In silico engineering of Pseudomonas metabolism reveals new biomarkers for increased biosurfactant production
Background Rhamnolipids, biosurfactants with a wide range of biomedical applications, are amphiphilic molecules produced on the surfaces of or excreted extracellularly by bacteria including Pseudomonas aeruginosa. However, Pseudomonas putida is a non-pathogenic model organism with greater metabolic versatility and potential for industrial applications. Methods We investigate in silico the metabolic capabilities of P. putida for rhamnolipids biosynthesis using statistical, metabolic and synthetic engineering approaches after introducing key genes (RhlA and RhlB) from P. aeruginosa into a genome-scale model of P. putida. This pipeline combines machine learning methods with multi-omic modelling, and drives the engineered P. putida model toward an optimal production and export of rhamnolipids out of the membrane. Results We identify a substantial increase in synthesis of rhamnolipids by the engineered model compared to the control model. We apply statistical and machine learning techniques on the metabolic reaction rates to identify distinct features on the structure of the variables and individual components driving the variation of growth and rhamnolipids production. We finally provide a computational framework for integrating multi-omics data and identifying latent pathways and genes for the production of rhamnolipids in P. putida. Conclusions We anticipate that our results will provide a versatile methodology for integrating multi-omics data for topological and functional analysis of P. putida toward maximization of biosurfactant production.
Occhipinti et al. (2018) In silico engineering of Pseudomonas metabolism reveals new biomarkers for increased biosurfactant production. PeerJ 6:e6046 https://doi.org/10.7717/peerj.6046
Transcriptome analysis of oil palm inflorescences revealed candidate genes for an auxin signaling pathway involved in parthenocarpy
Oil palm parthenocarpic fruits, which are produced without fertilization, can be targeted to increase oil content because the majority of the fruit is occupied by mesocarp, the part in which palm oil is stored. Consequently, gaining an understanding of the parthenocarpic mechanism would be instrumental for producing parthenocarpic oil palm. This study aims to determine effects of auxin treatment and analyze differentially expressed genes in oil palm pistils at the pollination/anthesis stage, using an RNA sequencing (RNA seq) approach. The auxin treatment caused 100% parthenocarpy when auxin was sprayed before stigmas opened. The parthenocarpy decreased to 55%, 8% and 5% when the auxin was sprayed 1, 2 and 3 days after the opening of stigmas, respectively. Oil palm plants used for RNA seq were plants untreated with auxin as controls and auxin-treated plants on the day before pollination and 1 day after pollination. The number of raw reads ranged from 8,425,859 to 11,811,166 reads, with an average size ranging from 99 to 137 base pairs (bp). When compared with the oil palm transcriptome, the mapped reads ranged from 8,179,948 to 11,320,799 reads, representing 95.85–98.01% of the oil palm matching. Based on five comparisons between RNA seq of treatments and controls, and confirmation using reverse transcription polymerase chain reaction and quantitative real-time RT-PCR expression, five candidate genes, including probable indole-3-acetic acid (IAA)-amido synthetase GH3.8 (EgGH3.8), IAA-amido synthetase GH3.1 (EgGH3.1), IAA induced ARG7 like (EgARG7), tryptophan amino transferase-related protein 3-like (EgTAA3) and flavin-containing monooxygenase 1 (EgFMO1), were differentially expressed between auxin-treated and untreated samples. This evidence suggests a pathway of parthenocarpic fruit development at the beginning of fruit development. However, more research is needed to identify which genes are definitely involved in parthenocarpy.
Somyong et al. (2018) Transcriptome analysis of oil palm inflorescences revealed candidate genes for an auxin signaling pathway involved in parthenocarpy. PeerJ 6:e5975 https://doi.org/10.7717/peerj.5975
Do longer sequences improve the accuracy of identification of forensically important Calliphoridae species?
Species identification is a crucial step in forensic entomology. In several cases the calculation of the larval age allows the estimation of the minimum Post-Mortem Interval (mPMI). A correct identification of the species is the first step for a correct mPMI estimation. To overcome the difficulties due to the morphological identification especially of the immature stages, a molecular approach can be applied. However, difficulties in separation of closely related species are still an unsolved problem. Sequences of 4 different genes (COI, ND5, EF-1α, PER) of 13 different fly species collected during forensic experiments (Calliphora vicina, Calliphora vomitoria, Lucilia sericata, Lucilia illustris, Lucilia caesar, Chrysomya albiceps, Phormia regina, Cynomya mortuorum, Sarcophaga sp., Hydrotaea sp., Fannia scalaris, Piophila sp., Megaselia scalaris) were evaluated for their capability to identify correctly the species. Three concatenated sequences were obtained combining the four genes in order to verify if longer sequences increase the probability of a correct identification. The obtained results showed that this rule does not work for the species L. caesar and L. illustris. Future works on other DNA regions are suggested to solve this taxonomic issue.
