bluuebunny/crossref_metadata_2025 huggingface dataset:
CRISPR/Cas9 gene drives in genetically variable and nonrandomly mating wild populations
Authors: Drury Douglas W. , Dapper Amy L. , Siniard Dylan J. , Zentner Gabriel E. , Wade Michael J.
Published: May 2017

Mating system and genetic variation impede the spread of gene drives, which target natural populations of disease-vectoring insects.
Field-induced magnetic instability within a superconducting condensate
Authors: Mazzone Daniel Gabriel , Raymond Stéphane , Gavilano Jorge Luis , Ressouche Eric , Niedermayer Christof , Birk Jonas Okkels , Ouladdiaf Bachir , Bastien Gaël , Knebel Georg , Aoki Dai , Lapertot Gérard , Kenzelmann Michel
Published: May 2017

Researchers found evidence for a quantum phase transition within a superconducting condensate.
A two-dimensional semiconductor transistor with boosted gate control and sensing ability
Authors: Xu Jing , Chen Lin , Dai Ya-Wei , Cao Qian , Sun Qing-Qing , Ding Shi-Jin , Zhu Hao , Zhang David Wei
Published: May 2017

We achieved record high photoresponsivity in both MoS
2
and ReS
2
on the structure of an Al
2
O
3
/ITO/SiO
2
/Si “stack.”
Molecular “surgery” on a 23-gold-atom nanoparticle
Authors: Li Qi , Luo Tian-Yi , Taylor Michael G. , Wang Shuxin , Zhu Xiaofan , Song Yongbo , Mpourmpakis Giannis , Rosi Nathaniel L. , Jin Rongchao
Published: May 2017

Changes to surface motifs provide precise tailoring of nanoparticle properties.
Argininosuccinate synthase 1 is an intrinsic Akt repressor transactivated by p53
Authors: Miyamoto Takafumi , Lo Paulisally Hau Yi , Saichi Naomi , Ueda Koji , Hirata Makoto , Tanikawa Chizu , Matsuda Koichi
Published: May 2017

ASS1 bridges a gap between p53 and Akt.
Increasing the revenue from lignocellulosic biomass: Maximizing feedstock utilization
Authors: Alonso David Martin , Hakim Sikander H. , Zhou Shengfei , Won Wangyun , Hosseinaei Omid , Tao Jingming , Garcia-Negron Valerie , Motagamwala Ali Hussain , Mellmer Max A. , Huang Kefeng , Houtman Carl J. , Labbé Nicole , Harper David P. , Maravelias Christos T. , Runge Troy , Dumesic James A.
Published: May 2017

Replacing petroleum by biomass can be economically feasible by generating revenue from the three primary biomass constituents.
Targeting latency-associated peptide promotes antitumor immunity
Authors: Gabriely Galina , da Cunha Andre P. , Rezende Rafael M. , Kenyon Brendan , Madi Asaf , Vandeventer Tyler , Skillin Nathaniel , Rubino Stephen , Garo Lucien , Mazzola Maria A. , Kolypetri Panagiota , Lanser Amanda J. , Moreira Thais , Faria Ana Maria C. , Lassmann Hans , Kuchroo Vijay , Murugaiyan Gopal , Weiner Howard L.
Published: May 2017

Anti-LAP antibody induces antitumor immunity by affecting both adaptive and innate immune mechanisms.
Intratumoral delivery of inactivated modified vaccinia virus Ankara (iMVA) induces systemic antitumor immunity via STING and Batf3-dependent dendritic cells
Authors: Dai Peihong , Wang Weiyi , Yang Ning , Serna-Tamayo Cristian , Ricca Jacob M. , Zamarin Dmitriy , Shuman Stewart , Merghoub Taha , Wolchok Jedd D. , Deng Liang
Published: May 2017

Inactivated vaccinia virus induces antitumor immunity that can overcome resistance to checkpoint inhibitors.
Development of Multiwell-Plate Methods Using Pure Cultures of Methanogens To Identify New Inhibitors for Suppressing Ruminant Methane Emissions
Authors: Weimar M. R. , Cheung J. , Dey D. , McSweeney C. , Morrison M. , Kobayashi Y. , Whitman W. B. , Carbone V. , Schofield L. R. , Ronimus R. S. , Cook G. M.
Published: May 2017

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H
2
and CO
2
that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen
Methanococcus maripaludis
strain S2 and the rumen methanogen
Methanobrevibacter
species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against
M. maripaludis
as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against
M. maripaludis
and
M.
AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions.

IMPORTANCE
Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.
Iron-Dependent Enzyme Catalyzes the Initial Step in Biodegradation of
N
-Nitroglycine by Variovorax sp. Strain JS1663
Authors: Mahan Kristina M. , Zheng Hangping , Fida Tekle T. , Parry Ronald J. , Graham David E. , Spain Jim C.
Published: May 2017

