bluuebunny/crossref_metadata_2025 huggingface dataset:
Coxiella burnetii Inhibits Neutrophil Apoptosis by Exploiting Survival Pathways and Antiapoptotic Protein Mcl-1
Authors: Cherla Rama , Zhang Yan , Ledbetter Lindsey , Zhang Guoquan
Published: January 2018

Our previous study demonstrated that neutrophils play an important role in host defense againstCoxiella burnetiiinfection in mice. In this study, avirulent strainC. burnetiiNine Mile phase II (NMII) was used to examine ifC. burnetiican modulate mouse bone marrow-derived neutrophil apoptosis. The results indicated that NMII can inhibit neutrophil apoptosis. Western blotting demonstrated that caspase-3 cleavage was decreased in NMII-infected neutrophils, while phosphorylated mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase 1 (Erk1) were increased. Additionally, p38, Erk1/2, phosphoinositide 3-kinase (PI3K), or NF-κB inhibitors reduced the ability of NMII to inhibit neutrophil apoptosis. These results suggest that NMII-mediated inhibition of neutrophil apoptosis depends on its ability to activate neutrophil MAPK pathways. Antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) was significantly increased in NMII-infected neutrophils, and an Mcl-1 inhibitor significantly reduced the ability of NMII to inhibit neutrophil apoptosis. Mcl-1 protein stability was enhanced by phosphorylation at Thr-163 by Erk, and the protein levels were regulated by p38, Erk, PI3K, and NF-κB. Furthermore, the observation that a type IV secretion system (T4SS)-deficientdotAmutant showed a significantly reduced ability to inhibit neutrophil apoptosis compared to wild-type (WT) NMII suggests that T4SS-secreted factors may be involved in NMII-induced inhibition of neutrophil apoptosis. Collectively, these results demonstrate that NMII inhibits neutrophil apoptosis through inhibition of caspase-3 cleavage and activation of MAPK survival pathways with subsequent expression and stabilization of antiapoptotic protein Mcl-1, a process that may partially require the T4SS.
The Drift in Molecular Testing for Influenza: Mutations Affecting Assay Performance
Authors: Stellrecht Kathleen A.
Published: January 2018

Influenza is associated with rapid evolution due to lack of RNA polymerase proofreading, immunogenic selection, and frequent rearrangement of gene segments. Evolutionary changes affecting the performance of diagnostic testing have long been recognized. Hence, it is not surprising that such challenges apply to nucleic acid amplification tests, even though they are designed to target highly conserved regions. Initially, case reports involved single isolates of A(H1N1)pdm09. Over the past 4 years, subtype H3N2 viruses evolved to viral clades with mutations in the WHO-recommended target region, such that almost all isolates worldwide have significantly reduced sensitivities with many commercial reverse transcription-PCR tests.
Development and Evaluation of a Broad Bead-Based Multiplex Immunoassay To Measure IgG Seroreactivity against Human Polyomaviruses
Authors: Kamminga Sergio , van der Meijden Els , Wunderink Herman F. , Touzé Antoine , Zaaijer Hans L. , Feltkamp Mariet C. W.
Published: January 2018

The family of polyomaviruses, which cause severe disease in immunocompromised hosts, has expanded substantially in recent years. To accommodate measurement of IgG seroresponses against all currently known human polyomaviruses (HPyVs), including the Lyon IARC polyomavirus (LIPyV), we extended our custom multiplex bead-based HPyV immunoassay and evaluated the performance of this pan-HPyV immunoassay. The VP1 proteins of 15 HPyVs belonging to 13
Polyomavirus
species were expressed as recombinant glutathione
S
-transferase (GST) fusion proteins and coupled to fluorescent Luminex beads. Sera from healthy blood donors and immunocompromised kidney transplant recipients were used to analyze seroreactivity against the different HPyVs. For BK polyomavirus (BKPyV), the GST-VP1 fusion protein-directed seroresponses were compared to those obtained against BKPyV VP1 virus-like particles (VLP). Seroreactivity against most HPyVs was common and generally high in both test populations. Low seroreactivity against HPyV9, HPyV12, New Jersey PyV, and LIPyV was observed. The assay was reproducible (Pearson's
r
2
> 0.84,
P
< 0.001) and specific. Weak but consistent cross-reactivity between the related viruses HPyV6 and HPyV7 was observed. The seroresponses measured by the GST-VP1-based immunoassay and a VP1 VLP-based enzyme-linked immunosorbent assay were highly correlated (Spearman's ρ = 0.823,
P
< 0.001). The bead-based pan-HPyV multiplex immunoassay is a reliable tool to determine HPyV-specific seroresponses with high reproducibility and specificity and is suitable for use in seroepidemiological studies.
Identification and Characterization of Sindbis Virus RNA-Host Protein Interactions
Authors: LaPointe Autumn T. , Gebhart Natasha N. , Meller Megan E. , Hardy Richard W. , Sokoloski Kevin J.
Published: January 2018

