bluuebunny/crossref_metadata_2025 huggingface dataset:
Photoinhibition in marine picocyanobacteria
Authors: Soitamo Arto , Havurinne Vesa , Tyystjärvi Esa
Published: April 2017

Marine Synechococcus and Prochlorococcus cyanobacteria have different antenna compositions although they are genetically near to each other, and different strains thrive in very different illumination conditions. We measured growth and photoinhibition of PSII in two low‐light and one high‐light Prochlorococcus strains and in one Synechococcus strain. All strains were found to be able to shortly utilize moderate or even high light, but the low‐light strains bleached rapidly in moderate light. Measurements of photoinhibition in the presence of the antibiotic lincomycin showed that a low‐light Prochlorococcus strain was more sensitive than a high‐light strain and both were more sensitive than the marine Synechococcus. The action spectrum of photoinhibition showed an increase from blue to ultraviolet wavelengths in all strains, suggesting contribution of manganese absorption to photoinhibition, but blue light caused less photoinhibition in marine cyanobacteria than expected on the basis of earlier results from plants and cyanobacteria. The visible‐light part of the action spectrum resembled the absorption spectrum of the organism, suggesting that photosynthetic antenna pigments, especially divinyl chlorophylls, have a more important role as photoreceptors of visible‐light photoinhibition in marine cyanobacteria than in other photoautotrophs.
Comparing the error‐related negativity across groups: The impact of error‐ and trial‐number differences
Authors: Fischer Adrian G. , Klein Tilmann A. , Ullsperger Markus
Published: April 2017

The error‐related negativity (ERN or Ne) is increasingly being investigated as a marker discriminating interindividual factors and moves toward a surrogate marker for disorders or interventions. Although reproducibility and validity of neuroscientific and psychological research has been criticized, clear data on how different quantification methods of the ERN and their relation to available trial numbers affect within‐ and across‐participant studies is sparse. Within a large sample of 863 healthy human participants, we demonstrate that, across participants, the number of errors correlates with the amplitude of the ERN independently of the number of errors included in ERN quantification per participant, constituting a possible confound when such variance is unaccounted for. Additionally, we find that ERN amplitudes reach high consistency within participants at lower trial numbers, yet when comparisons between groups of participants are desired, increasing error‐trial numbers lead to higher statistical power. We derive concrete suggestions for specific types of analyses, which may help researchers to more effectively design studies and analyze error‐related EEG data with the most appropriate measurement technique for the question at hand and trial number available.
Vertical ally‐or‐acquire choice and technological performance: a resource dependence perspective
Authors: Lin Liang‐Hung
Published: April 2017

In order to advance our understanding of technological firms’ vertical ally‐or‐acquire choices, this study applies resource dependence theory to explore how mutual dependence and dependence asymmetry between partners’ technologies affect governance choice. The collective effect of mutual dependence, dependence asymmetry and governance choice on technological performance of the transaction is also examined. Heckman's two‐stage analyses of 135 technology alliances and 91 technology acquisitions suggest that mutually dependent firms tend to adopt acquisitions, whereas asymmetrically dependent firms tend to adopt alliances. The fits between dependence asymmetry and alliances, and between mutual dependence and acquisitions consequently enhance technological performance.
Randomized cross‐over trial of ventilator modes during non‐invasive ventilation titration in amyotrophic lateral sclerosis
Authors: Vrijsen Bart , Buyse Bertien , Belge Catharina , Vanpee Goele , Van Damme Philip , Testelmans Dries
Published: April 2017

ABSTRACT

Background and objective

Non‐invasive ventilation (NIV) improves survival, quality of life and sleep in patients with amyotrophic lateral sclerosis (ALS). Nevertheless, NIV titration is conducted in different ways. We aim to provide more insight into NIV titration by comparing the effects of a spontaneous (S) and spontaneous‐timed (ST) modes on gas exchange, sleep architecture and patient‐ventilator asynchronies (PVAs).

Methods

After an initial night of NIV titration, patients were randomized to S or ST mode in a cross‐over design. NIV was titrated using polysomnography, oximetry (oxygen saturation, SpO2
%) and transcutaneous carbon dioxide (PtcCO2
) measurement. PVAs were analysed breath‐by‐breath.

Results

Thirteen patients were analysed after inclusion. ST mode showed better results in gas exchange (minimal SpO2
%: 83 (80–89)% vs 87 (84–89)%; oxygen desaturation index: 15 (5–28)/h sleep vs 7 (3–9)/h sleep; PtcCO2
>55 mm Hg: 20 (0–59)% vs 0 (0–27)% total sleep time for S and ST mode, respectively, all P < 0.05) and respiratory events (obstructive: 8.9 (1.2–18.3)/h sleep vs 1.8 (0.3–4.9)/h sleep and central: 2.6 (0.4–14.1)/h sleep vs 0.2 (0.0–1.1)/h sleep for S and ST mode, respectively, both P < 0.01). No differences in sleep architecture were found. Ineffective efforts and respiratory events were more frequently present in S mode.

Nevertheless, four patients were discharged on S mode as these patients showed clinically better results for sleep architecture and PVA during the night on S mode.