Nitramines are key constituents of most of the explosives currently in use and consequently contaminate soil and groundwater at many military facilities around the world. Toxicity from nitramine contamination poses a health risk to plants and animals. Thus, understanding how nitramines are biodegraded is critical to environmental remediation. The biodegradation of synthetic nitramine compounds such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been studied for decades, but little is known about the catabolism of naturally produced nitramine compounds. In this study, we report the isolation of a soil bacterium,
Variovorax
sp. strain JS1663, that degrades
N
-nitroglycine (NNG), a naturally produced nitramine, and the key enzyme involved in its catabolism.
Variovorax
sp. JS1663 is a Gram-negative, non-spore-forming motile bacterium isolated from activated sludge based on its ability to use NNG as a sole growth substrate under aerobic conditions. A single gene (
nnlA
) encodes an iron-dependent enzyme that releases nitrite from NNG through a proposed β-elimination reaction. Bioinformatics analysis of the amino acid sequence of NNG lyase identified a PAS (Per-Arnt-Sim) domain. PAS domains can be associated with heme cofactors and function as signal sensors in signaling proteins. This is the first instance of a PAS domain present in a denitration enzyme. The NNG biodegradation pathway should provide the basis for the identification of other enzymes that cleave the N—N bond and facilitate the development of enzymes to cleave similar bonds in RDX, nitroguanidine, and other nitramine explosives.

IMPORTANCE
The production of antibiotics and other allelopathic chemicals is a major aspect of chemical ecology. The biodegradation of such chemicals can play an important ecological role in mitigating or eliminating the effects of such compounds.
N
-Nitroglycine (NNG) is produced by the Gram-positive filamentous soil bacterium
Streptomyces noursei
. This study reports the isolation of a Gram-negative soil bacterium,
Variovorax
sp. strain JS1663, that is able to use NNG as a sole growth substrate. The proposed degradation pathway occurs via a β-elimination reaction that releases nitrite from NNG. The novel NNG lyase requires iron(II) for activity. The identification of a novel enzyme and catabolic pathway provides evidence of a substantial and underappreciated flux of the antibiotic in natural ecosystems. Understanding the NNG biodegradation pathway will help identify other enzymes that cleave the N—N bond and facilitate the development of enzymes to cleave similar bonds in synthetic nitramine explosives.
Effects of Arginine on Streptococcus mutans Growth, Virulence Gene Expression, and Stress Tolerance
Authors: Chakraborty Brinta , Burne Robert A.
Published: May 2017

Streptococcus mutans
is a common constituent of oral biofilms and a primary etiologic agent of human dental caries. The bacteria associated with dental caries have potent abilities to produce organic acids from dietary carbohydrates and to grow and metabolize in acidic conditions. By contrast, many commensal bacteria produce ammonia through the arginine deiminase system (ADS), which moderates the pH of oral biofilms. Arginine metabolism by the ADS is a significant deterrent to the initiation and progression of dental caries. In this study, we observed how exogenously provided
l
-arginine affects the growth, the virulence properties, and the tolerance of environmental stresses of
S. mutans
. Supplementation with 1.5% arginine (final concentration) had an inhibitory effect on the growth of
S. mutans
in complex and chemically defined media, particularly when cells were exposed to acid or oxidative stress. The genes encoding virulence factors required for attachment/accumulation (
gtfB
and
spaP
), bacteriocins (
nlmA
,
nlmB
,
nlmD
, and
cipB
), and the sigma factor required for competence development (
comX
) were downregulated during growth with 1.5% arginine. Deep sequencing of RNA (RNA-Seq) comparing the transcriptomes of
S. mutans
growing in chemically defined media with and without 1.5% arginine revealed differential expression of genes encoding ATP-binding cassette transporters, metal transporters, and constituents required for survival, metabolism, and biofilm formation. Therefore, the mechanisms of action by which arginine inhibits dental caries include direct adverse effects on multiple virulence-related properties of the most common human dental caries pathogen.

IMPORTANCE
Metabolism of the amino acid arginine by the arginine deiminase system (ADS) of certain oral bacteria raises the pH of dental plaque and provides a selective advantage to health-associated bacteria, thereby protecting the host from dental caries (cavities). Here, we examine the effects of arginine on the cavity-causing bacterium
Streptococcus mutans
. We find that arginine negatively impacts the growth, the pathogenic potential, and the tolerance of environmental stresses in a way that is likely to compromise the ability of
S. mutans
to cause disease. Using genetic and genomic techniques, multiple mechanisms by which arginine exerts its influence on virulence-related properties of
S. mutans
are discovered. This report demonstrates that a primary mechanism of action by which arginine inhibits the initiation and progression of dental caries may be by reducing the pathogenic potential of
S. mutans
.
Pseudomonas chlororaphis Produces Two Distinct R-Tailocins That Contribute to Bacterial Competition in Biofilms and on Roots
Authors: Dorosky Robert J. , Yu Jun Myoung , Pierson Leland S. , Pierson Elizabeth A.
Published: May 2017

R-type tailocins are high-molecular-weight bacteriocins that resemble bacteriophage tails and are encoded within the genomes of many
Pseudomonas
species. In this study, analysis of the
P. chlororaphis
30-84 R-tailocin gene cluster revealed that it contains the structural components to produce two R-tailocins of different ancestral origins. Two distinct R-tailocin populations differing in length were observed in UV-induced lysates of
P. chlororaphis
30-84 via transmission electron microscopy. Mutants defective in the production of one or both R-tailocins demonstrated that the killing spectrum of each tailocin is limited to
Pseudomonas
species. The spectra of pseudomonads killed by the two R-tailocins differed, although a few
Pseudomonas
species were either killed by or insusceptible to both tailocins. Tailocin release was disrupted by deletion of the holin gene within the tailocin gene cluster, demonstrating that the lysis cassette is required for the release of both R-tailocins. The loss of functional tailocin production reduced the ability of
P. chlororaphis
30-84 to compete with an R-tailocin-sensitive strain within biofilms and rhizosphere communities. Our study demonstrates that
Pseudomonas
species can produce more than one functional R-tailocin particle sharing the same lysis cassette but differing in their killing spectra. This study provides evidence for the role of R-tailocins as determinants of bacterial competition among plant-associated
Pseudomonas
in biofilms and the rhizosphere.