Arthropod-borne viruses, such as the members of the genus
Alphavirus
, are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish and complete their life cycles. Despite considerable efforts to define the host-pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA-protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized. The data presented here introduce a robust viral RNA-protein discovery method to elucidate the Sindbis virus (SINV) RNA-protein host interface. Cross-link-assisted mRNP purification (CLAMP) assessment revealed an extensive array of host-pathogen interactions centered on the viral RNAs (vRNAs). After prioritization of the host proteins associated with the vRNAs, we identified the site of protein-vRNA interaction by a UV cross-linking and immunoprecipitation sequencing (CLIP-seq) approach and assessed the consequences of the RNA-protein binding event of hnRNP K, hnRNP I, and hnRNP M in regard to viral infection. Here, we demonstrate that mutation of the prioritized hnRNP-vRNA interaction sites effectively disrupts hnRNP-vRNA interaction. Correlating with disrupted hnRNP-vRNA binding, SINV growth kinetics were reduced relative to wild-type parental viral infections in vertebrate and invertebrate tissue culture models of infection. The molecular mechanism leading to reduced viral growth kinetics was found to be dysregulated structural-gene expression. Collectively, this study further defines the scope and importance of the alphavirus host-pathogen vRNA-protein interactions.

IMPORTANCE
Members of the genus
Alphavirus
are widely recognized for their potential to cause severe disease. Despite this recognition, there are no antiviral therapeutics, or safe and effective vaccines, currently available to treat alphaviral infection. Alphaviruses utilize the host cell machinery to efficiently establish and complete their life cycle. However, the extent and importance of host-pathogen RNA-protein interactions are woefully undercharacterized. The efforts detailed in this study fill this critical gap, and the significance of this research is 3-fold. First, the data presented here fundamentally expand the scope and understanding of alphavirus host-pathogen interactions. Second, this study identifies the sites of interaction for several prioritized interactions and defines the contribution of the RNA-protein interaction at the molecular level. Finally, these studies build a strategy by which the importance of the given host-pathogen interactions may be assessed in the future, using a mouse model of infection.
Range Expansion and the Origin of USA300 North American Epidemic Methicillin-Resistant
Staphylococcus aureus
Authors: Challagundla Lavanya , Luo Xiao , Tickler Isabella A. , Didelot Xavier , Coleman David C. , Shore Anna C. , Coombs Geoffrey W. , Sordelli Daniel O. , Brown Eric L. , Skov Robert , Larsen Anders Rhod , Reyes Jinnethe , Robledo Iraida E. , Vazquez Guillermo J. , Rivera Raul , Fey Paul D. , Stevenson Kurt , Wang Shu-Hua , Kreiswirth Barry N. , Mediavilla Jose R. , Arias Cesar A. , Planet Paul J. , Nolan Rathel L. , Tenover Fred C. , Goering Richard V. , Robinson D. Ashley
Published: January 2018

The USA300 North American epidemic (USA300-NAE) clone of methicillin-resistant
Staphylococcus aureus
has caused a wave of severe skin and soft tissue infections in the United States since it emerged in the early 2000s, but its geographic origin is obscure. Here we use the population genomic signatures expected from the serial founder effects of a geographic range expansion to infer the origin of USA300-NAE and identify polymorphisms associated with its spread. Genome sequences from 357 isolates from 22 U.S. states and territories and seven other countries are compared. We observe two significant signatures of range expansion, including decreases in genetic diversity and increases in derived allele frequency with geographic distance from the Pennsylvania region. These signatures account for approximately half of the core nucleotide variation of this clone, occur genome wide, and are robust to heterogeneity in temporal sampling of isolates, human population density, and recombination detection methods. The potential for positive selection of a
gyrA
fluoroquinolone resistance allele and several intergenic regions, along with a 2.4 times higher recombination rate in a resistant subclade, is noted. These results are the first to show a pattern of genetic variation that is consistent with a range expansion of an epidemic bacterial clone, and they highlight a rarely considered but potentially common mechanism by which genetic drift may profoundly influence bacterial genetic variation.

IMPORTANCE
The process of geographic spread of an origin population by a series of smaller populations can result in distinctive patterns of genetic variation. We detect these patterns for the first time with an epidemic bacterial clone and use them to uncover the clone’s geographic origin and variants associated with its spread. We study the USA300 clone of methicillin-resistant
Staphylococcus aureus
, which was first noticed in the early 2000s and subsequently became the leading cause of skin and soft tissue infections in the United States. The eastern United States is the most likely origin of epidemic USA300. Relatively few variants, which include an antibiotic resistance mutation, have persisted during this clone’s spread. Our study suggests that an early chapter in the genetic history of this epidemic bacterial clone was greatly influenced by random subsampling of isolates during the clone’s geographic spread.
Role of Heat Shock Proteases in Quorum-Sensing-Mediated Regulation of Biofilm Formation by
Vibrio
Species
Authors: Lee Kyung-Jo , Jung You-Chul , Park Soon-Jung , Lee Kyu-Ho
Published: January 2018

Capsular polysaccharide (CPS) is essential for the dispersal of biofilms formed by the pathogenic bacterium
Vibrio vulnificus
. CPS production is induced by the quorum-sensing (QS) master regulator SmcR when biofilms mature. However,
V. vulnificus
biofilms formed under heat shock conditions did not exhibit the dispersion stage. Transcripts of the CPS gene cluster were at basal levels in the heat-exposed cell owing to reduced cellular levels of SmcR. At least two proteases induced by heat shock, ClpPA and Lon, were responsible for determining the instability of SmcR.
In vitro
and
in vivo
assays demonstrated that SmcR levels were regulated via proteolysis by these proteases, with preferential proteolysis of monomeric SmcR. Thus, CPS production was not induced by QS when bacteria were heat treated. Further studies performed with other
Vibrio
species demonstrated that high temperature deactivated the QS circuits by increased proteolysis of their QS master regulators, thus resulting in alterations to the QS-regulated phenotypes, including biofilm formation.