Conclusion

ST mode shows better results in gas exchange, respiratory events and PVA. Nevertheless, accurate NIV titration remains necessary as some patients show equal or better results when using the S mode.
Bayesian robustness modelling using theO‐regularly varying distributions
Authors: Andrade J. A. A. , Omey Edward
Published: April 2017

The theory of robustness modelling is essentially based on heavy‐tailed distributions, because longer tails are more prepared to deal with diverse information (such as outliers) because of the higher probabilities on the tails. There are many classes of distributions that can be regarded as heavy tails; some of them have interesting properties and are not explored in statistics. In the present work, we propose a robustness modelling approach based on theO‐regularly varying class (ORV), which is a generalization of the regular variation family; however, the ORV class allows more flexible tails behaviour, which can improve the way in which the outlying information is discarded by the model. We establish sufficient conditions in the location and in the scale parameter structures, which allow to resolve automatically the conflicts of information. We also provide a procedure for generating new distributions within the ORV class.
Chlorosis caused by two recessively interacting genes reveals a role of RNA helicase in hybrid breakdown in Arabidopsis thaliana
Authors: Plötner Björn , Nurmi Markus , Fischer Axel , Watanabe Mutsumi , Schneeberger Korbinian , Holm Svante , Vaid Neha , Schöttler Mark Aurel , Walther Dirk , Hoefgen Rainer , Weigel Detlef , Laitinen Roosa A. E.
Published: April 2017

Hybrids often differ in fitness from their parents. They may be superior, translating into hybrid vigour or heterosis, but they may also be markedly inferior, because of hybrid weakness or incompatibility. The underlying genetic causes for the latter can often be traced back to genes that evolve rapidly because of sexual or host–pathogen conflicts. Hybrid weakness may manifest itself only in later generations, in a phenomenon called hybrid breakdown. We have characterized a case of hybrid breakdown among two Arabidopsis thaliana accessions, Shahdara (Sha, Tajikistan) and Lövvik‐5 (Lov‐5, Northern Sweden). In addition to chlorosis, a fraction of the F2 plants have defects in leaf and embryo development, and reduced photosynthetic efficiency. Hybrid chlorosis is due to two major‐effect loci, of which one, originating from Lov‐5, appears to encode an RNA helicase (AtRH18). To examine the role of the chlorosis allele in the Lövvik area, in addition to eight accessions collected in 2009, we collected another 240 accessions from 15 collections sites, including Lövvik, from Northern Sweden in 2015. Genotyping revealed that Lövvik collection site is separated from the rest. Crosses between 109 accessions from this area and Sha revealed 85 cases of hybrid chlorosis, indicating that the chlorosis‐causing allele is common in this area. These results suggest that hybrid breakdown alleles not only occur at rapidly evolving loci, but also at genes that code for conserved processes.
Transfusion of 35‐day‐stored red blood cells does not alter lipopolysaccharide tolerance during human endotoxemia
Authors: Peters Anna L. , van Hezel Maike E. , Klanderman Robert B. , Tuip‐de Boer Anita M. , Wiersinga W. Joost , van der Spek Anne H. , van Bruggen Robin , de Korte Dirk , Juffermans Nicole P. , Vlaar Alexander P.J.
Published: April 2017

BACKGROUND

Transfusion‐related immunomodulation (TRIM) encompasses immunosuppressive and proinflammatory effects induced by red blood cell (RBC) transfusion. Changes that occur during storage in the RBC product have been hypothesized to underlie TRIM, mediated by tolerance of toll‐like receptors (TLR). We investigated whether transfusion of 35‐day‐stored autologous RBCs alters cytokine production in response to stimulation with lipopolysaccharide (LPS) or lipotheic acid (LTA), in a clinically relevant model of endotoxemia.

STUDY DESIGN AND METHODS

Eighteen volunteers received 2 ng/kg LPS intravenously, followed by normal saline or 2‐ or 35‐day‐stored autologous RBC transfusion. Before LPS, before transfusion, and 6 hours after transfusion blood was collected to measure cytokine gene expression. Whole blood was used for ex vivo stimulation with LPS and LTA, after which cytokine levels were measured with enzyme‐linked immunosorbent assay.

RESULTS

In vivo LPS induced a biphasic response in cytokine mRNA with peak values 2 hours after LPS infusion. Storage time of RBC transfusion did not influence cytokine mRNA levels. In vivo infusion of LPS resulted in tolerance for ex vivo stimulation with LPS and LTA. However, transfusion of either fresh or stored RBCs did not further affect the capacity to produce cytokines after ex vivo stimulation.