Bortolini et al. (2018) Do longer sequences improve the accuracy of identification of forensically important Calliphoridae species?. PeerJ 6:e5962 https://doi.org/10.7717/peerj.5962
Influenced but not determined by historical events: genetic, demographic and morphological differentiation in Heleobia ascotanensis from the Chilean Altiplano
In the present study, we focus on the phylogeographic pattern, demographic history and morphological differentiation of Heleobia ascotanensis, a freshwater gastropod restricted to the Ascotán saltpan in the Chilean Altiplano. The current distribution of the species is limited to twelve isolated or partially isolated springs that were affected by transitions between humid and arid periods during last glaciations. The genetic analysis of 322 specimens showed that H. ascotanensis is subdivided into three genetically divergent populations, with low and moderate degrees of historical gene flow among them and incipient morphological differentiation as a consequence of genetic and geographical isolation. Molecular analyses revealed different demographic histories among populations which seem to respond independently to climatic events, probably due to an environmental imposition and idiosyncratic strategies developed to cope with water availability. The results of this study and co-distributed taxa support the hypothesis that contemporary and historical events have influenced microevolutionary differentiation of these snails, although there is a need to complement further information to predict genetic or morphological divergence at microgeographic scale.
Valladares et al. (2018) Influenced but not determined by historical events: genetic, demographic and morphological differentiation in Heleobia ascotanensis from the Chilean Altiplano. PeerJ 6:e5802 https://doi.org/10.7717/peerj.5802
The Plasmodium knowlesi Pk41 surface protein diversity, natural selection, sub population and geographical clustering: a 6-cysteine protein family member
Introduction The zoonotic malaria parasite Plasmodium knowlesi has currently become the most dominant form of infection in humans in Malaysia and is an emerging infectious disease in most Southeast Asian countries. The P41 is a merozoite surface protein belonging to the 6-cysteine family and is a well-characterized vaccine candidate in P. vivax and P. falciparum; however, no study has been done in the orthologous gene of P. knowlesi. This study investigates the level of polymorphism, haplotypes and natural selection of pk41 genes in clinical isolates from Malaysia. Method Thirty-five full-length pk41 sequences from clinical isolates of Malaysia along with four laboratory lines (along with H-strain) were downloaded from public databases. For comparative analysis between species, orthologous P41 genes from P. falciparum, P. vivax, P. coatneyi and P. cynomolgi were also downloaded. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 software. Phylogenetic relationships between Pk41 genes were determined using MEGA 5.0 software. Results Analysis of 39 full-length pk41 sequences along with the H-strain identified 36 SNPs (20 non-synonymous and 16 synonymous substitutions) resulting in 31 haplotypes. Nucleotide diversity across the full-length gene was low and was similar to its ortholog in P. vivax; pv41. Domain-wise amino acid analysis of the two s48/45 domains indicated low level of polymorphisms for both the domains, and the glutamic acid rich region had extensive size variations. In the central domain, upstream to the glutamate rich region, a unique two to six (K-E)n repeat region was identified within the clinical isolates. Overall, the pk41 genes were indicative of negative/purifying selection due to functional constraints. Domain-wise analysis of the s48/45 domains also indicated purifying selection. However, analysis of Tajima’s D across the genes identified non-synonymous SNPs in the s48/45 domain II with high positive values indicating possible epitope binding regions. All the 6-cysteine residues within the s48/45 domains were conserved within the clinical isolates indicating functional conservation of these regions. Phylogenetic analysis of full-length pk41 genes indicated geographical clustering and identified three subpopulations of P. knowlesi; one originating in the laboratory lines and two originating from Sarawak, Malaysian Borneo. Conclusion This is the first study to report on the polymorphism and natural selection of pk41 genes from clinical isolates of Malaysia. The results reveal that there is low level of polymorphism in both s48/45 domains, indicating that this antigen could be a potential vaccine target. However, genetic and molecular immunology studies involving higher number of samples from various parts of Malaysia would be necessary to validate this antigen’s candidacy as a vaccine target for P. knowlesi.