IMPORTANCE
The term "quorum-sensing mechanism" is used to describe diverse bacterial cell density-dependent activities that are achieved by sensing of the signaling molecules and subsequent signal transduction to the master regulators. These well-known bacterial regulatory systems regulate the expression of diverse virulence factors and the construction of biofilms in pathogenic bacteria. There have been numerous studies designed to control bacterial quorum sensing by using small molecules to antagonize the quorum-sensing regulatory components or to interfere with the signaling molecules. In the present study, we showed that the quorum-sensing regulatory circuits of pathogenic
Vibrio
species were deactivated by heat shock treatment via highly increased proteolysis of the master transcription factors. Our results showed a new mode of quorum deactivation which can be achieved under conditions of high but nonlethal temperature even if the ambient signaling molecules may reach the levels representing high cell density.
Streptococcus pneumoniae
Modulates
Staphylococcus aureus
Biofilm Dispersion and the Transition from Colonization to Invasive Disease
Authors: Reddinger Ryan M. , Luke-Marshall Nicole R. , Sauberan Shauna L. , Hakansson Anders P. , Campagnari Anthony A.
Published: January 2018

Streptococcus pneumoniae
and
Staphylococcus aureus
are ubiquitous upper respiratory opportunistic pathogens. Individually, these Gram-positive microbes are two of the most common causative agents of secondary bacterial pneumonia following influenza A virus infection, and they constitute a significant source of morbidity and mortality. Since the introduction of the pneumococcal conjugate vaccine, rates of cocolonization with both of these bacterial species have increased, despite the traditional view that they are antagonistic and mutually exclusive. The interactions between
S. pneumoniae
and
S. aureus
in the context of colonization and the transition to invasive disease have not been characterized. In this report, we show that
S. pneumoniae
and
S. aureus
form stable dual-species biofilms on epithelial cells
in vitro
. When these biofilms are exposed to physiological changes associated with viral infection,
S. pneumoniae
disperses from the biofilm, whereas
S. aureus
dispersal is inhibited. These findings were supported by results of an
in vivo
study in which we used a novel mouse cocolonization model. In these experiments, mice cocolonized in the nares with both bacterial species were subsequently infected with influenza A virus. The coinfected mice almost exclusively developed pneumococcal pneumonia. These results indicate that despite our previous report that
S. aureus
disseminates into the lungs of mice stably colonized with these bacteria following influenza A virus infection, cocolonization with
S. pneumoniae in vitro
and
in vivo
inhibits
S. aureus
dispersal and transition to disease. This study provides novel insight into both the interactions between
S. pneumoniae
and
S. aureus
during carriage and the transition from colonization to secondary bacterial pneumonia.

IMPORTANCE
In this study, we demonstrate that
Streptococcus pneumoniae
can modulate the pathogenic potential of
Staphylococcus aureus
in a model of secondary bacterial pneumonia. We report that host physiological signals related to viral infection cease to elicit a dispersal response from
S. aureus
while in a dual-species setting with
S. pneumoniae
, in direct contrast to results of previous studies with each species individually. This study underscores the importance of studying polymicrobial communities and their implications in disease states.
Cyclic AMP Regulates Bacterial Persistence through Repression of the Oxidative Stress Response and SOS-Dependent DNA Repair in Uropathogenic
Escherichia coli
Authors: Molina-Quiroz Roberto C. , Silva-Valenzuela Cecilia , Brewster Jennifer , Castro-Nallar Eduardo , Levy Stuart B. , Camilli Andrew
Published: January 2018

Bacterial persistence is a transient, nonheritable physiological state that provides tolerance to bactericidal antibiotics. The stringent response, toxin-antitoxin modules, and stochastic processes, among other mechanisms, play roles in this phenomenon. How persistence is regulated is relatively ill defined. Here we show that cyclic AMP, a global regulator of carbon catabolism and other core processes, is a negative regulator of bacterial persistence in uropathogenic
Escherichia coli
, as measured by survival after exposure to a β-lactam antibiotic. This phenotype is regulated by a set of genes leading to an oxidative stress response and SOS-dependent DNA repair. Thus, persister cells tolerant to cell wall-acting antibiotics must cope with oxidative stress and DNA damage and these processes are regulated by cyclic AMP in uropathogenic
E. coli
.