CONCLUSION

In a clinically relevant model of human endotoxemia, autologous transfusion of 35‐day‐stored RBCs does not influence cytokine mRNA levels nor does it change the capacity of white blood cells in whole blood to produce cytokines after ex vivo stimulation with LPS or LTA.
Competition, firm size and returns to skills: Evidence from currency shocks and market liberalisations
Authors: Raitano Michele , Vona Francesco
Published: April 2017

We investigate the impact of product market competition on returns to skills in Italy using a longitudinal dataset on individual working histories. This impact is identified using three exogenous shocks affecting competition: the unforeseen devaluation of the Lira in 1992, its return to a fixed exchange regime in 1996 and the market liberalisation in the utility and transport sectors in the late 1990s–early 2000s. We analyse how firm heterogeneity and shocks of different types and signs affect the impact of competition on skill premia. We find that opposite shocks have opposite effects: an increase (resp. decrease) in international competition increases (resp. decreases) skill premia. Moreover, international shocks have greater effects on medium sized firms, while domestic liberalisation shocks have greater effects on large incumbents.
A tale of the nearly tail‐less: the effects of Plio‐Pleistocene climate change on the diversification of the African avian genus Sylvietta
Authors: Huntley Jerry W. , Voelker Gary
Published: April 2017

Sylvietta is a broadly distributed group of African species inhabiting a wide range of habitats and presents an interesting opportunity to investigate the historic mechanisms that have impacted the biogeography of African avian species. We collected sequence data from 50 individuals and used model‐based phylogenetic methods, molecular divergence estimates and ancestral area estimates to construct a time‐calibrated phylogeny and estimation of biogeographic history. We estimate a southern African origin for Sylvietta, with an initial divergence splitting the genus into two clades. The first consists of arid‐adapted species, with a southern African origin and subsequent diversification north into Ethiopia–Somalia. The second clade is estimated as having a Congolian forest origin with an eastward pattern of colonization and diversification as a result of Plio‐Pleistocene forest dynamics. Additionally, two members of the genus Sylvietta display interesting patterns of intraspecific diversification. Sylvietta rufescens is an arid‐adapted species inhabiting southern Africa, and we recover two subclades with a divergence dating to the Pleistocene, a unique pattern for avian species which may be explained via isolation in arid habitat fragments in the early Pleistocene. Second, Sylvietta virens, a species endemic to Afro‐tropical forests, is recovered with geographically structured genetic diversification across its broad range, an interesting result given that recent investigations of several avian forest species have found similar and substantial geographically structured genetic diversity relating to Plio‐Pleistocene forest fragmentation. Overall, Plio‐Pleistocene habitat cycling played a significant role in driving diversification in Sylvietta, and this investigation highlights the substantial impact of climate‐driven habitat dynamics on the history of sub‐Saharan species.
Species delimitation in the crypsis‐defended and polymorphic stick insects of the genusLibethra(Phasmatodea, Diapheromeridae)
Authors: Gutiérrez‐Valencia Juanita , Gutiérrez Yeisson , G. Dias Lucimar
Published: April 2017

Crypsis is a widespread defensive mechanism for prey ambushed by visually oriented predators. One well‐documented consequence of this interaction is the formation of search images by the predator that leads to the coexistence of multiple morphs in the prey population.Libethrais a genus of stick insects whose ornamentation and coloration are decidedly protective and exhibit exuberant intraspecific variation. Despite this fact, these traits have been extensively used to support the boundaries between the species it includes. This is the case in the north Andean speciesLibethra rabdotaandLibethra rabdotula, delimited on the basis of a specific arrangement of ornamental characters. To state whether such taxonomical practice is meaningful and convenient, we evaluated the grouping of specimens of both species by means of the independent inference of phylogenies obtained with mitochondrialDNAsequences and ootaxonomy. This article synonymizesL. rabdotaandL. rabdotula, redescribes the adults and describes the egg of the species. Additionally, we demonstrate that the characters related to crypsis used for the diagnosis ofL. rabdotaare not exclusive to this species by describingLibethra rioblancosp. n. andLibethra ucumariensissp. n. Our findings suggest that this genus requires extensive revision, but also provide support for the usage of different tools that might produce more accurate and stable classification. Evolutionary scenarios for both the extreme morphological variation and possible homoplastic state of camouflage‐related traits are discussed herein.
Examining the Psychometric Properties of Acceptance Scale for Kindergarten-Revised (ASK-R) in Turkish
Authors: Ersan Deniz Tekin , Ata Seda , Kaya Sinem
Published: April 2017

The aim of this study is to investigate the applicability of ASK-R among Turkish pre-school children and determine the psychometric properties (validity and reliability) of this scale. This study was conducted with 167 students from different pre-schools in Muğla. Within the aim of the study, three different methods were set down in order to investigate the validity of the scale. First of all, in order to get evidences about the construct validity of the scale, item analysis was conducted. In order to examine construct validity, confirmatory factor analysis was conducted. The fit statistics obtained from factor analysis showed that original form’s factor structure had been confirmed. Internal consistency was calculated and Cronbach’s alpha coefficient for the total scale was high. The test-retest correlation coefficient for the total score were significant (r= .78, p<0.01). As a result of the study, the Turkish version of ASK-R has good validity and reliability for Turkish pre-school children.
Teaching an Undergraduate Dynamics Course for Mechanical “Engineering Technology” Students: Successful Implementation for Students Learning
Authors: Hossain Awlad , Durfee Jason , Bae Heechang , Larsen Kyle
Published: April 2017

