope-based speech. This study measured masking release for two other speech signals in which level variations were minimal: peak clipping and TFS processing of an envelope signal. Consonant identification was measured for hearing-impaired listeners in backgrounds of continuous and fluctuating speech-shaped noise. The normalized masking release obtained using speech with normal variations in overall level was substantially less than that observed using speech processed to achieve highly restricted level variations. These results suggest that the performance of hearing-impaired listeners in fluctuating noise may be improved by signal processing that leads to a decrease in stimulus level variations.
A broadband polygonal cloak for acoustic wave designed with linear coordinate transformation
Authors: Zhu Rongrong , Zheng Bin , Ma Chu , Xu Jun , Fang Nicholas , Chen Hongsheng
Published: July 2016

Previous acoustic cloaks designed with transformation acoustics always involve inhomogeneous material. In this paper, a design of acoustic polygonal cloak is proposed using linear polygonal transformation method. The designed acoustic polygonal cloak has homogeneous and anisotropic parameters, which is much easier to realize in practice. Furthermore, a possible acoustic metamaterial structure to realize the cloak is proposed. Simulation results on the real structure show that the metamaterial acoustic cloak is effective to reduce the scattering of the object.
Radiocarbon dating reveals minimal collagen turnover in both healthy and osteoarthritic human cartilage
Authors: Heinemeier Katja M. , Schjerling Peter , Heinemeier Jan , Møller Mathias B. , Krogsgaard Michael R. , Grum-Schwensen Tomas , Petersen Michael M. , Kjaer Michael
Published: July 2016

The turnover of cartilage collagen matrix is minimal in human adults and is not influenced by disease.
The gut takes a “Toll” on the lung
Authors: Bhattacharya Mallar
Published: July 2016

A Toll-like receptor ligand secreted by injured intestine causes acute lung injury in necrotizing enterocolitis.
Lym(fat)ics
Authors: Thomas Susan Napier
Published: July 2016

Increased body weight negatively affects lymphatic function independent of diet.
Two Phase 1, Randomized, Double-Blind, Placebo-Controlled, Single-Ascending-Dose Studies To Investigate the Safety, Tolerability, and Pharmacokinetics of an Anti-Influenza A Virus Monoclonal Antibody, MHAA4549A, in Healthy Volunteers
Authors: Lim Jeremy J. , Deng Rong , Derby Michael A. , Larouche Richard , Horn Priscilla , Anderson Malia , Maia Mauricio , Carrier Stephanie , Pelletier Isabelle , Burgess Tracy , Kulkarni Priya , Newton Elizabeth , Tavel Jorge A.
Published: July 2016

Hospitalized patients with severe influenza are at significant risk for morbidity and mortality. MHAA4549A is a human monoclonal immunoglobulin (Ig) G1 antibody that binds to a highly conserved stalk region of the influenza A virus hemagglutinin protein and neutralizes all tested seasonal human influenza A virus strains. Two phase 1 trials examined the safety, tolerability, and pharmacokinetics of MHAA4549A in healthy volunteers. Both single ascending-dose trials were randomized, double blinded, and placebo controlled. Trial 1 randomized 21 healthy adults into four cohorts receiving a single intravenous dose of 1.5, 5, 15, or 45 mg/kg MHAA4549A or placebo. Trial 2 randomized 14 healthy adults into two cohorts receiving a single intravenous fixed dose of 8,400 mg or 10,800 mg of MHAA4549A or placebo. Subjects were followed for 120 days after dosing. No subject was discontinued in either trial, and no serious adverse events were reported. The most common adverse event in both studies was mild headache (trial 1, 4/16 subjects receiving MHAA4549A and 1/5 receiving placebo; trial 2, 4/8 subjects receiving MHAA4549A and 2/6 receiving placebo). MHAA4549A produced no relevant time- or dose-related changes in laboratory values or vital signs compared to those with placebo. No subjects developed an antitherapeutic antibody response following MHAA4549A administration. MHAA4549A showed linear serum pharmacokinetics, with a mean half-life of 22.5 to 23.7 days. MHAA4549A is safe and well tolerated in healthy volunteers up to a single intravenous dose of 10,800 mg and demonstrates linear serum pharmacokinetics consistent with those of a human IgG1 antibody lacking known endogenous targets in humans. (These trials have been registered at ClinicalTrials.gov under registration no. NCT01877785 and NCT02284607).
The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis
Authors: Hürlimann Lea M. , Corradi Valentina , Hohl Michael , Bloemberg Guido V. , Tieleman D. Peter , Seeger Markus A.
Published: July 2016

