[Experimental] List of manuscripts available for review volunteers
12 manuscripts available for review volunteers
January 25, 2017
Clustered regularly interspaced short palindromic repeat (CRISPR) systems are the adaptive immune systems of bacteria and archaea against viral infection. While CRISPRs have been exploited as a tool for genetic engineering, their spacer sequences can also provide valuable insights into microbial ecology by linking environmental viruses to their microbial hosts. Despite this importance, metagenomic CRISPR detection remains a major challenge. Here we present a reference-guided CRISPR spacer detection tool (Metagenomic CRISPR Reference-Aided Search Tool - MetaCRAST) that constrains searches based on user-specified direct repeats (DRs). These DRs could be expected from assembly or taxonomic profiles of metagenomes. We compared the performance of MetaCRAST to those of two existing metagenomic CRISPR detection tools – Crass and MinCED – using both real and simulated acid mine drainage (AMD) and enhanced biological phosphorus removal (EBPR) metagenomes. Our evaluation shows MetaCRAST improves CRISPR spacer detection in real metagenomes compared to the de novo CRISPR detection methods Crass and MinCED. Evaluation on simulated metagenomes show it performs better than de novo tools for Illumina metagenomes and comparably for 454 metagenomes. It also has comparable performance dependence on read length and community composition, run time, and accuracy to these toolsMetaCRAST is implemented in Perl, parallelizable through the Many Core Engine (MCE), and takes metagenomic sequence reads and direct repeat queries (FASTA) as input. It is freely available for download athttps://github.com/molleraj/MetaCRAST.
January 19, 2017
Site-specific evolutionary rates can be estimated from codon sequences or from amino-acid sequences. For codon sequences, the most popular methods use some variation of the dN/dS ratio. For amino-acid sequences, one widely-used method is called Rate4Site, and it assigns a relative conservation score to each site in an alignment. How site-wise dN/dS values relate to Rate4Site scores is not known. Here we elucidate the relationship between these two rate measurements. We simulate sequences with known dN/dS, using either dN/dS models or mutation--selection models for simulation. We then infer Rate4Site scores on the simulated alignments, and we compare those scores to either true or inferred dN/dS values on the same alignments. We find that Rate4Site scores generally correlate well with true dN/dS, and the correlation strengths increase in alignments with higher sequence divergence and higher number of taxa. Moreover, Rate4Site scores correlate nearly perfectly with inferred dN/dS values, even for small alignments with little divergence. Finally, we verify this relationship between Rate4Site and dN/dS in a variety of natural sequence alignments. We conclude that codon-level and amino-acid-level analysis frameworks are directly comparable and yield near-identical inferences.
January 18, 2017

Background: Food preservation is essential to the growing world population, food economy. Freezing is a commonly used method for food preservation. While extending the life of the product, freezing has detrimental effects. It is causing loss of food weight and is causing changes in food quality, e.g. enzymatic browning.

Method: Freezing of food is usually done under constant atmospheric pressure (isobaric). We have developed a new technology in which biological materials are preserved at subfreezing temperatures in an isochoric (constant volume) system. Experiments were performed with a food product, potato, in a thermodynamic isochoric device designed by us, that is robust and has no moving parts.

Results: We have shown that under similar storage conditions, freezing to -5°C, the isochoric preserved potato experienced no weight loss and limited enzymatic browning. In contrast the -5°C isobaric frozen potato experienced substantial weight loss and substantial enzymatic browning. Microscopic analysis, shows that the mechanism responsible for the different results is related to the integrity of the cell and the cell membrane, which are maintain during freezing in the isochoric system and lost during freezing in the isobaric system.

Discussion: The main mechanism of cell damage during isobaric freezing are the increase in extracellular osmolality and the mechanical damage by ice crystals. In contrast, during isochoric freezing the cells in the preserved material are under conditions in which the intracellular osmolality is comparable to the extracellular osmolality and they are not affected by ice mechanical damage. The conditions during isochoric freezing result in improved quality of the preserved food products.

Conclusion: We have shown that the quality of food products preserved by isochoric freezing is better than the quality of food preserved to the same temperature in isobaric conditions. This is only a preliminary study on isochoric preservation of food. However, it illustrates the potential of the technology.