Applied mechanics is a branch of the physical sciences that describes the response of bodies (solids and fluids) or systems of bodies to external forces. It deals with the basic concepts of force, moment and its effects on the bodies at rest or in motion. It helps engineers or engineering students to understand how different bodies behave under the application of different types of loads. Mechanics can be broadly divided into two branches as called Statics and Dynamics. Statics deals with the bodies at rest whereas dynamics involves studies related to bodies in motion. In particular, the major emphasis of a dynamics course is to provide the details of the principles of applied mechanics or physics with the studies of motion of objects caused by forces or torques. It is an important course to develop a method of stripping a problem to its essentials and solving it in a logical, organized manner. In our institution, we offer a one-quarter long Dynamics class for Mechanical Engineering Technology (MET) curriculum. This course teaches several topics of solving dynamics problems that belong to Kinematics in Rectilinear & Angular Motions, Plane Motion, Kinetics, Work & Energy, and Impulse & Momentum. This course is designed for the MET students, who are more “hands-on” and have mathematical knowledge up to Calculus II. However, the prerequisite of this course is Tech Statics, not Calculus II. On the other hand, the prerequisites of Tech Statics are Physics and Pre-Cal-II. Therefore, MET students enrolled in Dynamics course solve problems using algebra rather than using calculus. As a whole, this course becomes challenging to convey different concepts of dynamics to our students within 10 weeks’ time frame. To facilitate the overall learning, the course instructors solve different interesting realistic dynamics problems, besides solving the conventional problems from the text book. Solving these realistic dynamics problem helps our students to enhance their conceptual understanding, and motivate them to pursue further in subsequent chapters. The paper presents in details several interesting problems related to different chapters and how they are linked to convey the targeted message related to course objectives. The paper also presents how different topics taught in this class fulfill the targeted course objectives, which are mapped with ABET Engineering Technology criteria. While a course in Dynamics could be a common offering in many universities, the authors of this paper presents the pedagogical approaches undertaken to successfully teach or implement the course objectives to the undergraduate engineering technology students.
Introducing Renewable Energy Through Projects
Authors: Celik Emine
Published: April 2017

Applied Energy Systems is an elective class in Thermal-Fluids area. The course focuses on energy and fluids engineering by covering topics such as current and developing energy sources and their impact on the environment. One of the outcomes of this class is to identify and compare energy resources such as solar, wind, ocean, geothermal energy, and hydropower by covering their working principle, system components, cost analysis, benefits and drawbacks. During the semester, several small scale projects were introduced to students. These projects included a debate session on wind energy, a feasibility study on wind energy, research on a Tesla turbine, and testing of a hydro turbine. This study provides the description of each project and shows examples of student work, survey results and the assessment of the work.
Student Presentation Based Effective Teaching (SPET) Approach for Advanced Courses
Authors: Tyagi Pawan
Published: April 2017

A student activity based effective teaching approach can significantly improve student learning. However, implementing student activity based teaching for the advanced level courses can be very challenging. Incomplete course coverage and the amount of time required by an instructor for designing active teaching strategies are cited as the common inhibiting factors in the adoption of active student teaching. This paper discusses a student presentation based effective teaching (SPET) approach that covers more course material than that covered in the conventional or other student-active teaching methods. Moreover, SPET approach requires less preparation time on instructor behalf. This paper is based on the effective teaching experiments conducted on senior level science and technology courses at University of the District of Columbia. Under the SPET approach, students are given reading assignment to prepare ∼ 10–20 minutes long power point presentation on well-defined conceptual topics, questions, or chapter modules. In every class typically three presentations take place on the same questions or topics. However, non-presenter students are required to generate conceptual questions. These questions were asked during or after the presentation by the designated students. Students’ presentations were graded according to the rubric focusing on coverage of suggested topics, quality of presentation, and questions and answers. Hence, the whole class is engaged in understanding the topic either for making the presentation or for creating conceptual questions. These grades were posted right after the class in the Blackboard’s online grade center to provide quick feedback. The following are key advantages of this approach. (1) Students understand 50–100% about the intended topic during self-reading and while making a presentation or participating in class discussion. (2) Repeating same concepts thrice during a class period and occasionally with instructor’s insights enable deep learning. (3) Students get quick quantitative feedback after each class and qualitative feedback during the class from instructor and peers. (4) This approach allowed coverage of very complex topics. (5) Students improved their communication skills by making coherent presentations and doing class discussion. In the survey, students reflected a higher degree of satisfaction with their learning as compared to instructor’s lecture-based classroom education system. This approach is highly suitable for advanced-level elective courses with small enrollment.
Learning System on Surgical Technique of Laparoscopic Cholecystectomy
Authors: Shiomi Hisanori , Yamashiro Kazuaki , Takai Yuka , Goto Akihiko , Hamada Hiroyuki
Published: April 2017