Nosocomial infections with
Enterococcus faecalis
are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of
E. faecalis
that are annotated as drug efflux pumps. Deletion of
ef0789-ef0790
on the chromosome of
E. faecalis
resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of
E. faecalis
. In contrast, heterologous expression in
Lactococcus lactis
revealed that EfrAB, EfrCD, and the product of
ef2226-ef2227
(EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in
L. lactis
. Since all seven transporters were purified as heterodimers after overexpression in
L. lactis
, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell.
Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System
Authors: Dinkel Kelcey D. , Stanton James B. , Boykin David W. , Stephens Chad E. , Madsen-Bouterse Sally A. , Schneider David A.
Published: July 2016

The transmissible spongiform encephalopathies are fatal neurodegenerative disorders characterized by the misfolding of the native cellular prion protein (PrP
C
) into the accumulating, disease-associated isoform (PrP
Sc
). Despite extensive research into the inhibition of prion accumulation, no effective treatment exists. Previously, we demonstrated the inhibitory activity of DB772, a monocationic phenyl-furan-benzimidazole, against PrP
Sc
accumulation in sheep microglial cells. In an effort to determine the effect of structural substitutions on the antiprion activity of DB772, we employed an
in vitro
strategy to survey a library of structurally related, monothiophene- and furan-based compounds for improved inhibitory activity. Eighty-nine compounds were screened at 1 μM for effects on cell viability and prion accumulation in a persistently infected ovine microglia culture system. Eleven compounds with activity equivalent to or higher than that of DB772 were identified as preliminary hit compounds. For the preliminary hits, cytotoxicities and antiprion activities were compared to calculate the tissue culture selectivity index. A structure-activity relationship (SAR) analysis was performed to determine molecular components contributing to antiprion activity. To investigate potential mechanisms of inhibition, effects on PrP
C
and PrP
Sc
were examined. While inhibition of total PrP
C
was not observed, the results suggest that a potential target for inhibition at biologically relevant concentrations is through PrP
C
misfolding to PrP
Sc
. Further, SAR analysis suggests that two structural elements were associated with micromolar antiprion activity. Taken together, the described data provide a foundation for deeper investigation into untested DB compounds and in the design of effective therapeutics.
Safe and Sensitive Antiviral Screening Platform Based on Recombinant Human Coronavirus OC43 Expressing the Luciferase Reporter Gene
Authors: Shen Liang , Yang Yang , Ye Fei , Liu Gaoshan , Desforges Marc , Talbot Pierre J. , Tan Wenjie
Published: July 2016

Human coronaviruses (HCoVs) cause 15 to 30% of mild upper respiratory tract infections. However, no specific antiviral drugs are available to prevent or treat HCoV infections to date. Here, we developed four infectious recombinant HCoVs-OC43 (rHCoVs-OC43) which express the
Renilla
luciferase (Rluc) reporter gene. Among these four rHCoVs-OC43, rOC43-ns2DelRluc (generated by replacing ns2 with the Rluc gene) showed robust luciferase activity with only a slight impact on its growth characteristics. Additionally, this recombinant virus remained stable for at least 10 passages in BHK-21 cells. rOC43-ns2DelRluc was comparable to its parental wild-type virus (HCoV-OC43-WT) with respect to the quantity of the antiviral activity of chloroquine and ribavirin. We showed that chloroquine strongly inhibited HCoV-OC43 replication
in vitro
, with a 50% inhibitory concentration (IC
50
) of 0.33 μM. However, ribavirin showed inhibition of HCoV-OC43 replication only at high concentrations which may not be applicable to humans in clinical treatment, with an IC
50
of 10 μM. Furthermore, using a luciferase-based small interfering RNA (siRNA) screening assay, we identified double-stranded-RNA-activated protein kinase (PKR) and DEAD box RNA helicases (DDX3X) that exhibited antiviral activities, which were further verified by the use of HCoV-OC43-WT. Therefore, rOC43-ns2DelRluc represents a promising safe and sensitive platform for high-throughput antiviral screening and quantitative analysis of viral replication.
The DNA Polymerase Gamma R953C Mutant Is Associated with Antiretroviral Therapy-Induced Mitochondrial Toxicity
Authors: Li Min , Mislak Andrea C. , Foli Yram , Agbosu Esinam , Bose Vivek , Bhandari Shreya , Szymanski Michal R. , Shumate Christie K. , Yin Y. Whitney , Anderson Karen S. , Paintsil Elijah
Published: July 2016