IMPORTANCE
Recent studies have identified R-tailocin gene clusters potentially encoding more than one R-tailocin within the genomes of plant-associated
Pseudomonas
but have not demonstrated that more than one particle is produced or the ecological significance of the production of multiple R-tailocins. This study demonstrates for the first time that
Pseudomonas
strains can produce two distinct R-tailocins with different killing spectra, both of which contribute to bacterial competition between rhizosphere-associated bacteria. These results provide new insight into the previously uncharacterized role of R-tailocin production by plant-associated
Pseudomonas
species in bacterial population dynamics within surface-attached biofilms and on roots.
Raffinose Induces Biofilm Formation by Streptococcus mutans in Low Concentrations of Sucrose by Increasing Production of Extracellular DNA and Fructan
Authors: Nagasawa Ryo , Sato Tsutomu , Senpuku Hidenobu
Published: May 2017

Streptococcus mutans
is the primary etiological agent of dental caries and causes tooth decay by forming a firmly attached biofilm on tooth surfaces. Biofilm formation is induced by the presence of sucrose, which is a substrate for the synthesis of extracellular polysaccharides but not in the presence of oligosaccharides. Nonetheless, in this study, we found that raffinose, which is an oligosaccharide with an intestinal regulatory function and antiallergic effect, induced biofilm formation by
S. mutans
in a mixed culture with sucrose, which was at concentrations less than those required to induce biofilm formation directly. We analyzed the possible mechanism behind the small requirement for sucrose for biofilm formation in the presence of raffinose. Our results suggested that sucrose contributed to an increase in bacterial cell surface hydrophobicity and biofilm formation. Next, we examined how the effects of raffinose interacted with the effects of sucrose for biofilm formation. We showed that the presence of raffinose induced fructan synthesis by fructosyltransferase and aggregated extracellular DNA (eDNA, which is probably genomic DNA released from dead cells) into the biofilm. eDNA seemed to be important for biofilm formation, because the degradation of DNA by DNase I resulted in a significant reduction in biofilm formation. When assessing the role of fructan in biofilm formation, we found that fructan enhanced eDNA-dependent cell aggregation. Therefore, our results show that raffinose and sucrose have cooperative effects and that this induction of biofilm formation depends on supportive elements that mainly consist of eDNA and fructan.

IMPORTANCE
The sucrose-dependent mechanism of biofilm formation in
Streptococcus mutans
has been studied extensively. Nonetheless, the effects of carbohydrates other than sucrose are inadequately understood. Our findings concerning raffinose advance the understanding of the mechanism underlying the joint effects of sucrose and other carbohydrates on biofilm formation. Since raffinose has been reported to have positive effects on enterobacterial flora, research on the effects of raffinose on the oral flora are required prior to its use as a beneficial sugar for human health. Here, we showed that raffinose induced biofilm formation by
S. mutans
in low concentrations of sucrose. The induction of biofilm formation generally generates negative effects on the oral flora. Therefore, we believe that this finding will aid in the development of more effective oral care techniques to maintain oral flora health.
Making Time: Conservation of Biological Clocks from Fungi to Animals
Authors: Dunlap Jay C. , Loros Jennifer J.
Published: May 2017

The capacity for biological timekeeping arose at least three times through evolution, in prokaryotic cyanobacteria, in cells that evolved into higher plants, and within the group of organisms that eventually became the fungi and the animals.
Neurospora
is a tractable model system for understanding the molecular bases of circadian rhythms in the last of these groups, and is perhaps the most intensively studied circadian cell type. Rhythmic processes described in fungi include growth rate, stress responses, developmental capacity, and sporulation, as well as much of metabolism; fungi use clocks to anticipate daily environmental changes. A negative feedback loop comprises the core of the circadian system in fungi and animals. In
Neurospora
, the best studied fungal model, it is driven by two transcription factors, WC-1 and WC-2, that form the White Collar Complex (WCC). WCC elicits expression of the
frq
gene. FRQ complexes with other proteins, physically interacts with the WCC, and reduces its activity; the kinetics of these processes is strongly influenced by progressive phosphorylation of FRQ. When FRQ becomes sufficiently phosphorylated that it loses the ability to influence WCC activity, the circadian cycle starts again. Environmental cycles of light and temperature influence
frq
and FRQ expression and thereby reset the internal circadian clocks. The molecular basis of circadian output is also becoming understood. Taken together, molecular explanations are emerging for all the canonical circadian properties, providing a molecular and regulatory framework that may be extended to many members of the fungal and animal kingdoms, including humans.
Water for Agriculture: the Convergence of Sustainability and Safety
Authors: Markland Sarah M. , Ingram David , Kniel Kalmia E. , Sharma Manan
Published: May 2017