IMPORTANCE
Bacterial persister cells are important in relapsing infections in patients treated with antibiotics and also in the emergence of antibiotic resistance. Our results show that in uropathogenic
E. coli
, the second messenger cyclic AMP negatively regulates persister cell formation, since in its absence much more persister cells form that are tolerant to β-lactams antibiotics. We reveal the mechanism to be decreased levels of reactive oxygen species, specifically hydroxyl radicals, and SOS-dependent DNA repair. Our findings suggest that the oxidative stress response and DNA repair are relevant pathways to target in the design of persister-specific antibiotic compounds.
A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease
Authors: Alfson Kendra J. , Avena Laura E. , Delgado Jenny , Beadles Michael W. , Patterson Jean L. , Carrion Ricardo , Griffiths Anthony
Published: January 2018

Marburg virus (MARV) causes disease with a high case fatality rate, and there are no approved vaccines or therapies. Serial amplification of viruses in cell culture often results in accumulation of mutations, but the effect of such cell culture passage on MARV is unclear. Serial passages of MARV resulted in a single mutation in the region encoding the glycoprotein (GP). This is a region where mutations can have important consequences on outbreaks and human disease [S. Mahanty and M. Bray, Lancet Infect Dis 4:487–498, 2004,
https://doi.org/10.1016/S1473-3099(04)01103-X
]. We thus investigated whether this mutation impacted disease by using a cynomolgus macaque model of MARV infection. Monkeys exposed to virus containing the mutation had better clinical outcomes than monkeys exposed to virus without the mutation. We also observed that a remarkably low number of MARV particles was sufficient to cause death. Our results could have a significant impact on how future studies are designed to model MARV disease and test vaccines and therapeutics.
Maternal HIV-1 Env Vaccination for Systemic and Breast Milk Immunity To Prevent Oral SHIV Acquisition in Infant Macaques
Authors: Eudailey Joshua A. , Dennis Maria L. , Parker Morgan E. , Phillips Bonnie L. , Huffman Tori N. , Bay Camden P. , Hudgens Michael G. , Wiseman Roger W. , Pollara Justin J. , Fouda Genevieve G. , Ferrari Guido , Pickup David J. , Kozlowski Pamela A. , Van Rompay Koen K. A. , De Paris Kristina , Permar Sallie R.
Published: January 2018

Without novel strategies to prevent mother-to-child HIV-1 transmission, more than 5% of HIV-1-exposed infants will continue to acquire HIV-1, most through breastfeeding. This study of rhesus macaque dam-and-infant pairs is the first preclinical study to investigate the protective role of transplacentally transferred HIV-1 vaccine-elicited antibodies and HIV-1 vaccine-elicited breast milk antibody responses in infant oral virus acquisition. It revealed highly variable placental transfer of potentially protective antibodies and emphasized the importance of pregnancy immunization timing to reach peak antibody levels prior to delivery. While there was no discernible impact of maternal immunization on late infant oral virus acquisition, we observed a strong correlation between the percentage of activated CD4
+
T cells in infant peripheral blood and a reduced number of challenges to infection. This finding highlights an important consideration for future studies evaluating alternative strategies to further reduce the vertical HIV-1 transmission risk.
Cooperating H3N2 Influenza Virus Variants Are Not Detectable in Primary Clinical Samples
Authors: Xue Katherine S. , Greninger Alexander L. , Pérez-Osorio Ailyn , Bloom Jesse D.
Published: January 2018

Viruses mutate rapidly, and recent studies of RNA viruses have shown that related viral variants can sometimes cooperate to improve each other’s growth. We previously described two variants of H3N2 influenza virus that cooperate in cell culture. The mutation responsible for cooperation is often observed when human samples of influenza virus are grown in the lab before sequencing, but it is unclear whether the mutation also exists in human infections or is exclusively the result of lab passage. We identified nine human isolates of influenza virus that had developed the cooperating mutation after being grown in the lab and performed highly sensitive deep sequencing of the unpassaged clinical samples to determine whether the mutation existed in the original human infections. We found no evidence of the cooperating mutation in the unpassaged samples, suggesting that the cooperation arises primarily under laboratory conditions.
Integrating wood fuels into agriculture and food security agendas and research in sub-Saharan Africa
Authors: Mendum Ruth , Njenga Mary
Published: January 2018

In sub-Saharan Africa (SSA), food security can be influenced by many factors including farmer productivity, access to soil amendments, labor availability, and family incomes (just to name a few). In this paper, we suggest that an additional issue contributes to food insecurity and has been historically absent from the discussion, namely access to cooking energy, particularly for very low income, food insecure individuals. This paper examines the most recent literature that describes the central role played by wood fuels, in particular firewood and charcoal, as a vital, though controversial, source of fuel used by the vast majority of rural and urban sub-Saharan Africans. We explore the reality that although the health risks of collecting and using firewood and charcoal in traditional manners are real, policy makers, researchers, and donors need to address the sustainability and viability of the current fuel types used by the majority of people. We end the paper with a series of practical suggestions for improving the wood fuel systems as they currently exist in the region.
Particle Swarm Optimization for Split Delivery Vehicle Routing Problem
Authors: Shi Jianli , Zhang Jin , Wang Kun , Fang Xin
Published: January 2018

The split delivery vehicle routing problem (SDVRP) is a variation of the capacitated vehicle routing problem in which some customers may be served by more than one vehicle. We have proposed a particle swarm optimization approach that incorporates a local search to solve the SDVRP. An integer coding method was presented, and a decoding method based on Bellman’s equation was modified for the SDVRP. A way to address the differences in the length of the velocity vector, the position vector, the personal best position vector, the local best position vector and the global best position vector was designed. Two groups of local searches for top solutions were incorporated into the algorithm, with the ability to control whether they are executed on a given solution. The algorithm was initially tested using the modified Solomon’s instances to verify the parameters used, including the local search probability, the size of the swarm, the velocity equation and the length of the vectors. Extensive computational experiments were carried out on 131 benchmark instances available in the literature. The results obtained were competitive. More precisely, equally good solutions were found in 32 instances, and improved solutions were found in 35 instances, with an average improvement of 0.02% and a maximum improvement of 1.12%.
Integrability of the coupled cubic–quintic complex Ginzburg–Landau equations and multiple-soliton solutions via mathematical methods
Authors: Selima Ehab S. , Seadawy Aly R. , Yao Xiaohua , Essa F. A.
Published: January 2018

