title: PeerJ description: Articles published in PeerJ link: https://peerj.com/articles/index.rss3?journal=peerj&page=1519 creator: info@peerj.com PeerJ errorsTo: info@peerj.com PeerJ language: en title: Comparative description and ossification patterns of Dendropsophus labialis (Peters, 1863) and Scinax ruber (Laurenti, 1758) (Anura: Hylidae) link: https://peerj.com/articles/4525 last-modified: 2018-06-06 description: Although comparative studies of anuran ontogeny have provided new data on heterochrony in the life cycles of frogs, most of them have not included ossification sequences. Using differential staining techniques, we observe and describe differences and similarities of cranial and postcranial development in two hylid species, Scinax ruber (Scinaxinae) and Dendropsophus labialis (Hylinae), providing new data of ontogenetic studies in these Colombian species. We examined tadpoles raining from Gosner Stages 25 to 45. We found differences between species in the infrarostral and suprarostral cartilages, optic foramen, planum ethmoidale, and gill apparatus. In both species, the first elements to ossify were the atlas and transverse processes of the vertebral column and the parasphenoid. Both species exhibited suprascapular processes as described in other hylids. Although the hylids comprise a large group (over 700 species), postcranial ossification sequence is only known for 15 species. Therefore, the descriptions of the skeletal development and ossification sequences provided herein will be useful for future analyses of heterochrony in the group. creator: Angélica Arenas-Rodríguez creator: Juan Francisco Rubiano Vargas creator: Julio Mario Hoyos uri: https://doi.org/10.7717/peerj.4525 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2018 Arenas-Rodríguez et al. title: Coupling GIS spatial analysis and Ensemble Niche Modelling to investigate climate change-related threats to the Sicilian pond turtle Emys trinacris, an endangered species from the Mediterranean link: https://peerj.com/articles/4969 last-modified: 2018-06-05 description: The pond turtle Emys trinacris is an endangered endemic species of Sicily showing a fragmented distribution throughout the main island. In this study, we applied “Ensemble Niche Modelling”, combining more classical statistical techniques as Generalized Linear Models and Multivariate Adaptive Regression Splines with machine-learning approaches as Boosted Regression Trees and Maxent, to model the potential distribution of the species under current and future climatic conditions. Moreover, a “gap analysis” performed on both the species’ presence sites and the predictions from the Ensemble Models is proposed to integrate outputs from these models, in order to assess the conservation status of this threatened species in the context of biodiversity management. For this aim, four “Representative Concentration Pathways”, corresponding to different greenhouse gases emissions trajectories were considered to project the obtained models to both 2050 and 2070. Areas lost, gained or remaining stable for the target species in the projected models were calculated. E. trinacris’ potential distribution resulted to be significantly dependent upon precipitation-linked variables, mainly precipitation of wettest and coldest quarter. Future negative effects for the conservation of this species, because of more unstable precipitation patterns and extreme meteorological events, emerged from our analyses. Further, the sites currently inhabited by E. trinacris are, for more than a half, out of the Protected Areas network, highlighting an inadequate management of the species by the authorities responsible for its protection. Our results, therefore, suggest that in the next future the Sicilian pond turtle will need the utmost attention by the scientific community to avoid the imminent risk of extinction. Finally, the gap analysis performed in GIS environment resulted to be a very informative post-modeling technique, potentially applicable to the management of species at risk and to Protected Areas’ planning in many contexts. creator: Mattia Iannella creator: Francesco Cerasoli creator: Paola D’Alessandro creator: Giulia Console creator: Maurizio Biondi uri: https://doi.org/10.7717/peerj.4969 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2018 Iannella et al. title: Molecular attributes and apoptosis-inducing activities of a putative serine protease isolated from Tiger Milk mushroom (Lignosus rhinocerus) sclerotium against breast cancer cells in vitro link: https://peerj.com/articles/4940 last-modified: 2018-06-05 description: BackgroundThe highly valued medicinal tiger milk mushroom (also known as Lignosus rhinocerus) has the ability to cure numerous ailments. Its anticancer activities are well explored, and recently a partially purified cytotoxic protein fraction termed F5 from the mushroom’s sclerotial cold water extract consisting mainly of fungal serine proteases was found to exhibit potent selective cytotoxicity against a human breast adenocarcinoma