Agricultural water is a precious and limited resource. Increasingly more water types and sources are being explored for use in irrigation within the United States and across the globe. As outlined in this chapter, the Produce Safety Rule (PSR) in the Food Safety and Modernization Act (FSMA) provide irrigation water standards for application of water to fruits and vegetables consumed raw. These rules for production and use of water will continue to develop and be required as the world experiences aspects of a changing climate including flooding as well as drought conditions. Research continues to assess the use of agricultural water types. The increased use of reclaimed water in the United States as well as for selected irrigation water needs for specific crops may provide increased water availability. The use of surface water can be used in irrigation as well, but several studies have shown the presence of some enteric bacterial pathogens (enterohemorrhagic
E. coli
,
Salmonella
spp. and
Listeria monocytogenes
) in these waters that may contaminate fruits and vegetables. There have been outbreaks of foodborne illness in the U.S., South America, Europe, and Australia related to the use of contaminated water in fruit and vegetable irrigation or washing. Unreliable water supplies, more stringent microbial water standards, mitigation technologies and expanded uses of reclaimed waters have all increased interest in agricultural water.
Design of the Subpopulations and Intermediate Outcome Measures in COPD (SPIROMICS) AIR Study
Authors: Hansel Nadia N , Paulin Laura M , Gassett Amanda J , Peng Roger D , Alexis Neil , Fan Vincent S , Bleecker Eugene , Bowler Russell , Comellas Alejandro P , Dransfield Mark , Han MeiLan K , Kim Victor , Krishnan Jerry A , Pirozzi Cheryl , Cooper Christopher B , Martinez Fernando , Woodruff Prescott G , Breysse Patrick J , Barr R Graham , Kaufman Joel D
Published: May 2017

Introduction

Population-based epidemiological evidence suggests that exposure to ambient air pollutants increases hospitalisations and mortality from chronic obstructive pulmonary disease (COPD), but less is known about the impact of exposure to air pollutants on patient-reported outcomes, morbidity and progression of COPD.

Methods and analysis

The Subpopulations and Intermediate Outcome Measures in COPD (SPIROMICS) Air Pollution Study (SPIROMICS AIR) was initiated in 2013 to investigate the relation between individual-level estimates of short-term and long-term air pollution exposures, day-to-day symptom variability and disease progression in individuals with COPD. SPIROMICS AIR builds on a multicentre study of smokers with COPD, supplementing it with state-of-the-art air pollution exposure assessments of fine particulate matter, oxides of nitrogen, ozone, sulfur dioxide and black carbon. In the parent study, approximately 3000 smokers with and without airflow obstruction are being followed for up to 3 years for the identification of intermediate biomarkers which predict disease progression. Subcohorts undergo daily symptom monitoring using comprehensive daily diaries. The air monitoring and modelling methods employed in SPIROMICS AIR will provide estimates of individual exposure that incorporate residence-specific infiltration characteristics and participant-specific time-activity patterns. The overarching study aim is to understand the health effects of short-term and long-term exposures to air pollution on COPD morbidity, including exacerbation risk, patient-reported outcomes and disease progression.

Ethics and dissemination

The institutional review boards of all the participating institutions approved the study protocols. The results of the trial will be presented at national and international meetings and published in peer-reviewed journals.
Canine dystocia in 50 UK first‐opinion emergency‐care veterinary practices: prevalence and risk factors
Authors: O'Neill D. G. , O'Sullivan A. M. , Manson E. A. , Church D. B. , Boag A. K. , McGreevy P. D. , Brodbelt D. C.
Published: May 2017

Dystocia can represent a major welfare issue for dogs of certain breeds and morphologies. First‐opinion emergency‐care veterinary caseloads represent a useful data resource for epidemiological research because dystocia can often result in emergency veterinary care. The study analysed a merged database of clinical records from 50 first‐opinion emergency‐care veterinary practices participating in the VetCompass Programme. Multivariable logistic regression modelling was used for risk factors analysis. There were 701 dystocia cases recorded among 18,758 entire female dogs, resulting in a dystocia prevalence of 3.7 per cent (95 per cent CI 3.5–4.0 per cent). Breeds with the highest odds of dystocia compared with crossbred bitches were French Bulldog (OR: 15.9, 95 per cent CI 9.3 to 27.2, P<0.001), Boston Terrier (OR: 12.9, 95 per cent CI 5.6 to 29.3, P<0.001), Chihuahua (OR: 10.4, 95 per cent CI 7.0 to 15.7, P<0.001) and Pug (OR: 11.3, 95 per cent CI 7.1 to 17.9, P<0.001). Bitches aged between 3.0 and 5.9 years had 3.1 (95 per cent CI 2.6 to 3.7, P<0.001) times the odds of dystocia compared with bitches aged under 3.0years. Certain breeds, including some brachycephalic and toy breeds, appeared at high risk of dystocia. Opportunities to improve this situation are discussed.
Preliminary evaluation of cytosine‐phosphate‐guanine oligodeoxynucleotides bound to gelatine nanoparticles as immunotherapy for canine atopic dermatitis
Authors: Wagner I. , Geh K. J. , Hubert M. , Winter G. , Weber K. , Classen J. , Klinger C. , Mueller R. S.
Published: May 2017