January 17, 2017
Cytokines are fundamental for a functioning immune system, and thus, potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a novel probe-based quantitative gene expression assay for immunological assessment of captive beluga whales ( Delphinapterusleucas ) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.
January 14, 2017
Background. Software maintenance is an important activity in the process of software engineering where over time maintenance team members leave and new members join. The identification of files being changes together frequently has been proposed several times. Yet, existing studies about these file changes ignore the feedback from developers as well as the impact on the performance of maintenance and rely on the analysis findings and expert evaluation.
Methods. We conducted an experiment with the goal to investigate the usefulness of coupled file changes during maintenance tasks when developers are inexperienced in programming or when they are new on the project. Using data mining on software repositories we can identify files that changed most frequently together in the past. We extract coupled file changes from the Git repository of a Java software system and join them with corresponding attributes from the versioning and issue tracking system and the project documentation. We present a controlled experiment involving 36 student participants where we investigate if coupled file change suggestions influence the correctness of the task solutions and the time to complete them.
Results. The results show that coupled file change suggestions significantly increase the correctness of the solutions. However, there is only a small effect on the time to complete the tasks. We also derived a set of the most useful attributes based on the developers feedback.
Discussion. Coupled file changes and a limited number of the proposed attributes are useful for inexperienced developers working on maintenance tasks whereby although the developers using these suggestions solved more tasks, they still need time to organize and understand and implement this information.
January 11, 2017
Background. Theory predicts strong bottom-up control in detritus-based food webs, yet field experiments with detritus-based terrestrial systems have uncovered contradictory evidence regarding the strength and pervasiveness of bottom-up control processes. Two factors likely leading to contradictory results are experiment duration, which influences exposure to temporal variation in abiotic factors such as rainfall and affects the likelihood of detecting approach to a new equilibrium; and openness of the experimental units to immigration and migration. To investigate the contribution of these two factors, we conducted a long-term experiment with open and fenced plots in the forest that was the site of an earlier, short-term experiment (3.5 months) with open plots (Chen & Wise 1999) that produced evidence of strong bottom-up control for 14 taxonomic groupings of primary consumers of litter and fungi (microbi-detritivores) and their predators. Methods. We added artificial high-quality detritus to ten 2 x 2-m forest-floor plots at bi-weekly intervals from April through September in three consecutive years (Supplemented treatment). Ten comparable Ambient plots were controls. Half of the Supplemented and Ambient plots were enclosed by metal fencing. Results. Arthropod community structure (based upon 18 response variables) diverged over time between Supplemented and Ambient treatments, with no effect of Fencing on the multivariate response pattern. Fencing possibly influenced only ca. 20% of the subsequent univariate analyses. Multi- and univariate analyses revealed bottom-up control by fall of Year 1 of some, but not all, microbi-detritivores and predators. During the following two years the pattern of responses became more complex than that observed by Chen & Wise (1999). Some taxa showed consistent bottom-up control whereas many did not. Variation across years could not be explained completely by differences in rainfall because some taxa exhibited negative, not positive, responses to detrital supplementation. Discussion. Our 3-yr experiment did not confirm the conclusion of strong, pervasive bottom-up control of microbi-detritivores and predators reported by Chen and Wise (1999). Our longer-term experiment revealed a more complex pattern of responses, a pattern much closer to the range of outcomes reported in the literature for many short-term experiments. Much of the variation in responses across studies likely reflects variation in factors such as rainfall and the quality of added detritus. Nevertheless, it is also possible that long-term resource enhancement can drive a community towards a new equilibrium state that differs from what would have been predicted from the initial short-term responses exhibited by primary and secondary consumers.
January 11, 2017

Phytochemicals produced by plants, including at flowers, function in protection against plant diseases, and have a long history of use against trypanosome infection. Floral nectar and pollen, the sole food sources for many species of insect pollinators, contain phytochemicals that have been shown to reduce trypanosome infection in bumble and honey bees when fed as isolated compounds. Nectar and pollen, however, consist of phytochemical mixtures, which can have greater antimicrobial activity than do single compounds. This study tested the hypothesis that pollen extracts would inhibit parasite growth.Extracts of six different pollens were tested for direct inhibitory activity against cell cultures of the bumble bee trypanosome gut parasite Crithidia bombi.