This paper is devoted to study the (1+1)-dimensional coupled cubic–quintic complex Ginzburg–Landau equations (cc–qcGLEs) with complex coefficients. This equation can be used to describe the nonlinear evolution of slowly varying envelopes of periodic spatial–temporal patterns in a convective binary fluid. Dispersion relation and properties of cc–qcGLEs are constructed. Painlevé analysis is used to check the integrability of cc–qcGLEs and to establish the Bäcklund transformation form. New traveling wave solutions and a general form of multiple-soliton solutions of cc–qcGLEs are obtained via the Bäcklund transformation and simplest equation method with Bernoulli, Riccati and Burgers’ equations as simplest equations.
Impact of Patient-Specific In Vivo Vessel Material Properties on Carotid Atherosclerotic Plaque Stress/Strain Calculations
Authors: Wang Qingyu , Tang Dalin , Canton Gador , Hatsukami Thomas S. , Billiar Kristen L. , Wu Zheyang , Yuan Chun
Published: January 2018

Patient-specific vessel material properties are in general lacking in image-based computational models. Carotid plaque stress and strain conditions with in vivo material and old material models were investigated (8 patients, 16 plaques). Plaque models using patient-specific in vivo vessel material properties showed significant differences from models using old material properties from the literature on stress and strain calculations. These differences demonstrated that models using in vivo material properties could improve the accuracy of stress and strain calculations which could potentially lead to more accurate plaque vulnerability assessment.
The Spiral of Deinstitutionalization Untangled: An Observation about the Field Opinion within Kuomintang against Public Opinion during the 2016 Presidential Election Campaign
Authors: LIU FRANK C. S. , CHANG RYAN Y. P. , TAN ALEXANDER C.
Published: January 2018

As political scientists start applying the complex-system approach to study party politics and as business scholars start to apply communication theories to study deinstitutionalization, we prospect a new possibility to study and explain politics within a political party. This study employs a systematically collected field observation data to evaluate Clemente and Roulet’s (2015) “the spiral of deinstitutionalization” framework. Based on analysis of news events and internal reports within Kuomintang from April 20 to October 17, 2015, we believe that this framework facilitates explanation about how the decision of nominating Hung Hsiu-Chu as the party’s first female presidential candidate was replaced three months before the Election Day. We interpret the whole story and provide details that contribute to enriching the framework for future organizational and political party research.
Environmental Information Disclosure in the Hotel Sector: Global Reporting Initiative Application
Authors: Kang Sung Wook , Hahn Song Ee
Published: January 2018

The purpose of this study is to identify core Environmental Information Disclosure (EID) contents and to evaluate the current level of EID proactiveness of the hotel companies on the Global Reporting Initiative (GRI) database. We confirmed 38 reports as a sample from 2008 to 2014. Utilising content analysis and a case study approach, we comprehensively investigated the quality and extent of what is being reported and evaluated in respect to each hotel’s EID proactiveness. Six core EID categories which had received significant attention from the hotels were identified, and we found the sampled hotels emphasised cost reduction activities of their operations in their EID. EID proactiveness was addressed and there was significant difference between the EID proactiveness groups (top, middle, bottom) in terms of property, quality and quantity. This study provides an overview of the current level of EID proactiveness of the hotels in the GRI database and also provides a measure for EID proactiveness for the hotel sector.
A Chemical Perspective of Pharmacology and Toxicology
Authors: Cho Arthur K.
Published: January 2018

My chemical training provided a somewhat different perspective of biolo-gical problems, in the problem itself and approaches to its solution. I was fortunate to have in my laboratory postdocs and students who shared this perspective and used appropriate tools to address problems in amphetamine pharmacology and air pollution toxicology. These apparently disparate areas of research shared two chemical reactions: prooxidant-based generation of reactive oxygen and formation of covalent bonds between electrophiles and biological nucleophiles. This article is an attempt to summarize that research and to identify those individuals who made the contributions.
Physiologically Based Pharmacokinetic and Pharmacodynamic Analysis Enabled by Microfluidically Linked Organs-on-Chips
Authors: Prantil-Baun Rachelle , Novak Richard , Das Debarun , Somayaji Mahadevabharath R. , Przekwas Andrzej , Ingber Donald E.
Published: January 2018

Physiologically based pharmacokinetic (PBPK) modeling and simulation approaches are beginning to be integrated into drug development and approval processes because they enable key pharmacokinetic (PK) parameters to be predicted from in vitro data. However, these approaches are hampered by many limitations, including an inability to incorporate organ-specific differentials in drug clearance, distribution, and absorption that result from differences in cell uptake, transport, and metabolism. Moreover, such approaches are generally unable to provide insight into pharmacodynamic (PD) parameters. Recent development of microfluidic Organ-on-a-Chip (Organ Chip) cell culture devices that recapitulate tissue-tissue interfaces, vascular perfusion, and organ-level functionality offer the ability to overcome these limitations when multiple Organ Chips are linked via their endothelium-lined vascular channels. Here, we discuss successes and challenges in the use of existing culture models and vascularized Organ Chips for PBPK and PD modeling of human drug responses, as well as in vitro to in vivo extrapolation (IVIVE) of these results, and how these approaches might advance drug development and regulatory review processes in the future.
Visceral hypersensitivity induced by optogenetic activation of the amygdala in conscious rats
Authors: Johnson Anthony C. , Latorre Rocco , Ligon Casey O. , Greenwood-Van Meerveld Beverley
Published: January 2018