In this research, eye movement measurement was performed for the process of the operation under laparoscopy to two or more persons. The knack of advanced technique was understood through numerical method and the difference in technology were evaluated, and it aimed at showing the influence of years of experience on eye movement by comparing the operation for the operators with different level of skill. The target operation was laparoscopic cholecystectomy. We decided to carry out under the same conditions using a simulator, and the subject was taken as two experts and one unskilled operator from which years of experience differ so that comparison between two or more subjects might be attained. According to the procedure in which it is most worked by the whole operation by actual laparoscopic cholecystectomy, it classified into nine items and measured the factor about working hours and a view at each process.
A finite element study on the cause of vocal fold vertical stiffness variation
Authors: Geng Biao , Xue Qian , Zheng Xudong
Published: April 2017

A finite element method based numerical indentation technique was used to quantify the effect of the material stiffness variation and the subglottal convergence angle of the vocal fold on the vertical stiffness difference of the medial surface. It was found that the vertical stiffness difference increased with the increasing subglottal angle, and it tended to saturate beyond a subglottal angle of about 50°. The material stiffness variation could be as important as the subglottal angle depending on the actual material properties.
LAG3 limits regulatory T cell proliferation and function in autoimmune diabetes
Authors: Zhang Qianxia , Chikina Maria , Szymczak-Workman Andrea L. , Horne William , Kolls Jay K. , Vignali Kate M. , Normolle Daniel , Bettini Maria , Workman Creg J. , Vignali Dario A. A.
Published: April 2017

Regulatory T cell–specific deletion of LAG3 in nonobese diabetic mice reduces the incidence of autoimmune diabetes.
Benign Rabbit Calicivirus in New Zealand
Authors: Nicholson Leila J. , Mahar Jackie E. , Strive Tanja , Zheng Tao , Holmes Edward C. , Ward Vernon K. , Duckworth Janine A.
Published: April 2017

The Czech v351 strain of rabbit hemorrhagic disease virus (RHDV1) is used in Australia and New Zealand as a biological control agent for rabbits, which are important and damaging introduced vertebrate pests in these countries. However, nonpathogenic rabbit caliciviruses (RCVs) can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with effective rabbit biocontrol. Antibodies that cross-reacted against RHDV antigens were found in wild rabbits before the release of RHDV1 in New Zealand in 1997, suggesting that nonpathogenic RCVs were already present in New Zealand. The aim of this study was to confirm the presence of nonpathogenic RCV in New Zealand and describe its geographical distribution. RCV and RHDV antibody assays were used to screen serum samples from 350 wild rabbits from 14 locations in New Zealand. The serological survey indicated that both RCV and RHDV are widespread in New Zealand wild rabbits, with antibodies detected in 10 out of 14 and 12 out of 14 populations, respectively. Two closely related RCV strains were identified in the duodenal tissue from a New Zealand wild rabbit (RCV Gore-425A and RCV Gore-425B). Both variants are most closely related to Australian RCV strains, but with 88% nucleotide identity, they are genetically distinct. Phylogenetic analysis revealed that the New Zealand RCV strains fall within the genetic diversity of the Australian RCV isolates, indicating a relatively recent movement of RCVs between Australia and New Zealand.

IMPORTANCE
Wild rabbits are important and damaging introduced vertebrate pests in Australia and New Zealand. Although RHDV1 is used as a biological control agent, some nonpathogenic RCVs can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with its effectiveness for rabbit control. The presence of nonpathogenic RCVs in New Zealand wild rabbits has been long hypothesized, but earlier attempts to isolate a New Zealand RCV strain have been unsuccessful. Therefore, it is important to determine if such nonpathogenic viruses exist in New Zealand rabbits, especially considering the proposed introduction of new RHDV strains into New Zealand as biocontrols.
Genetic (In)stability of 2,6-Dichlorobenzamide Catabolism in Aminobacter sp. Strain MSH1 Biofilms under Carbon Starvation Conditions
Authors: Horemans Benjamin , Raes Bart , Brocatus Hannelore , T'Syen Jeroen , Rombouts Caroline , Vanhaecke Lynn , Hofkens Johan , Springael Dirk
Published: April 2017

Aminobacter
sp. strain MSH1 grows on and mineralizes the groundwater micropollutant 2,6-dichlorobenzamide (BAM) and is of interest for BAM removal in drinking water treatment plants (DWTPs). The BAM-catabolic genes in MSH1 are located on plasmid pBAM1, carrying
bbdA
, which encodes the conversion of BAM to 2,6-dichlorobenzoic acid (2,6-DCBA) (BbdA
+
phenotype), and plasmid pBAM2, carrying gene clusters encoding the conversion of 2,6-DCBA to tricarboxylic acid (TCA) cycle intermediates (Dcba
+
phenotype). There are indications that MSH1 easily loses its BAM-catabolic phenotype. We obtained evidence that MSH1 rapidly develops a population that lacks the ability to mineralize BAM when grown on nonselective (R2B medium) and semiselective (R2B medium with BAM) media. Lack of mineralization was explained by loss of the Dcba
+
phenotype and corresponding genes. The ecological significance of this instability for the use of MSH1 for BAM removal in the oligotrophic environment of DWTPs was explored in lab and pilot systems. A higher incidence of BbdA
+
Dcba
−
MSH1 cells was also observed when MSH1 was grown as a biofilm in flow chambers under C and N starvation conditions due to growth on nonselective residual assimilable organic carbon. Similar observations were made in experiments with a pilot sand filter reactor bioaugmented with MSH1. BAM conversion to 2,6-DCBA was not affected by loss of the DCBA-catabolic genes. Our results show that MSH1 is prone to BAM-catabolic instability under the conditions occurring in a DWTP. While conversion of BAM to 2,6-DCBA remains unaffected, BAM mineralization activity is at risk, and monitoring of metabolites is warranted.