Surprisingly, pollen extracts increased parasite growth rather than inhibiting it. Experimental manipulations of growth media showed that supplemental monosaccharides (glucose and fructose) were sufficient to promote growth, while a common floral phytochemical (caffeic acid) with inhibitory activity against other trypanosomes had only weak inhibitory effects on Crithidia bombi. These results indicate that, although pollen is essential for bees and other pollinators, pollen may promote growth of intestinal parasites that are uninhibited by pollen phytochemicals and, as a result, can benefit from the nutrients that pollen provides.

January 6, 2017
In order to understand the distribution and prevalence of Ommatissus lybicus (Homoptera: Tropiduchidae) as well as analyse their current biographical patterns and predict their future spread, comprehensive and highly sophisticated information on the environmental, climatic, and agricultural practices are essential. The analytical techniques available in modern spatial analysis packages, such as Remote Sensing and Geographical Information Systems, can help detect and model spatial links and correlations between the presence, absence and density of O. lybicus in response to climatic, environmental and human factors. The main objective of this paper is to review remote sensing and geographical information analytical techniques that can be applied in mapping and modelling the habitat and population density of O. lybicus in Oman. An exhaustive search of related literature revealed that there are few studies linking location-based infestation levels of pests like the O. lybicus with climatic, environmental and human practice related variables in the Middle East. Our review also highlights the accumulated knowledge and addresses the gaps in this area of research. Furthermore, it makes recommendations for future studies, and gives suggestions on monitoring and surveillance sites that are necessary in designing both local and regional level integrated pest management (IPM) policing of palm tree and other affected cultivated crops.
December 15, 2016
Downy mildew pathogens affect several economically important crops worldwide but due to their obligate nature, few genetic resources are available for genomic and population analyses. Draft genomes for emergent downy mildew pathogens such as the oomycete Pseudoperonospora cubensis, causal agent of cucurbit downy mildew, have been published and can be used to perform comparative genomic analysis and develop tools such as microsatellites to characterize pathogen population structure. We used bioinformatics to identify 2,738 microsatellites in the P. cubensis predicted transcriptome and evaluate them for transferability to the hop downy mildew pathogen, Pseudoperonospora humuli, since no draft genome is available for this species. We also compared the microsatellite repertoire of P. cubensis to that of the model organism Hyaloperonospora arabidopsidis, which causes downy mildew in Arabidopsis. Although trends in frequency of motif-type were similar, the percentage of SSRs identified from P. cubensis transcripts differed significantly from H. arabidopsidis. The majority of a subset of microsatellites selected for laboratory validation (92%) produced a product in P. cubensis isolates, and 83 microsatellites demonstrated transferability to P. humuli. Eleven microsatellites were found to be polymorphic and consistently amplified in P. cubensis isolates. Analysis of Pseudoperonospora isolates from diverse hosts and locations revealed higher diversity in P. cubensis compared to P. humuli isolates. These microsatellites will be useful in efforts to better understand relationships within Pseudoperonospora species and P. cubensis on a population level.
December 13, 2016
The evaluation of multisegment coordination is important for gaining a better understanding of gait and physical activities in humans. Therefore, the aim of this study was to verify whether the use of knee sleeves affected the coordination of lower-limb segments during level walking and one-leg hopping. Eleven healthy male adults participated in this study. They were asked to walk 10 m on a level ground and perform one-leg hops without wearing a knee sleeve and while wearing a knee sleeve. The segment angles and the response velocities of the thigh, shank, and foot were measured and calculated by using a motion analysis system. Then, the phases between the segment angle and the velocity were calculated; the continuous relative phase (CRP) was calculated as the phase of the distal segment subtracted from the phase of the proximal segment and was denoted as CRPTS (thigh–shank), CRPSF (shank–foot), and CRPTF (thigh–foot). Root mean square (RMS) values were used for the evaluation of in-phase or out-of-phase states, and standard deviation (SD) values were used for the evaluation of variability in the stance and swing phases during level walking and in the preflight, flight, and landing phases during one-leg hopping. The walking velocity and flight time improved when the knee sleeve was worn (p < 0.05). The segment angles of the thigh and shank changed when the knee sleeve was worn during level walking and one-leg hopping. The RMS values of CRPTS and CRPSF in the stance phase and the RMS values of CRPSF in the preflight and landing phases changed (p < 0.05 in all cases). The SD values of CRPTS in the landing phase and the SD values of CRPSF in the preflight and landing phases increased (p < 0.05 in all cases). These results indicated that wearing a knee sleeve caused changes in not only segment kinematics but also segment coordination. In conclusion, wearing a knee sleeve might be an effective technique to prevent injuries without any inhibitory effects.