In vivo optogenetics identifies brain circuits controlling behaviors in conscious animals by using light to alter neuronal function and offers a novel tool to study the brain-gut axis. Using adenoviral-mediated expression, we aimed to investigate whether photoactivation with channelrhodopsin (ChR2) or photoinhibition with halorhodopsin (HR3.0) of fibers originating from the central nucleus of the amygdala (CeA) at the bed nucleus of the stria terminalis (BNST) had any effect on colonic sensitivity. We also investigated whether there was any deleterious effect of the adenovirus on the neuronal population or the neuronal phenotype within the CeA-BNST circuitry activated during the optogenetic stimulation. In male rats, the CeA was infected with vectors expressing ChR2 or HR3.0 and fiber optic cannulae were implanted on the BNST. After 8–10 wk, the response to graded, isobaric colonic distension was measured with and without laser stimulation of CeA fibers at the BNST. Immunohistochemistry and histology were used to evaluate vector expression, neuronal integrity, and neurochemical phenotype. Photoactivation of CeA fibers at the BNST with ChR2 induced colonic hypersensitivity, whereas photoinhibition of CeA fibers at the BNST with HR3.0 had no effect on colonic sensitivity. Control groups treated with virus expressing reporter proteins showed no abnormalities in neuronal morphology, neuronal number, or neurochemical phenotype following laser stimulation. Our experimental findings reveal that optogenetic activation of discrete brain nuclei can be used to advance our understanding of complex visceral nociceptive circuitry in a freely moving rat model.

NEW & NOTEWORTHY Our findings reveal that optogenetic technology can be employed as a tool to advance understanding of the brain-gut axis. Using adenoviral-mediated expression of opsins, which were activated by laser light and targeted by fiber optic cannulae, we examined central nociceptive circuits mediating visceral pain in a freely moving rat. Photoactivation of amygdala fibers in the stria terminalis with channelrhodopsin induced colonic hypersensitivity, whereas inhibition of the same fibers with halorhodopsin did not alter colonic sensitivity.
Extracellular oxidation in cystic fibrosis airway epithelium causes enhanced EGFR/ADAM17 activity
Authors: Stolarczyk Marta , Veit Guido , Schnúr Andrea , Veltman Mieke , Lukacs Gergely L. , Scholte Bob J.
Published: January 2018

The EGF receptor (EGFR)/a disintegrin and metalloproteinase 17 (ADAM17) signaling pathway mediates the shedding of growth factors and secretion of cytokines and is involved in chronic inflammation and tissue remodeling. Since these are hallmarks of cystic fibrosis (CF) lung disease, we hypothesized that CF transmembrane conductance regulator (CFTR) deficiency enhances EGFR/ADAM17 activity in human bronchial epithelial cells. In CF bronchial epithelial CFBE41o− cells lacking functional CFTR (iCFTR−) cultured at air-liquid interface (ALI) we found enhanced ADAM17-mediated shedding of the EGFR ligand amphiregulin (AREG) compared with genetically identical cells with induced CFTR expression (iCFTR+). Expression of the inactive G551D-CFTR did not have this effect, suggesting that active CFTR reduces EGFR/ADAM17 activity. This was confirmed in CF compared with normal differentiated primary human bronchial epithelial cells (HBEC-ALI). ADAM17-mediated AREG shedding was tightly regulated by the EGFR/MAPK pathway. Compared with iCFTR+ cells, iCFTR− cells displayed enhanced apical presentation and phosphorylation of EGFR, in accordance with enhanced EGFR/ADAM17 activity in CFTR-deficient cells. The nonpermeant natural antioxidant glutathione (GSH) strongly inhibited AREG release in iCFTR and in primary HBEC-ALI, suggesting that ADAM17 activity is directly controlled by extracellular redox potentials in differentiated airway epithelium. Furthermore, the fluorescent redox probe glutaredoxin 1-redox-sensitive green fluorescent protein-glycosylphosphatidylinositol (Grx1-roGFP-GPI) indicated more oxidized conditions in the extracellular space of iCFTR− cells, consistent with the role of CFTR in GSH transport. Our data suggest that in CFTR-deficient airway epithelial cells a more oxidized state of the extracellular membrane, likely caused by defective GSH secretion, leads to enhanced activity of the EGFR/ADAM17 signaling axis. In CF lungs this could contribute to tissue remodeling and hyperinflammation.
Measurements of pulmonary gas exchange efficiency using expired gas and oximetry: results in normal subjects
Authors: West John B. , Wang Daniel L. , Prisk G. Kim
Published: January 2018