IMPORTANCE
Bioaugmentation of dedicated biofiltration units with bacterial strains that grow on and mineralize micropollutants was suggested as an alternative for treating micropollutant-contaminated water in drinking water treatment plants (DWTPs). Organic-pollutant-catabolic genes in bacteria are often easily lost, especially under nonselective conditions, which affects the bioaugmentation success. In this study, we provide evidence that
Aminobacter
sp. strain MSH1, which uses the common groundwater micropollutant 2,6-dichlorobenzamide (BAM) as a C source, shows a high frequency of loss of its BAM-mineralizing phenotype due to the loss of genes that convert 2,6-DCBA to Krebs cycle intermediates when nonselective conditions occur. Moreover, we show that catabolic-gene loss also occurs in the oligotrophic environment of DWTPs, where growth of MSH1 depends mainly on the high fluxes of low concentrations of assimilable organic carbon, and hence show the ecological relevance of catabolic instability for using strain MSH1 for BAM removal in DWTPs.
Mechanisms for Pseudoalteromonas piscicida-Induced Killing of Vibrios and Other Bacterial Pathogens
Authors: Richards Gary P. , Watson Michael A. , Needleman David S. , Uknalis Joseph , Boyd E. Fidelma , Fay Johnna P.
Published: April 2017

Pseudoalteromonas piscicida
is a Gram-negative gammaproteobacterium found in the marine environment. Three strains of pigmented
P. piscicida
were isolated from seawater and partially characterized by inhibition studies, electron microscopy, and analysis for proteolytic enzymes. Growth inhibition and death occurred around colonies of
P. piscicida
on lawns of the naturally occurring marine pathogens
Vibrio vulnificus
,
Vibrio parahaemolyticus
,
Vibrio cholerae
,
Photobacterium damselae
, and
Shewanella algae
. Inhibition also occurred on lawns of
Staphylococcus aureus
but not on
Escherichia coli
O157:H7 or
Salmonella enterica
serovar Typhimurium. Inhibition was not pH associated, but it may have been related to the secretion of a cysteine protease with strong activity, as detected with a synthetic fluorogenic substrate. This diffusible enzyme was secreted from all three
P. piscicida
strains. Direct overlay of the
Pseudoalteromonas
colonies with synthetic fluorogenic substrates demonstrated the activity of two aminopeptidase Bs, a trypsin-like serine protease, and enzymes reactive against substrates for cathepsin G-like and caspase 1-like proteases. In seawater cultures, scanning electron microscopy revealed numerous vesicles tethered to the outer surface of
P. piscicida
and a novel mechanism of direct transfer of these vesicles to
V. parahaemolyticus
. Vesicles digested holes in
V. parahaemolyticus
cells, while the
P. piscicida
congregated around the vibrios in a predatory fashion. This transfer of vesicles and vesicle-associated digestion of holes were not observed in other bacteria, suggesting that vesicle binding may be mediated by host-specific receptors. In conclusion, we show two mechanisms by which
P. piscicida
inhibits and/or kills competing bacteria, involving the secretion of antimicrobial substances and the direct transfer of digestive vesicles to competing bacteria.

IMPORTANCE
Pseudoalteromonas
species are widespread in nature and reduce competing microflora by the production of antimicrobial compounds. We isolated three strains of
P. piscicida
and characterized secreted and cell-associated proteolytic enzymes, which may have antimicrobial properties. We identified a second method by which
P. piscicida
kills
V. parahaemolyticus
. It involves the direct transfer of apparently lytic vesicles from the surface of the
Pseudoalteromonas
strains to the surface of
Vibrio
cells, with subsequent digestion of holes in the
Vibrio
cell walls. Enzymes associated with these vesicles are likely responsible for the digestion of holes in the cell walls.
Pseudoalteromonas piscicida
has potential applications in aquaculture and food safety, in control of the formation of biofilms in the environment, and in food processing. These findings may facilitate the probiotic use of
P. piscicida
to inactivate pathogens and may lead to the isolation of enzymes and other antimicrobial compounds of pharmacological value.
Microbiota Analysis of an Environmental Slurry and Its Potential Role as a Reservoir of Bovine Digital Dermatitis Pathogens
Authors: Klitgaard Kirstine , Strube Mikael L. , Isbrand Anastasia , Jensen Tim K. , Nielsen Martin W.
Published: April 2017