December 12, 2016
Background. Stroke survivors often have multiple cardiovascular risk factors that necessitate promoting engagement in multiple healthy behaviors. However, observational studies of stroke survivors have typically focused on promoting a single health behavior. Thus, there is a poor understanding of linkages between healthy behaviors and the circumstances in which factors, such as stroke impairments, may influence a single or multiple health behaviors. Methods. We conducted a mixed methods convergent parallel study of 25 stroke survivors to examine the relationships between stroke impairments and physical activity, sleep, and nutrition. Our goal was to gain further insight into possible strategies to promote multiple health behaviors among stroke survivors. This study focused on physical activity, sleep, and nutrition because of their importance in achieving energy balance, maintaining a healthy weight, and reducing cardiovascular risks. Qualitative and quantitative data were collected concurrently, with the former being prioritized over the latter in order to develop a conceptual model of multiple health behaviors. Qualitative and quantitative data were analyzed independently and then were integrated during the inference stage to identify meta-inferences. The 25 stroke survivors completed closed-ended questionnaires on healthy behaviors and physical function. They also participated in face-to-face focus groups and one-to-one phone interviews. Results. We found statistically significant and moderate correlations between hand function and healthy eating habits (r = 0.45), sleep disturbances and limitations in activities of daily living (r = -0.55), BMI and limitations in activities of daily living (r = -0.49), physical activity and limitations in activities of daily living (r = 0.41), mobility impairments and BMI (r = -0.41), sleep disturbances and physical activity (r = -0.48), sleep disturbances and BMI (r = 0.48), and physical activity and BMI (r = -0.45). We identified five qualitative themes: (1) Impairments: reduced autonomy, (2) Environmental forces: caregivers and information, (3) Re-evaluation: priorities and attributions, (4) Resiliency: finding motivation and solutions, and (5) Negative affectivity: stress and self-consciousness.Three meta-inferences and a conceptual model described circumstances in which factors could influence single or multiple health behaviors. Discussion. This is the first mixed methods study of stroke survivors to elaborate on relationships between multiple health behaviors, BMI, and physical function. A conceptual model illustrates addressing sleep disturbances, activity limitations, self-image, and emotions to promote multiple health behaviors. We discuss the relevance of the meta-inferences in designing multiple behavior change interventions for stroke survivors.
December 10, 2016
Mixed-species shoals of fish are frequently found in the field; however, little is known about individual-level interactions within these groups. We analyzed the collective motion of two cyprinids (Chinese bream, Parabramis pekinensis, and qingbo, Spinibarbus sinensis) that occupy partially overlapping habitats but differ in social behavior (high vs low aggressiveness) and preferred flow regime (fast vs slow water velocity). The spontaneous swimming activity, group cohesion (suggested by group member distance), synchronization of speed, coordination of orientation among members (suggested by separation swimming angle), occupied position (suggested by percentage of time a given individual swam at the front of a shoal), and responses to an artificial fright stimulus were measured with eight repetitions in four individuals of either Chinese bream or qingbo (conspecific group) and two Chinese bream plus two qingbo (heterospecific group). Chinese bream showed lower swimming activity and coordination than qingbo in conspecific groups, possibly due to the difference between their preferred habitats. However, this difference vanished as Chinese bream elevated their swimming activity to coordinate with heterospecific groups. This finding plus the similar separation swimming angle (SSA) values among the three experimental groups suggested that both species may share similar ineraction rules regarding shoaling behavior. However, qingbo showed greater separation distances than Chinese bream even in heterospecific groups, suggesting that there is an underlying difference between the two species’ shoaling mechanisms. Chinese bream showed higher variation in spatial distribution than qingbo, possibly due to the difference in behavior and/or habitat, whereas there was an unexpected lack of difference between the two species in the percentage of time spent at the front of a heterospecific group. Both species’ decreased response ratio to fright stimuli in a heterospecific group and the decreased consistency of responses between species compared with those within species suggest that information is transferred less efficiently in heterospecific groups. In conclusion, Chinese bream adjusted their swimming activity to that of qingbo in heterospecific groups to coordinate group movement; however, the ecological benefits of shoaling, such as defense against predators, might decrease due to the decreased information transfer.

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