We are developing a novel, noninvasive method for measuring the efficiency of pulmonary gas exchange in patients with lung disease. The patient wears an oximeter, and we measure the partial pressures of oxygen and carbon dioxide in inspired and expired gas using miniature analyzers. The arterial Po2 is then calculated from the oximeter reading and the oxygen dissociation curve, using the end-tidal Pco2 to allow for the Bohr effect. This calculation is only accurate when the oxygen saturation is <94%, and therefore, these normal subjects breathed 12.5% oxygen. When the procedure is used in patients with hypoxemia, they breathe air. The Po2 difference between the end-tidal and arterial values is called the “oxygen deficit.” Preliminary data show that this index increases substantially in patients with lung disease. Here we report measurements of the oxygen deficit in 20 young normal subjects (age 19 to 31 yr) and 11 older normal subjects (47 to 88 yr). The mean value of the oxygen deficit in the young subjects was 2.02 ± 3.56 mmHg (means ± SD). This mean is remarkably small. The corresponding value in the older group was 7.53 ± 5.16 mmHg (means ± SD). The results are consistent with the age-related trend of the traditional alveolar-arterial difference, which is calculated from the calculated ideal alveolar Po2 minus the measured arterial Po2. That measurement requires an arterial blood sample. The present study suggests that this noninvasive procedure will be valuable in assessing the degree of impaired gas exchange in patients with lung disease.
Activated human T lymphocytes inhibit TGFβ-induced fibroblast to myofibroblast differentiation via prostaglandins D2and E2
Authors: Lacy Shannon H. , Epa Amali P. , Pollock Stephen J. , Woeller Collynn F. , Thatcher Thomas H. , Phipps Richard P. , Sime Patricia J.
Published: January 2018

In pulmonary fibrosis (PF), fibroblasts and myofibroblasts proliferate and deposit excessive extracellular matrix in the interstitium, impairing normal lung function. Because most forms of PF have a poor prognosis and limited treatment options, PF represents an urgent unmet need for novel, effective therapeutics. Although the role of immune cells in lung fibrosis is unclear, recent studies suggest that T lymphocyte (T cell) activation may be impaired in PF patients. Furthermore, we have previously shown that activated T cells can produce prostaglandins with anti-scarring potential. Here, we test the hypothesis that activated T cells directly inhibit myofibroblast differentiation using a coculture system. Coculture with activated primary blood-derived T cells, from both healthy human donors and PF patients, inhibited transforming growth factor β-induced myofibroblast differentiation in primary human lung fibroblasts isolated from either normal or PF lung tissue. Coculture supernatants contained anti-fibrotic prostaglandins D2and E2, and the inhibitory effect of coculture on myofibroblast differentiation was largely reversed when prostaglandin production was abrogated either by resting the T cells before coculture or via specific pharmacological inhibitors. Moreover, coculture conditions induced COX-2 in HLFs but not in T cells, suggesting that T cells deliver an activating signal to HLFs, which in turn produce anti-fibrotic prostaglandins. We show for the first time that coculture with activated primary human T lymphocytes strongly inhibits myofibroblast differentiation, revealing a novel cell-to-cell communication network with therapeutic implications for fibrotic lung diseases.
Vascular endothelial cell changes in postcardiac surgery acute kidney injury
Authors: Qureshi Saqib H. , Patel Nishith N. , Murphy Gavin J.
Published: January 2018

Acute kidney injury (AKI) is common complication of cardiac surgery; however, the phenotype of this condition is poorly defined. The aim of this study was to characterize changes in endothelial structure and function that underlie postcardiopulmonary bypass (post-CPB) AKI. Adult pigs ( n = 16) were randomized to undergo the following procedures ( n = 8 per group): group 1: sham operation, neck dissection with 2.5 h of general anesthesia; and group 2: CPB, 2.5 h of cardiopulmonary bypass. CPB resulted in the depletion of specific epitopes of glycosaminoglycans side chains of the endothelial glycocalyx: Dolichos biflorus agglutinin: mean difference (MD) [95% confidence interval (CI)], P value: −0.26 (−0.42, −0.09), P = 0.0024, Triticum vulgaris (wheat germ) agglutinin: −0.83 (−1.2, −0.38), P = 0.0005, and Ulex europaeus agglutinin 1: −0.25 (−0.49, −0.009), P = 0.041; endothelial membrane protein: thrombomodulin: −3.13 (−5.6, −0.65), P = 0.02; and adherens junction: VE-cadherin: −1.06 (−1.98, −0.145), P = 0.02. CPB also resulted in reductions in microvascular cortical perfusion: −0.62 (−1.02, −0.22), P = 0.006, and increased renal cortex adenosine levels: 2.32 (0.83, 3.8), P = 0.0059. These changes were accompanied by significant reduction in creatinine clearance at 1.5 h postintervention, MD 95% CI; −51.7 (−99.7, −3.7), P = 0.037, and at 24 h, MD (95% CI): −47.3 (−87.7, −7.6), P = 0.023, and proteinuria immediately postintervention MD (95% CI): 18.79 (2.17, 35.4), P = 0.03 vs. sham. In our experimental CPB model, endothelial injury was associated with loss of autoregulation, increase in microvascular permeability, and reduced glomerular filtration. Interventions that promote endothelial homeostasis may have clinical utility in the prevention of postcardiac surgery AKI.
Mechanism for higher urine pH in normal women compared with men
Authors: Worcester Elaine M. , Bergsland Kristin J. , Gillen Daniel L. , Coe Fredric L.
Published: January 2018