At present, very little information exists regarding what role the environmental slurry may play as an infection reservoir and/or route of transmission for bovine digital dermatitis (DD), a disease which is a global problem in dairy herds. To investigate whether DD-related bacteria belong to the indigenous microbiota of the dairy herd environment, we used deep amplicon sequencing of the 16S rRNA gene in 135 slurry samples collected from different sites in 22 dairy farms, with and without DD-infected cows. Both the general bacterial populations and digital dermatitis-associated
Treponema
were targeted in this study. The results revealed significant differences in the bacterial communities between the herds, with only 12 bacterial taxa shared across at least 80% of all the individual samples. These differences in the herd microbiota appeared to reflect mainly between-herd variation. Not surprisingly, the slurry was dominated by ubiquitous gastrointestinal bacteria, such as
Ruminococcaceae
and
Lachnospiraceae
. Despite the low relative abundance of spirochetes, which ranged from 0 to 0.6%, we were able to detect small amounts of bacterial DNA from DD-associated treponemes in the slurry. However, the DD-associated
Treponema
spp. were detected only in samples from herds with reported DD problems. These data indicate that treponemes involved in the pathogenesis of DD are not part of the normal environmental microflora in dairy herds without clinical DD and, consequently, that slurry is not a primary reservoir of infection.

IMPORTANCE
Bovine digital dermatitis (DD), a dermal disease which causes lameness in dairy cattle, is a serious problem worldwide. To control this disease, the infection reservoirs and transmission routes of DD pathogens need to be clarified. The dairy herd slurry may be a pathogen reservoir of DD-associated bacteria. The rationale for the present study was, therefore, to examine whether DD-associated bacteria are always present in slurry or if they are found only in DD-afflicted herds. The results strongly indicated that DD
Treponema
spp. are not part of the indigenous slurry and, therefore, do not comprise an infection reservoir in healthy herds. This study applied next-generation sequencing technology to decipher the microbial compositions of environmental slurry of dairy herds with and without digital dermatitis.
Evolution of a Biomass-Fermenting Bacterium To Resist Lignin Phenolics
Authors: Cerisy Tristan , Souterre Tiffany , Torres-Romero Ismael , Boutard Magali , Dubois Ivan , Patrouix Julien , Labadie Karine , Berrabah Wahiba , Salanoubat Marcel , Doring Volker , Tolonen Andrew C.
Published: April 2017

Increasing the resistance of plant-fermenting bacteria to lignocellulosic inhibitors is useful to understand microbial adaptation and to develop candidate strains for consolidated bioprocessing. Here, we study and improve inhibitor resistance inClostridium phytofermentans(also calledLachnoclostridium phytofermentans), a model anaerobe that ferments lignocellulosic biomass. We survey the resistance of this bacterium to a panel of biomass inhibitors and then evolve strains that grow in increasing concentrations of the lignin phenolic, ferulic acid, by automated, long-term growth selection in an anaerobic GM3 automat. Ultimately, strains resist multiple inhibitors and grow robustly at the solubility limit of ferulate while retaining the ability to ferment cellulose. We analyze genome-wide transcription patterns during ferulate stress and genomic variants that arose along the ferulate growth selection, revealing how cells adapt to inhibitors through changes in gene dosage and regulation, membrane fatty acid structure, and the surface layer. Collectively, this study demonstrates an automated framework forin vivodirected evolution of anaerobes and gives insight into the genetic mechanisms by which bacteria survive exposure to chemical inhibitors.

IMPORTANCEFermentation of plant biomass is a key part of carbon cycling in diverse ecosystems. Further, industrial biomass fermentation may provide a renewable alternative to fossil fuels. Plants are primarily composed of lignocellulose, a matrix of polysaccharides and polyphenolic lignin. Thus, when microorganisms degrade lignocellulose to access sugars, they also release phenolic and acidic inhibitors. Here, we study how the plant-fermenting bacteriumClostridium phytofermentansresists plant inhibitors using the lignin phenolic, ferulic acid. We examine how the cell responds to abrupt ferulate stress by measuring changes in gene expression. We evolve increasingly resistant strains by automated, long-term cultivation at progressively higher ferulate concentrations and sequence their genomes to identify mutations associated with acquired ferulate resistance. Our study develops an inhibitor-resistant bacterium that ferments cellulose and provides insights into genomic evolution to resist chemical inhibitors.
Enhanced Antimicrobial Activity Based on a Synergistic Combination of Sublethal Levels of Stresses Induced by UV-A Light and Organic Acids
Authors: de Oliveira Erick F. , Cossu Andrea , Tikekar Rohan V. , Nitin Nitin
Published: April 2017

The reduction of microbial load in food and water systems is critical for their safety and shelf life. Conventionally, physical processes such as heat or light are used for the rapid inactivation of microbes, while natural compounds such as lactic acid may be used as preservatives after the initial physical process. This study demonstrates the enhanced and rapid inactivation of bacteria based on a synergistic combination of sublethal levels of stresses induced by UV-A light and two food-grade organic acids. A reduction of 4.7 ± 0.5 log CFU/ml inEscherichia coliO157:H7 was observed using a synergistic combination of UV-A light, gallic acid (GA), and lactic acid (LA), while the individual treatments and the combination of individual organic acids with UV-A light resulted in a reduction of less than 1 log CFU/ml. Enhanced inactivation of bacteria on the surfaces of lettuce and spinach leaves was also observed based on the synergistic combination. Mechanistic investigations suggested that the treatment with a synergistic combination of GA plus LA plus UV-A (GA+LA+UV-A) resulted in significant increases in membrane permeability and intracellular thiol oxidation and affected the metabolic machinery ofE. coli. In addition, the antimicrobial activity of the synergistic combination of GA+LA+UV-A was effective only against metabolically activeE. coliO157:H7. In summary, this study illustrates the potential of simultaneously using a combination of sublethal concentrations of natural antimicrobials and a low level of physical stress in the form of UV-A light to inactivate bacteria in water and food systems.