Cytosine‐phosphate‐guanine oligodeoxynucleotides (CpG ODN) are a promising new immunotherapeutic treatment option for canine atopic dermatitis (AD). The aim of this uncontrolled pilot study was to evaluate clinical and immunological effects of gelatine nanoparticle (GNP)‐bound CpG ODN (CpG GNP) on atopic dogs. Eighteen dogs with AD were treated for 8 weeks (group 1, n=8) or 18 weeks (group 2, n=10). Before inclusion and after 2 weeks, 4 weeks, 6 weeks (group 1+2), 8 weeks, 12 weeks and 16 weeks (group 2) 75 µg CpG ODN/dog (bound to 1.5 mg GNP) were injected subcutaneously. Pruritus was evaluated daily by the owner. Lesions were evaluated and serum concentrations and mRNA expressions of interferon‐γ, tumour necrosis factor‐α, transforming growth factor‐β, interleukin (IL) 10 and IL‐4 (only mRNA expression) were determined at inclusion and after 8 weeks (group 1+2) and 18 weeks (group 2). Lesions and pruritus improved significantly from baseline to week 8. Mean improvements from baseline to week 18 were 23 per cent and 44 per cent for lesions and pruritus, respectively, an improvement of ≥50 per cent was seen in six out of nine and three out of six dogs, respectively. IL‐4 mRNA expression decreased significantly. The results of this study show a clinical improvement of canine AD with CpG GNP comparable to allergen immunotherapy. Controlled studies are needed to confirm these findings.
Social Networks and Macrosocial Change
Authors: Erikson Emily , Occhiuto Nicholas
Published: May 2017

Social networks are heavily implicated in large-scale social transformations. They are both transformed and transformative. We review the ways in which social networks act as agents of change in macrohistorical processes, stressing two distinct theoretical approaches. Formalism analyzes the structure of networks. Relationalism evaluates the linking properties of networks. Using these two approaches to organize the literature, we present the current state of knowledge on the effects of social networks for four central macrohistorical outcomes: civil uprising, state formation, global and national policy formation and diffusion, and economic development and increasing inequality. We then consider new theoretical advances in institutional emergence and methodological innovations in computational modeling and their potential for reconciling and advancing existing findings and approaches on the effects of social networks on macrosocial change.
A simple laboratory exercise with rat isolated esophagus and stomach fundus to reveal functional differences between striated and smooth muscle cells
Authors: Batista-Lima Francisco José , Gadelha Kalinne Kelly Lima , Oliveira Daniel Maia , Vasconcelos Thiago Brasileiro , Brito Teresinha Silva , Magalhães Pedro Jorge Caldas
Published: May 2017

This study describes an undergraduate student laboratory activity using isolated preparations from rat gastrointestinal tissues that possess contractile profiles typically exhibited by striated and smooth muscle cells. While students are introduced to an ex vivo methodology, they can compare differences in trace experiments, twitch aspects, phasic and tonic properties, force-frequency relationships, and pharmacological responsiveness of esophageal (striated) and fundic (smooth muscle) segments. Muscle strips were subjected to electrical field stimulation (EFS) applied by platinum electrodes immersed in the physiological solution. The contractile profile of EFS responses varied between these two types of gut preparations. Atropine and tubocurarine revealed differential inhibitory influences in esophagus or fundus tissues; caffeine and procaine produced similar effects, i.e., potentiation and blockade of the EFS-induced contractile response in these tissues, respectively. Experimental results obtained during the activity helped the improvement of student learning about basic concepts previously discussed in theoretical lectures. To measure student learning with this laboratory exercise, a questionnaire was applied before and after the activity, and the number of expected correct answers, concerning the mechanisms of contraction in striated and smooth muscle, could be clearly evidenced.
Understanding the interrelationship between the synthesis of urea and gluconeogenesis by formulating an overall balanced equation
Authors: Ipata Piero L. , Pesi Rossana
Published: May 2017

It is well known that a strong metabolic interrelationship exists between ureagenesis and gluconeogenesis. In this paper, we present a detailed, overall equation, describing a possible metabolic link between ureagenesis and gluconeogenesis. We adopted a guided approach in which we strongly suggest that students, when faced with the problem of obtaining the overall equation of a metabolic pathway, carefully account for all atoms and charges of the single reactions, as well as the cellular localizations of the substrates, and the related transport systems. If this suggestion is always taken into account, a balanced, overall equation of a metabolic pathway will be obtained, which strongly facilitates the discussion of its physiological role. Unfortunately, textbooks often report unbalanced overall equations of metabolic pathways, including ureagenesis and gluconeogenesis. Most likely the reason is that metabolism and enzymology have been neglected for about three decades, owing to the remarkable advances of molecular biology and molecular genetics. In this paper, we strongly suggest that students, when faced with the problem of obtaining the overall reaction of a metabolic pathway, carefully control if the single reactions are properly balanced for atoms and charges. Following this suggestion, we were able to obtain an overall equation describing the metabolic interrelationship between ureagenesis and gluconeogenesis, in which urea and glucose are the final products. The aim is to better rationalize this topic and to convince students and teachers that metabolism is an important and rewarding chapter of human physiology.
Loss of the anion exchanger DRA (Slc26a3), or PAT1 (Slc26a6), alters sulfate transport by the distal ileum and overall sulfate homeostasis
Authors: Whittamore Jonathan M. , Hatch Marguerite
Published: May 2017