Regulation of acid-base metabolism maintains the pH of body fluids within a tight range. Urine pH (UpH) is also regulated under normal conditions. Median pH of 24-h urines is ~6, but others have noted that UpH in women is higher than men, which has been attributed to differences in diet. If true, it would help to explain the fact that calcium phosphate stones, which form at higher urine pH, are much more common in women than in men. We studied 14 normal subjects (7 men and 7 women) fed identical meals in a Clinical Research Center. Urine and blood samples were collected during fasting and after meals. UpH of women (6.74 ± 0.11) exceeded that of men (6.07 ± 0.17) fed, but not fasting, and UpH rose significantly with meals in women but not men. Serum and urine total CO2rose with meals in women but not men, and in women net acid excretion fell to zero during the fed period. In a general linear model adjusted for age, sex, and weight, net gastrointestinal anion uptake was the main predictor of UpH and was significantly higher in women (3.9 ± 0.6) than men (1.8 ± 0.7) in the fed period. Urine citrate, an anion absorbed by the gastrointestinal tract, was higher in women than men in the fed state, and fractional excretion of citrate was higher in women than men. The higher fed UpH in women is related to a greater absorption of food anions and raises 24-h UpH.
Involvement of cAMP/EPAC/Akt signaling in the antiproteolytic effects of pentoxifylline on skeletal muscles of diabetic rats
Authors: Arcaro Carlos Alberto , Assis Renata Pires , Zanon Neusa Maria , Paula-Gomes Silvia , Navegantes Luiz Carlos Carvalho , Kettelhut Isis Carmo , Brunetti Iguatemy Lourenço , Baviera Amanda Martins
Published: January 2018

Advances in the knowledge of the mechanisms controlling protein breakdown in skeletal muscles have allowed the exploration of new options for treating muscle-wasting conditions. Pentoxifylline (PTX), a nonselective phosphodiesterase (PDE) inhibitor, attenuates the loss of muscle mass during catabolic conditions, mainly via inhibiting protein breakdown. The aim of this study was to explore the mechanisms by which PTX inhibits proteolysis in the soleus and extensor digitorum longus (EDL) muscles of streptozotocin-induced diabetic rats. The levels of atrogin-1 and muscle RING finger-1 were decreased, as were the activities of caspase-3 (EDL) and calpains (soleus and EDL), in diabetic rats treated with PTX, which at least partly explains the drop in the ubiquitin conjugate (EDL) levels and in proteasome activity (soleus and EDL). Treatment with PTX decreased PDE activity and increased cAMP content in muscles of diabetic rats; moreover, it also increased both the protein levels of exchange protein directly activated by cAMP (EPAC, a cAMP effector) and the phosphorylation of Akt. The loss of muscle mass was practically prevented in diabetic rats treated with PTX. These findings advance our understanding of the mechanisms underlying the antiproteolytic effects of PTX and suggest the use of PDE inhibitors as a strategy to activate cAMP signaling, which is emerging as a promising target for treating muscle mass loss during atrophic conditions.

NEW & NOTEWORTHY cAMP signaling has been explored as a strategy to attenuate skeletal muscle atrophies. Therefore, in addition to β2AR agonists, phosphodiesterase inhibitors such as pentoxifylline (PTX) can be an interesting option. This study advances the understanding of the mechanisms related to the antiproteolytic effects of PTX on skeletal muscles of diabetic rats, which involve the activation of both exchange protein directly activated by cAMP and Akt effectors, inhibiting the expression of atrogenes and calpain/caspase-3-proteolytic machinery.
Modeling sources of interlaboratory variability in electrophysiological properties of mammalian neurons
Authors: Tebaykin Dmitry , Tripathy Shreejoy J. , Binnion Nathalie , Li Brenna , Gerkin Richard C. , Pavlidis Paul
Published: January 2018

Patch-clamp electrophysiology is widely used to characterize neuronal electrical phenotypes. However, there are no standard experimental conditions for in vitro whole cell patch-clamp electrophysiology, complicating direct comparisons between data sets. In this study, we sought to understand how basic experimental conditions differ among laboratories and how these differences might impact measurements of electrophysiological parameters. We curated the compositions of external bath solutions (artificial cerebrospinal fluid), internal pipette solutions, and other methodological details such as animal strain and age from 509 published neurophysiology articles studying rodent neurons. We found that very few articles used the exact same experimental solutions as any other, and some solution differences stem from recipe inheritance from advisor to advisee as well as changing trends over the years. Next, we used statistical models to understand how the use of different experimental conditions impacts downstream electrophysiological measurements such as resting potential and action potential width. Although these experimental condition features could explain up to 43% of the study-to-study variance in electrophysiological parameters, the majority of the variability was left unexplained. Our results suggest that there are likely additional experimental factors that contribute to cross-laboratory electrophysiological variability, and identifying and addressing these will be important to future efforts to assemble consensus descriptions of neurophysiological phenotypes for mammalian cell types.

NEW & NOTEWORTHY This article describes how using different experimental methods during patch-clamp electrophysiology impacts downstream physiological measurements. We characterized how methodologies and experimental solutions differ across articles. We found that differences in methods can explain some, but not all, of the study-to-study variance in electrophysiological measurements. Explicitly accounting for methodological differences using statistical models can help correct downstream electrophysiological measurements for cross-laboratory methodology differences.