We found a heterozygous C2857T mutation (R953C) in polymerase gamma (Pol-γ) in an HIV-infected patient with mitochondrial toxicity. The R953C Pol-γ mutant binding affinity for dCTP is 8-fold less than that of the wild type. The R953C mutant shows a 4-fold decrease in discrimination of analog nucleotides relative to the wild type. R953 is located on the “O-helix” that forms the substrate deoxynucleoside triphosphate (dNTP) binding site; the interactions of R953 with E1056 and Y986 may stabilize the O-helix and affect polymerase activity.
Whole-Genome Sequencing for Detecting Antimicrobial Resistance in Nontyphoidal Salmonella
Authors: McDermott Patrick F. , Tyson Gregory H. , Kabera Claudine , Chen Yuansha , Li Cong , Folster Jason P. , Ayers Sherry L. , Lam Claudia , Tate Heather P. , Zhao Shaohua
Published: July 2016

Laboratory-based
in vitro
antimicrobial susceptibility testing is the foundation for guiding anti-infective therapy and monitoring antimicrobial resistance trends. We used whole-genome sequencing (WGS) technology to identify known antimicrobial resistance determinants among strains of nontyphoidal
Salmonella
and correlated these with susceptibility phenotypes to evaluate the utility of WGS for antimicrobial resistance surveillance. Six hundred forty
Salmonella
of 43 different serotypes were selected from among retail meat and human clinical isolates that were tested for susceptibility to 14 antimicrobials using broth microdilution. The MIC for each drug was used to categorize isolates as susceptible or resistant based on Clinical and Laboratory Standards Institute clinical breakpoints or National Antimicrobial Resistance Monitoring System (NARMS) consensus interpretive criteria. Each isolate was subjected to whole-genome shotgun sequencing, and resistance genes were identified from assembled sequences. A total of 65 unique resistance genes, plus mutations in two structural resistance loci, were identified. There were more unique resistance genes (
n =
59) in the 104 human isolates than in the 536 retail meat isolates (
n =
36). Overall, resistance genotypes and phenotypes correlated in 99.0% of cases. Correlations approached 100% for most classes of antibiotics but were lower for aminoglycosides and beta-lactams. We report the first finding of extended-spectrum β-lactamases (ESBLs) (
bla
CTX-M1
and
bla
SHV2a
) in retail meat isolates of
Salmonella
in the United States. Whole-genome sequencing is an effective tool for predicting antibiotic resistance in nontyphoidal
Salmonella
, although the use of more appropriate surveillance breakpoints and increased knowledge of new resistance alleles will further improve correlations.
Population Pharmacokinetics of Isavuconazole from Phase 1 and Phase 3 (SECURE) Trials in Adults and Target Attainment in Patients with Invasive Infections Due to Aspergillus and Other Filamentous Fungi
Authors: Desai Amit , Kovanda Laura , Kowalski Donna , Lu Qiaoyang , Townsend Robert , Bonate Peter L.
Published: July 2016