Ahmed et al. (2018) The Plasmodium knowlesi Pk41 surface protein diversity, natural selection, sub population and geographical clustering: a 6-cysteine protein family member. PeerJ 6:e6141 https://doi.org/10.7717/peerj.6141
Shaping ability of the profile 25/0.06 and protaper F2 in rotary motion, and reciproc in simulated canals
Background Since the introduction of nickel–titanium (Ni–Ti) instruments to dentistry, a wide variety of Ni–Ti instruments have become commercially available. These Ni–Ti instruments are expensive, which limits their usage in developing countries and forces practitioners to use instruments repeatedly. Another problem is the possible prion cross-contamination associated with the multiple usage of endodontic instruments. In addition, the use of these instruments requires new skills and experience. In this article, the shaping capacities of two conventional rotary file systems, ProFile 25/0.06 and ProTaper F2, were reviewed and compared with the Reciproc single-file system. Methods A total of 45 simulated canals with 40° curvature, in clear resin blocks, were prepared using conventional rotary systems consisting of ProFile orifice shaping (OS) #3 and final flaring #25/.06, Reciproc R25, and ProTaper shaping file SX and finishing file F2. Pre-and post-instrumentation images were analyzed at ten different levels, using AutoCAD 2007 software. The measurement positions were defined in 1-mm intervals: positions 0–3 established the apical part, positions 4–6 constituted the middle part, and positions 7–10 established the coronal part of the canal. The amount of removed resin, the transportation, instrumentation time, change in working length (WL), instrumentation fractures, and the presence of ledge were evaluated. Data were analyzed using ANOVA, Kruskal–Wallis and independent t-test (p < 0.001). Results ProFile removed the least resin (p < 0.001) and caused less transportation than Reciproc and ProTaper, in total (p < 0.001). ProTaper caused more transportation ProFile and Reciproc in the apical part (p < 0.000). Reciproc caused more transportation than ProTaper and ProFile (p < 0.001), and the transportation tendency toward the inner aspect of the curvature in the middle part. Reciproc caused the less transportation than ProFile and ProTaper in the coronal part. The transportations tended to occur toward the outside of the curvature, except the middle part with Reciproc and at points 5 and 6 with ProTaper. There were no significant differences among the groups in terms of maintaining the original WL. Reciproc was significantly faster than the others group (p < 0.001). Only one instrument fracture (25/0.06 ProFile) was noted. All groups showed one ledge each. Discussion The results of the present study showed that both ProFile 25/06 and ProTaper F2, combined with a file used for coronal enlargement (OS3 and SX), have the potential to create satisfactory canal shape in the curved root canals. Further studies using real human teeth are needed to confirm our results.
Çelik et al. (2018) Shaping ability of the profile 25/0.06 and protaper F2 in rotary motion, and reciproc in simulated canals. PeerJ 6:e6109 https://doi.org/10.7717/peerj.6109
Comparing enzyme activity modifier equations through the development of global data fitting templates in Excel
The classical way of defining enzyme inhibition has obscured the distinction between inhibitory effect and the inhibitor binding constant. This article examines the relationship between the simple binding curve used to define biomolecular interactions and the standard inhibitory term (1 + ([I]∕Ki)). By understanding how this term relates to binding curves which are ubiquitously used to describe biological processes, a modifier equation which distinguishes between inhibitor binding and the inhibitory effect, is examined. This modifier equation which can describe both activation and inhibition is compared to standard inhibitory equations with the development of global data fitting templates in Excel and via the global fitting of these equations to simulated and previously published datasets. In both cases, this modifier equation was able to match or outperform the other equations by providing superior fits to the datasets. The ability of this single equation to outperform the other equations suggests an over-complication of the field. This equation and the template developed in this article should prove to be useful tools in the study of enzyme inhibition and activation.