The ileum is considered the primary site of inorganic sulfate ([Formula: see text]) absorption. In the present study, we explored the contributions of the apical chloride/bicarbonate (Cl−/[Formula: see text]) exchangers downregulated in adenoma (DRA; Slc26a3), and putative anion transporter 1 (PAT1; Slc26a6), to the underlying transport mechanism. Transepithelial35[Formula: see text] and36Cl−fluxes were determined across isolated, short-circuited segments of the distal ileum from wild-type (WT), DRA-knockout (KO), and PAT1-KO mice, together with measurements of urine and plasma sulfate. The WT distal ileum supported net sulfate absorption [197.37 ± 13.61 (SE) nmol·cm−2·h−1], but neither DRA nor PAT1 directly contributed to the unidirectional mucosal-to-serosal flux ([Formula: see text]), which was sensitive to serosal (but not mucosal) DIDS, dependent on Cl−, and regulated by cAMP. However, the absence of DRA significantly enhanced net sulfate absorption by one-third via a simultaneous rise in [Formula: see text] and a 30% reduction to the secretory serosal-to-mucosal flux ([Formula: see text]). We propose that DRA, together with PAT1, contributes to [Formula: see text] by mediating sulfate efflux across the apical membrane. Associated with increased ileal sulfate absorption in vitro, plasma sulfate was 61% greater, and urinary sulfate excretion ( USO4) 2.2-fold higher, in DRA-KO mice compared with WT controls, whereas USO4was increased 1.8-fold in PAT1-KO mice. These alterations to sulfate homeostasis could not be accounted for by any changes to renal sulfate handling suggesting that the source of this additional sulfate was intestinal. In summary, we characterized transepithelial sulfate fluxes across the mouse distal ileum demonstrating that DRA (and to a lesser extent, PAT1) secretes sulfate with significant implications for intestinal sulfate absorption and overall homeostasis.

NEW & NOTEWORTHY Sulfate is an essential anion that is actively absorbed from the small intestine involving members of the Slc26 gene family. Here, we show that the main intestinal chloride transporter Slc26a3, known as downregulated in adenoma (DRA), also handles sulfate and contributes to its secretion into the lumen. In the absence of functional DRA (as in the disease congenital chloride diarrhea), net intestinal sulfate absorption was significantly enhanced resulting in substantial alterations to overall sulfate homeostasis.
S-nitrosylation of VASP at cysteine 64 mediates the inflammation-stimulated increase in microvascular permeability
Authors: Zamorano Patricia , Marín Natalie , Córdova Francisco , Aguilar Alejandra , Meininger Cynthia , Boric Mauricio P. , Golenhofen Nikola , Contreras Jorge E. , Sarmiento José , Durán Walter N. , Sánchez Fabiola A.
Published: May 2017

We tested the hypothesis that platelet-activating factor (PAF) induces S-nitrosylation of vasodilator-stimulated phosphoprotein (VASP) as a mechanism to reduce microvascular endothelial barrier integrity and stimulate hyperpermeability. PAF elevated S-nitrosylation of VASP above baseline levels in different endothelial cells and caused hyperpermeability. To ascertain the importance of endothelial nitric oxide synthase (eNOS) subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced S-nitrosylation of VASP in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Reconstitution of VASP knockout myocardial endothelial cells with cysteine mutants of VASP demonstrated that S-nitrosylation of cysteine 64 is associated with PAF-induced hyperpermeability. We propose that regulation of VASP contributes to endothelial cell barrier integrity and to the onset of hyperpermeability. S-nitrosylation of VASP inhibits its function in barrier integrity and leads to endothelial monolayer hyperpermeability in response to PAF, a representative proinflammatory agonist.

NEW & NOTEWORTHY Here, we demonstrate that S-nitrosylation of vasodilator-stimulated phosphoprotein (VASP) on C64 is a mechanism for the onset of platelet-activating factor-induced hyperpermeability. Our results reveal a dual role of VASP in endothelial permeability. In addition to its well-documented function in barrier integrity, we show that S-nitrosylation of VASP contributes to the onset of endothelial permeability.
Contribution of Primate Magnocellular Red Nucleus to Timing of Hand Preshaping During Reaching to Grasp
Authors: Van Kan Peter L. E. , McCurdy Martha L.
Published: May 2017

Magnocellular red nucleus (RNm) is involved in controlling goal-directed limb movements such as reaching to grasp. We tested two hypotheses related to RNm's role in controlling reach-to-grasp movements. One hypothesis is that forelimb RNm neurons are grasp specific, and the other is that they specify the timing of metacarpi-phalangeal (MCP) extension to preshape the hand during the appropriate phase of the reach. We recorded single-unit discharge while monkeys performed two behavioral tasks that elicited similar reaches but differed in grasp. One task consisted of a reach with a precision grasp that elicited independent use of thumb and forefinger; the other included a whole-hand grasp that elicited concerted use of the four fingers. Most RNm neurons tested were engaged strongly during both the whole-hand and precision tasks, and the magnitude of discharge modulation did not differ between tasks. Thus most RNm neurons are not grasp specific but, instead, may contribute to behavioral features common to the two tasks. Two methods were used to investigate relations between single-unit discharge and kinematic data from the same individual trials of the whole-hand and precision tasks for a subset of forelimb RNm neurons. One method focused on correlations between parameters of RNm discharge and the duration, amplitude, and velocity of rotation of forelimb joints for each of the tasks. The second method compared between-task differences in times of peak neuronal discharge to between-task differences in times of rotations of forelimb joints. Parameters of reach-related RNm discharge were more frequently correlated with parameters of MCP extension than with parameters of rotation of wrist, elbow, and shoulder joints. Analyses of temporal relations between discharge and kinematic data during both the whole-hand and precision tasks indicate that discharge was time locked most frequently to MCP extension and, to a lesser extent, elbow extension during both tasks. We conclude that RNm may command muscle synergies that provide a basic preshape of the hand at the appropriate phase of limb transport. In addition, the timing of RNm's contribution to hand preshaping varies with the behavioral requirements of the task.
Nociceptive Quality of the Laser-Evoked Blink Reflex in Humans
Authors: Romaniello A. , Valls-Solé J. , Iannetti G. D. , Truini A. , Manfredi M. , Cruccu G.
Published: May 2017