Isavuconazole, the active moiety of the water-soluble prodrug isavuconazonium sulfate, is a triazole antifungal agent used for the treatment of invasive fungal infections. The objective of this analysis was to develop a population pharmacokinetic (PPK) model to identify covariates that affect isavuconazole pharmacokinetics and to determine the probability of target attainment (PTA) for invasive aspergillosis patients. Data from nine phase 1 studies and one phase 3 clinical trial (SECURE) were pooled to develop the PPK model (NONMEM, version 7.2). Stepwise covariate modeling was performed in Perl-speaks-NONMEM, version 3.7.6. The area under the curve (AUC) at steady state was calculated for 5,000 patients by using Monte Carlo simulations. The PTA using the estimated pharmacodynamic (PD) target value (total AUC/MIC ratio) estimated from
in vivo
PD studies of invasive aspergillosis over a range of MIC values was calculated using simulated patient AUC values. A two-compartment model with a Weibull absorption function and a first-order elimination process adequately described plasma isavuconazole concentrations. The mean estimate for isavuconazole clearance was 2.360 liters/h (percent coefficient of variation [%CV], 34%), and the mean AUC from 0 to 24 h (AUC
0–24
) was ∼100 mg·h/liter. Clearance was approximately 36% lower in Asians than in Caucasians. The PTA calculated over a range of MIC values by use of the nonneutropenic murine efficacy index corresponding to 90% survival indicated that adequate isavuconazole exposures were achieved in >90% of simulated patients to treat infections with MICs up to and including 1 mg/liter according to European Committee on Antimicrobial Susceptibility Testing methodology and in >90% of simulated patients for infections with MICs up to and including 0.5 mg/liter according to Clinical and Laboratory Standards Institute methodology. The highest MIC result for PTA was the same for Caucasian and Asian patients.
The Lantibiotic NAI-107 Efficiently Rescues Drosophila melanogaster from Infection with Methicillin-Resistant Staphylococcus aureus USA300
Authors: Thomsen Thomas T. , Mojsoska Biljana , Cruz João C. S. , Donadio Stefano , Jenssen Håvard , Løbner-Olesen Anders , Rewitz Kim
Published: July 2016

We used the fruit fly
Drosophila melanogaster
as a cost-effective
in vivo
model to evaluate the efficacy of novel antibacterial peptides and peptoids for treatment of methicillin-resistant
Staphylococcus aureus
(MRSA) infections. A panel of peptides with known antibacterial activity
in vitro
and/or
in vivo
was tested in
Drosophila
. Although most peptides and peptoids that were effective
in vitro
failed to rescue lethal effects of
S. aureus
infections
in vivo
, we found that two lantibiotics, nisin and NAI-107, rescued adult flies from fatal infections. Furthermore, NAI-107 rescued mortality of infection with the MRSA strain USA300 with an efficacy equivalent to that of vancomycin, a widely applied antibiotic for the treatment of serious MRSA infections. These results establish
Drosophila
as a useful model for
in vivo
drug evaluation of antibacterial peptides.
Affinity-Based Screening of Tetravalent Peptides Identifies Subtype-Selective Neutralizers of Shiga Toxin 2d, a Highly Virulent Subtype, by Targeting a Unique Amino Acid Involved in Its Receptor Recognition
Authors: Mitsui Takaaki , Watanabe-Takahashi Miho , Shimizu Eiko , Zhang Baihao , Funamoto Satoru , Yamasaki Shinji , Nishikawa Kiyotaka
Published: July 2016