IMPORTANCEThere is a critical unmet need to improve the microbial safety of the food supply, while retaining optimal nutritional and sensory properties of food. Furthermore, there is a need to develop novel technologies that can reduce the impact of food processing operations on energy and water resources. Conventionally, physical processes such as heat and light are used for inactivating microbes in food products, but these processes often significantly reduce the sensory and nutritional properties of food and are highly energy intensive. This study demonstrates that the combination of two natural food-grade antimicrobial agents with a sublethal level of physical stress in the form of UV-A light can greatly increase microbial load inactivation. In addition, this report elucidates the potential mechanisms for this synergistic interaction among physical and chemical stresses. Overall, these results provide a novel approach to develop antimicrobial solutions for food and water systems.
Structure-Based Engineering of an Artificially Generated NADP
+
-Dependent
d
-Amino Acid Dehydrogenase
Authors: Hayashi Junji , Seto Tomonari , Akita Hironaga , Watanabe Masahiro , Hoshino Tamotsu , Yoneda Kazunari , Ohshima Toshihisa , Sakuraba Haruhiko
Published: April 2017

A stable NADP
+
-dependent
d
-amino acid dehydrogenase (DAADH) was recently created from
Ureibacillus thermosphaericus
meso
-diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized
d
-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward
d
-leucine,
d
-norleucine,
d
-methionine,
d
-isoleucine, and
d
-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP
+
and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic
d
-amino acids with bulky side chains.

IMPORTANCE
In recent years, the potential uses for
d
-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of
d
-amino acids, but all include tedious steps. The use of NAD(P)
+
-dependent
d
-amino acid dehydrogenase (DAADH) makes single-step production of
d
-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of
d
-amino acids, such as
d
-leucine and
d
-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of
d
-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs.
The R263K Dolutegravir Resistance-Associated Substitution Progressively Decreases HIV-1 Integration
Authors: Mesplède Thibault , Leng Jing , Pham Hanh Thi , Liang Jiaming , Quan Yudong , Han Yingshan , Wainberg Mark A.
Published: April 2017

Human immunodeficiency virus (HIV) infection persists despite decades of active antiretroviral therapy (ART), effectively preventing viral eradication. Treatment decreases plasma viral RNA, but viral DNA persists, mostly integrated within the genome of nucleated blood cells. Viral DNA blood levels correlate with comorbidities and the rapidity of viral rebound following treatment interruption. To date, no intervention aiming at decreasing HIV DNA levels below those attained through ART has been successful. This includes use of some integrase inhibitors either as part of ART or in treatment intensification studies. We have argued that using the integrase inhibitor dolutegravir (DTG) in similar studies may yield better results, but this remains to be studied. In treatment-experienced individuals, the most frequent substitution associated with failure with dolutegravir is R263K in integrase. R263K decreases integration both in cell-free and tissue culture assays. We investigated here how integrated DNA levels evolve over time during prolonged infections with R263K viruses. To investigate a potential defect in reverse transcription with R263K, the levels of reverse transcripts were measured by quantitative PCR. We measured HIV type 1 (HIV-1) integration in Jurkat cells over the course of 4-week infections using Alu-mediated quantitative PCR. The results show that R263K did not decrease reverse transcription. Prolonged infections with R263K mutant viruses led to less HIV-1 integrated DNA over time compared to wild-type viruses. These tissue culture results help to explain the absence of the R263K substitution in most individuals experiencing failure with DTG and support studies aiming at longitudinally measuring the levels of integrated DNA in individuals treated with this drug.

IMPORTANCEAntiretroviral treatment decreases plasma viral RNA, but HIV DNA persists for decades within infected cells. Studies of nonhuman primates have suggested that reducing retroviral DNA levels might represent a path to eradication. The integrase inhibitor dolutegravir is less susceptible than any other anti-HIV drug to the emergence of resistance in treatment-naive individuals. In treatment-experienced individuals, in contrast, rare cases of treatment failure were commonly associated with emergence of an R263K integrase substitution that confers low-level resistance to dolutegravir. It is unclear why this substitution is not more common in individuals experiencing failure with dolutegravir. We report here that R263K progressively diminishes the levels of integrated HIV-1 DNA in tissue culture over multiple cycles of infection. Our results help to explain aspects of the clinical efficacy of dolutegravir and suggest that this drug may be able to reduce HIV DNA levels within infected individuals compared to other drugs.