Walsh (2018) Comparing enzyme activity modifier equations through the development of global data fitting templates in Excel. PeerJ 6:e6082 https://doi.org/10.7717/peerj.6082
Detection of bacterial endosymbionts in freshwater crustaceans: the applicability of non-degenerate primers to amplify the bacterial 16S rRNA gene
Bacterial endosymbionts of aquatic invertebrates remain poorly studied. This is at least partly due to a lack of suitable techniques and primers for their identification. We designed a pair of non-degenerate primers which enabled us to amplify a fragment of ca. 500 bp of the 16S rRNA gene from various known bacterial endosymbiont species. By using this approach, we identified four bacterial endosymbionts, two endoparasites and one uncultured bacterium in seven, taxonomically diverse, freshwater crustacean hosts from temporary waters across a wide geographical area. The overall efficiency of our new WOLBSL and WOLBSR primers for amplification of the bacterial 16S rRNA gene was 100%. However, if different bacterial species from one sample were amplified simultaneously, sequences were illegible, despite a good quality of PCR products. Therefore, we suggest using our primers at the first stage of bacterial endosymbiont identification. Subsequently, genus specific primers are recommended. Overall, in the era of next-generation sequencing our method can be used as a first simple and low-cost approach to identify potential microbial symbionts associated with freshwater crustaceans using simple Sanger sequencing. The potential to detected bacterial symbionts in various invertebrate hosts in such a way will facilitate studies on host-symbiont interactions and coevolution.
Mioduchowska et al. (2018) Detection of bacterial endosymbionts in freshwater crustaceans: the applicability of non-degenerate primers to amplify the bacterial 16S rRNA gene. PeerJ 6:e6039 https://doi.org/10.7717/peerj.6039
Evidence for the Cretaceous shark Cretoxyrhina mantelli feeding on the pterosaur Pteranodon from the Niobrara Formation
A cervical vertebra of the large, pelagic pterodactyloid pterosaur Pteranodon sp. from the Late Cretaceous Niobrara Formation of Kansas, USA is significant for its association with a tooth from the large lamniform shark, Cretoxyrhina mantelli. Though the tooth does not pierce the vertebral periosteum, the intimate association of the fossils—in which the tooth is wedged below the left prezygapophysis—suggests their preservation together was not mere chance, and the specimen is evidence of Cretoxyrhina biting Pteranodon. It is not possible to infer whether the bite reflects predatory or scavenging behaviour from the preserved material. There are several records of Pteranodon having been consumed by other fish, including other sharks (specifically, the anacoracid Squalicorax kaupi), and multiple records of Cretoxyrhina biting other vertebrates of the Western Interior Seaway, but until now interactions between Cretoxyrhina and Pteranodon have remained elusive. The specimen increases the known interactions between large, pelagic, vertebrate carnivores of the Western Interior Seaway of North America during the Late Cretaceous, in addition to bolstering the relatively small fossil record representing pterosaurian interactions with other species.
Hone et al. (2018) Evidence for the Cretaceous shark Cretoxyrhina mantelli feeding on the pterosaur Pteranodon from the Niobrara Formation. PeerJ 6:e6031 https://doi.org/10.7717/peerj.6031
Exploring the mechanistic link between corticosterone and insulin-like growth factor-1 in a wild passerine bird
Background Physiological regulators of life history trade-offs need to be responsive to sudden changes of resource availability. When homeostasis is challenged by unpredictable stressors, vertebrates respond through a set of physiological reactions, which can promote organismal survival. Glucocorticoids have been traditionally recognized as one of the main regulators of the physiological stress response, but the role of an evolutionarily more conserved pathway, the hypothalamic-pituitary-somatotropic (HPS) axis producing insulin-like growth factor-1 (IGF-1) has received much less attention. Although IGF-1 is known to affect several life history traits, little is known about its role in the physiological stress response and it has never been studied directly in adult wild animals. Methods In this study, we combined field observations with a controlled experiment to investigate how circulating levels of IGF-1 change in response to stress and whether this change is due to concomitant change in glucocorticoids in a free-living songbird, the bearded reedling Panurus biarmicus. We used a standard capture-restraint protocol in field observation, in which we took first and second (stress induced: 15 minutes later) samples. In a follow-up experiment, we used a minimally invasive oral corticosterone manipulation. Results We showed that corticosterone levels significantly increased while IGF-1 levels significantly decreased during capture and handling stress. However, change in corticosterone levels were not related to change in IGF-1 levels. We found that experimentally elevated corticosterone levels did not affect IGF-1 levels. Discussion Our results are the first to highlight that circulating IGF-1 levels are responsive to stress independently from glucocorticoids and suggest that the HPS axis is an autonomous physiological pathway that may play an important role as regulator of life-history decisions.
Tóth et al. (2018) Exploring the mechanistic link between corticosterone and insulin-like growth factor-1 in a wild passerine bird. PeerJ 6:e5936 https://doi.org/10.7717/peerj.5936