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic
Escherichia coli
(EHEC), can be classified into two subgroups, Stx1 and Stx2, each consisting of various closely related subtypes. Stx2 subtypes Stx2a and Stx2d are highly virulent and linked with serious human disorders, such as acute encephalopathy and hemolytic-uremic syndrome. Through affinity-based screening of a tetravalent peptide library, we previously developed peptide neutralizers of Stx2a in which the structure was optimized to bind to the B-subunit pentamer. In this study, we identified Stx2d-selective neutralizers by targeting Asn16 of the B subunit, an amino acid unique to Stx2d that plays an essential role in receptor binding. We synthesized a series of tetravalent peptides on a cellulose membrane in which the core structure was exactly the same as that of peptides in the tetravalent library. A total of nine candidate motifs were selected to synthesize tetravalent forms of the peptides by screening two series of the tetravalent peptides. Five of the tetravalent peptides effectively inhibited the cytotoxicity of Stx2a and Stx2d, and notably, two of the peptides selectively inhibited Stx2d. These two tetravalent peptides bound to the Stx2d B subunit with high affinity dependent on Asn16. The mechanism of binding to the Stx2d B subunit differed from that of binding to Stx2a in that the peptides covered a relatively wide region of the receptor-binding surface. Thus, this highly optimized screening technique enables the development of subtype-selective neutralizers, which may lead to more sophisticated treatments of infections by Stx-producing EHEC.
A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain
Authors: González-Rivera Christian , Campbell Anne M. , Rutherford Stacey A. , Pyburn Tasia M. , Foegeding Nora J. , Barke Theresa L. , Spiller Benjamin W. , McClain Mark S. , Ohi Melanie D. , Lacy D. Borden , Cover Timothy L.
Published: July 2016

Helicobacter pylori
secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly β-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by
H. pylori
. The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the β-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the β-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly.
The Chlamydia-Secreted Protease CPAF Promotes Chlamydial Survival in the Mouse Lower Genital Tract
Authors: Yang Zhangsheng , Tang Lingli , Shao Lili , Zhang Yuyang , Zhang Tianyuan , Schenken Robert , Valdivia Raphael , Zhong Guangming
Published: July 2016

Despite the extensivein vitrocharacterization of CPAF (chlamydialprotease/proteasome-likeactivityfactor), its role in chlamydial infection and pathogenesis remains unclear. We now report that aChlamydia trachomatisstrain deficient in expression of CPAF (L2-17) is no longer able to establish a successful infection in the mouse lower genital tract following an intravaginal inoculation. The L2-17 organisms were cleared from the mouse lower genital tract within a few days, while a CPAF-sufficientC. trachomatisstrain (L2-5) survived in the lower genital tract for more than 3 weeks. However, both the L2-17 and L2-5 organisms maintained robust infection courses that lasted up to 4 weeks when they were directly delivered into the mouse upper genital tract. The CPAF-dependent chlamydial survival in the lower genital tract was confirmed in multiple strains of mice. Thus, we have demonstrated a critical role of CPAF in promotingC. trachomatissurvival in the mouse lower genital tracts. It will be interesting to further investigate the mechanisms of the CPAF-dependent chlamydial pathogenicity.
Burkholderia pseudomallei Rapidly Infects the Brain Stem and Spinal Cord via the Trigeminal Nerve after Intranasal Inoculation
Authors: St. John James A. , Walkden Heidi , Nazareth Lynn , Beagley Kenneth W. , Ulett Glen C. , Batzloff Michael R. , Beacham Ifor R. , Ekberg Jenny A. K.
Published: July 2016

Infection with
Burkholderia pseudomallei
causes melioidosis, a disease with a high mortality rate (20% in Australia and 40% in Southeast Asia). Neurological melioidosis is particularly prevalent in northern Australian patients and involves brain stem infection, which can progress to the spinal cord; however, the route by which the bacteria invade the central nervous system (CNS) is unknown. We have previously demonstrated that
B. pseudomallei
can infect the olfactory and trigeminal nerves within the nasal cavity following intranasal inoculation. As the trigeminal nerve projects into the brain stem, we investigated whether the bacteria could continue along this nerve to penetrate the CNS. After intranasal inoculation of mice,
B. pseudomallei
caused low-level localized infection within the nasal cavity epithelium, prior to invasion of the trigeminal nerve in small numbers.
B. pseudomallei
rapidly invaded the trigeminal nerve and crossed the astrocytic barrier to enter the brain stem within 24 h and then rapidly progressed over 2,000 μm into the spinal cord. To rule out that the bacteria used a hematogenous route, we used a capsule-deficient mutant of
B. pseudomallei
that does not survive in the blood and found that it also entered the CNS via the trigeminal nerve. This suggests that the primary route of entry is via the nerves that innervate the nasal cavity. We found that actin-mediated motility could facilitate initial infection of the olfactory epithelium. Thus, we have demonstrated that
B. pseudomallei
can rapidly infect the brain and spinal cord via the trigeminal nerve branches that innervate the nasal cavity.
Regulation of Ribosomal Protein Operons
rplM-rpsI
,
rpmB-rpmG
, and
rplU-rpmA
at the Transcriptional and Translational Levels
Authors: Aseev Leonid V. , Koledinskaya Ludmila S. , Boni Irina V.
Published: July 2016