Laser radiant-heat pulses selectively excite the free nerve endings in the superficial layers of the skin and activate mechano-thermal nociceptive afferents; when directed to the perioral or supraorbital skin, high-intensity laser pulses evoke a blink-like response in the orbicularis oculi muscle (the laser blink reflex, LBR). We investigated the functional properties (startle or nociceptive origin) of the LBR and sought to characterize its central pathways. Using high-intensity CO2-laser stimulation of the perioral or supraorbital regions and electromyographic (EMG) recordings from the orbicularis oculi muscles, we did five experiments in 20 healthy volunteers. First, to investigate whether the LBR is a startle response, we studied its habituation to expected rhythmic stimuli and to unexpected arrhythmic stimuli. To assess its possible nociceptive quality, we studied changes in the LBR and the R2 component of the electrical blink reflex after a lidocaine-induced supraorbital nerve block and after intramuscular injection of the opiate fentanyl and the opiate-antagonist naloxone. To characterize the central pathways for the LBR, we investigated the interaction between the LBR and the three components of the blink reflex (R1, R2, and R3) by delivering laser pulses to the perioral or supraorbital regions before or after electrical stimulation of the supraorbital nerve at various interstimulus intervals. Finally, to gain further information on the central LBR pathways, using two identical CO2-laser stimulators, we studied the LBR recovery curves with paired laser pulses delivered to adjacent forehead points at interstimulus intervals from 250 ms to 1.5 s. The LBR withstood relatively high-frequency rhythmic stimulations, and unexpected laser pulses failed to evoke larger responses. When lidocaine began to induce hypoalgesia (about 5 min after the injection), the LBR was abolished, whereas R2 was only partly suppressed 10 min after the injection. Fentanyl injection induced strong, naloxone-reversible, LBR suppression (the response decreased to 25.3% of predrug values at 10 min and to 4% at 20 min), whereas R2 remained appreciably unchanged. Whether directed to the perioral or supraorbital regions, preceding laser pulses strongly suppressed R2 and R3 though not R1. Conversely, preceding electrical stimuli to the supraorbital nerve suppressed the LBR. In response to paired stimuli, the LBR recovered significantly faster than R2. These findings indicate that the LBR is a nociceptive reflex, which shares part of the interneuron chain mediating the nonnociceptive R2 blink reflex, probably in the medullary reticular formation. The LBR may prove useful for studying the pathophysiology of orofacial pain syndromes.
GABAB Presynaptic Inhibition Has an In Vivo Time Constant Sufficiently Rapid to Allow Modulation at Theta Frequency
Authors: Molyneaux Bradley J. , Hasselmo Michael E.
Published: May 2017

Cyclical activity of GABAergic interneurons during theta rhythm could mediate phasic changes in strength of glutamatergic synaptic transmission in the hippocampal formation if presynaptic inhibition from activation of GABAB receptors is sufficiently rapid to change within a theta cycle. The experiments described here analyzed the time course of GABABmodulation using a heterosynaptic depression paradigm in anesthetized rats at physiological temperatures. Heterosynaptic depression of the slope of evoked potentials decayed with a time constant that would allow significant changes in transmission across different phases of the theta cycle. This heterosynaptic depression was significantly reduced by local infusion of the GABAB receptor antagonist CGP55845A.
Spinal Inhibitory Effects of Cardiopulmonary Afferent Inputs in Monkeys: Neuronal Processing in High Cervical Segments
Authors: Chandler Margaret J. , Zhang Jianhua , Qin Chao , Foreman Robert D.
Published: May 2017