It is widely assumed that in the best-characterized model bacterium
Escherichia coli
, transcription units encoding ribosomal proteins (r-proteins) and regulation of their expression have been already well defined. However, transcription start sites for several
E. coli
r-protein operons have been established only very recently, so that information concerning the regulation of these operons at the transcriptional or posttranscriptional level is still missing. This paper describes for the first time the
in vivo
regulation of three r-protein operons,
rplM-rpsI
,
rpmB-rpmG
, and
rplU-rpmA
. The results demonstrate that transcription of all three operons is subject to ppGpp/DksA-dependent negative stringent control under amino acid starvation, in parallel with the rRNA operons. By using single-copy translational fusions with the chromosomal
lacZ
gene, we show here that at the translation level only one of these operons,
rplM-rpsI
, is regulated by the mechanism of autogenous repression involving the 5′ untranslated region (UTR) of the operon mRNA, while
rpmB-rpmG
and
rplU-rpmA
are not subject to this type of regulation. This may imply that translational feedback control is not a general rule for modulating the expression of
E. coli
r-protein operons. Finally, we report that L13, a primary protein in 50S ribosomal subunit assembly, serves as a repressor of
rplM-rpsI
expression
in vivo
, acting at a target within the
rplM
translation initiation region. Thus, L13 represents a novel example of regulatory r-proteins in bacteria.

IMPORTANCE
It is important to obtain a deeper understanding of the regulatory mechanisms responsible for coordinated and balanced synthesis of ribosomal components. In this paper, we highlight the major role of a stringent response in regulating transcription of three previously unexplored r-protein operons, and we show that only one of them is subject to feedback regulation at the translational level. Improved knowledge of the regulatory pathways controlling ribosome biogenesis may promote the development of novel antibacterial agents.
The Last
r
Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin
Authors: Chen Yi , Young Ry
Published: July 2016

The latent period of phage T4, normally ∼25 min, can be extended indefinitely if the infected cell is superinfected after 5 min. This phenomenon, designated lysis inhibition (LIN), was first described in the 1940s and is genetically defined by mutations in diverse T4
r
genes. RI, the main effector of LIN, has been shown to be secreted to the periplasm, where, upon activation by superinfection with a T-even virion, it binds to the C-terminal periplasmic domain of the T4 holin T and blocks its lethal permeabilization of the cytoplasmic membrane. Another
r
locus,
rIII
, has been the subject of conflicting reports. In this study, we show that RIII, an 82-amino-acid protein, is also required for LIN in both
Escherichia coli
B strains and
E. coli
K-12 strains. In T4Δ
rIII
infections, LIN was briefly established but was unstable. The overexpression of a cloned
rIII
gene alone impeded T-mediated lysis temporarily. However, coexpression of
rIII
and
rI
resulted in a stable LIN state. Bacterial two-hybrid assays and pulldown assays showed that RIII interacts with the cytoplasmic N terminus of T, which is a critical domain for holin function. We conclude that RIII is a T4 antiholin that blocks membrane hole formation by interacting directly with the holin. Accordingly, we propose an augmented model for T4 LIN that involves the stabilization of a complex of three proteins in two compartments of the cell: RI interacting with the C terminus of T in the periplasm and RIII interacting with the N terminus of T in the cytoplasm.

IMPORTANCE
Lysis inhibition is a unique feature of phage T4 in response to environmental conditions, effected by the antiholin RI, which binds to the periplasmic domain of the T holin and blocks its hole-forming function. Here we report that the T4 gene
rIII
encodes a cytoplasmic antiholin that, together with the main antiholin, RI, inhibits holin T by forming a complex of three proteins spanning two cell compartments.