Noxious stimulation of spinal afferents inhibits primate spinothalamic tract (STT) neurons in segments distant from the region of afferent entry. Inhibitory effects of cardiopulmonary sympathetic afferent (CPSA) stimulation remain after C1 transection but disappear with spinal transection between C3 and C7. We hypothesized that spinal inhibitory effects produced by CPSA stimulation are processed by neurons in C1–C3 segments. One purpose of this study in anesthetized monkeys was to determine whether chemical activation of high cervical neurons reduced sacral STT cell responses to colorectal distension (CRD) and urinary bladder distension (UBD). First, effects and interactions of pelvic and cardiopulmonary visceral afferent inputs were determined in 10 monkeys on extracellular activity of sacral STT neurons recorded in deep dorsal horn. CRD and UBD increased activity in 95 and 91% of sacral STT neurons, respectively. CPSA and cardiopulmonary vagal stimulation decreased activity in 84 and 56% of STT neurons, respectively. CPSA stimulation decreased CRD-evoked activity in six of eight sacral STT neurons and decreased UBD-evoked activity in five of eight STT neurons tested. Excitatory amino acid application at C2 segment decreased CRD-evoked responses in 7 of 10 sacral STT neurons and decreased UBD-evoked responses in 9 of 12 STT neurons. The second purpose of this study was to examine responses of C1–C3 descending propriospinal neurons to stimulation of cardiopulmonary afferent fibers. If C1–C3 neurons process CPSA input to suppress STT transmission, then CPSA stimulation should excite C1–C3neurons with descending projections. Effects of thoracic vagus nerve stimulation also were examined. Vagal stimulation inhibits STT neurons in segments below C3 but excites C1–C3 STT neurons; we theorized that vagal inhibition of sensory transmission might relay in high cervical segments and, therefore, excite C1–C3 descending propriospinal neurons. Extracellular discharge rate was recorded for C1–C3 neurons antidromically activated from thoracic or lumbar spinal cord in 24 monkeys. CPSA stimulation increased activity of 16 of 45 neurons and inhibited one cell. Thoracic vagus stimulation increased activity of 20 of 43 neurons and inhibited one cell; stimulation of abdominal vagus fibers did not affect activity of six of six cells that were excited by thoracic vagal input. Mechanical stimulation of somatic fields excited 30 of 41 neurons tested. All neurons activated by visceral input received convergent somatic input from noxious pinch of somatic receptive fields that generally included the neck and upper body; 11 C1–C3 propriospinal neurons did not respond to any afferent input examined. Results of these studies were consistent with the idea that modulation of spinal nociceptive transmission might involve neuronal connections in high cervical segments.
Characterization of Mitotic Neurons Derived From Adult Rat Hypothalamus and Brain Stem
Authors: Evans Jenafer , Sumners Colin , Moore Jennifer , Huentelman Matthew J. , Deng Jie , Gelband Craig H. , Shaw Gerry
Published: May 2017

Embryonic or neonatal rat neurons retain plasticity and are readily grown in tissue culture, but neurons of the adult brain were thought to be terminally differentiated and therefore difficult to culture. Recent studies, however, suggest that it may be possible to culture differentiated neurons from the hippocampus of adult rats. We modified these procedures to grow differentiated neurons from adult rat hypothalamus and brain stem. At day 7 in tissue culture and beyond, the predominant cell types in hypothalamic and brain stem cultures had a stellate morphology and could be subdivided into two distinct groups, one of which stained with antibodies to the immature neuron marker α-internexin, while the other stained with the astrocyte marker GFAP. The α-internexin positive cells were mitotic and grew to form a characteristic two-dimensional cellular network. These α-internexin positive cells coimmunostained for the neuronal markers MAP2, type III β-tubulin, and tau, and also bound tetanus toxin, but were negative for the oligodendrocyte marker GalC and also for the neurofilament triplet proteins NF-L, NF-M, and NF-H, markers of more mature neurons. Patch-clamp analysis of these α-internexin positive cells revealed small Ca2+ currents with a peak current of −0.5 ± 0.1 pA/pF at a membrane potential of −20 mV ( n = 5) and half-maximal activation at −30 mV ( n = 5). Na+ currents with a peak current density of −154.5 ± 49.8 pA/pF at a membrane potential of −15 mV ( n = 5) were also present. We also show that these cells can be frozen and regrown in tissue culture and that they can be efficiently infected by viral vectors. These cells therefore have the immunological and electrophysiological properties of immature mitotic neurons and should be useful in a variety of future studies of neuronal differentiation and function.
Role of Ca2+ in the Synchronization of Transmitter Release at Calyceal Synapses in the Auditory System of Rat
Authors: Chuhma Nao , Ohmori Harunori
Published: May 2017

The synchronization of transmitter release in the synapse of the medial nucleus of the trapezoid body (MNTB) is achieved during early postnatal development as a consequence of elimination of delayed asynchronous releases and appears to reflect changes in the dynamics of Ca2+ entry and clearance. To examine the role of Ca2+ in regulating synchronization of transmitter release in the mature synapse (after postnatal day 9, P9), we perturbed Ca2+ dynamics systematically. Replacement of external Ca2+ (2 mM) with Sr2+ induced delayed asynchronous release following the major EPSC. We tried to reproduce asynchronous releases without using Sr2+ and instead by manipulating the time course and the size of Ca2+ transient in the presynaptic terminal, under the assumption that replacement of external Na+ with Li+ or application of eosin-Y would prolong the lifetime of Ca2+ transient by reducing the rate of Ca2+ extrusion from the terminal. With application of Li+, Ca2+ transient in the terminal was prolonged, the EPSC decay time course was prolonged, and the EPSC amplitude increased. However, these EPSCs were not followed by delayed asynchronous release. When Ca2+ influx was reduced, either by partial Ca2+ channel blockade with a low concentration of Cd2+ or ω-agatoxin IVA, a marked asynchronous release resulted. This was further enhanced by the combined application of Li+ or eosin-Y. These results suggest that cooperative increases of both Ca2+ influx and Ca2+ clearance capacities leading to a sharper Ca2+ spike in the presynaptic terminal underlie synchronized transmitter release in the presynaptic terminal of the MNTB.