cell line (MCF7) with IC50 value of 3.00 μg/ml. However, characterization of its cell death-inducing activity has yet to be established.MethodsThe mechanism involved in the cytotoxic activities of F5 against MCF7 cells was elucidated by flow cytometry-based apoptosis detection, caspases activity measurement, and expression profiling of apoptosis markers by western blotting. Molecular attributes of F5 were further mined from L. rhinocerus’s published genome and transcriptome for future exploration.Results and DiscussionApoptosis induction in MCF7 cells by F5 may involve a cross-talk between the extrinsic and intrinsic apoptotic pathways with upregulation of caspase-8 and -9 activities and a marked decrease of Bcl-2. On the other hand, the levels of pro-apoptotic Bax, BID, and cleaved BID were increased accompanied by observable actin cleavage. At gene level, F5 composed of three predicted non-synonymous single nucleotide polymorphisms (T > C) and an alternative 5′ splice site.ConclusionsFindings from this study provide an advanced framework for further investigations on cancer therapeutics development from L. rhinocerus. creator: Hui Yeng Y. Yap creator: Nget Hong Tan creator: Szu Ting Ng creator: Chon Seng Tan creator: Shin Yee Fung uri: https://doi.org/10.7717/peerj.4940 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2018 Yap et al. title: An inexpensive, customizable microscopy system for the automated quantification and characterization of multiple adherent cell types link: https://peerj.com/articles/4937 last-modified: 2018-06-05 description: Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated microscopy-based cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated microscopy-based cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the microscopy-based cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity toward tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our microscopy-based cytometer is successfully able to elucidate. creator: Vishwaratn Asthana creator: Yuqi Tang creator: Adam Ferguson creator: Pallavi Bugga creator: Anantratn Asthana creator: Emily R. Evans creator: Allen L. Chen creator: Brett S. Stern creator: Rebekah A. Drezek uri: https://doi.org/10.7717/peerj.4937 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2018 Asthana et al. title: The mitochondrial genomes of two walnut pests, Gastrolina depressa depressa and G. depressa thoracica (Coleoptera: Chrysomelidae), and phylogenetic analyses link: https://peerj.com/articles/4919 last-modified: 2018-06-05 description: In this study, the mitochondrial genomes (mitogenomes) of two walnut leaf insect pests, Gastrolina depressa depressa and G. depressa thoracica, were sequenced by Sanger sequencing technology. The mitogenome of G. depressa thoracica was complete at 16,109 bp in length, while the mitogenome of G. depressa depressa (14,277 bp) was partial. The genomic analyses indicated that both mitogenomes have the typical gene content and arrangement. The formerly identified elements, ‘TAGTA’ between trnSer(UCN) and nad2, and ‘ATGATAA’ between atp8 and atp6, were more conserved than that between nad4L and nad4, which was ‘ATGTTAA’ in Coleoptera excluding Polyphaga. Phylogenetic analyses of the 13 protein-coding genes from 36 coleopteran species well supported a close affinity between the subfamily Chrysomelinae including G. depressa thoracica and G. depressa depressa and Galerucinae, as well as a sister relationship of ((Eumolpinae + Cryptocephalinae) + Cassidinae) within Chrysomelidae. creator: Qiqi Wang creator: Guanghui Tang uri: https://doi.org/10.7717/peerj.4919 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2018 Wang and Tang title: Shift in VEGFA isoform balance towards more angiogenic variants is associated with tumor stage and differentiation of human hepatocellular carcinoma link: https://peerj.com/articles/4915 last-modified: 2018-06-05 description: BackgroundHepatocellular carcinoma (HCC) is the most common and aggressive type of malignant liver tumor. HCC progression depends significantly on its vascularization and formation of new blood vessels. Vascular endothelial growth factor A (VEGFA) is a crucial regulator of tumor vascularization and components of VEGF-induced cell signaling pathways are important targets of therapeutical drugs that demonstrated the highest efficiency in case of advanced HCC (sorafenib and regorafenib). VEGFA is expressed as a set of isoforms with different functional properties, thus VEGFA isoform expression pattern may affect tumor sensitivity to anti-angiogenic drugs. However, information about VEGFA isoforms expression in HCC is still incomplete and contradictory. The present study aims to quantitatively investigate VEGFA isoform expression aberrations in HCC tissue.MethodsA total of 50 pairs of HCC and non-tumor tissue samples were used to evaluate the VEGFA isoform spectrum using RT-PCR and quantitatively estimate changes in isoform expression using RT-qPCR. Correlations between these changes and tumor clinicopathological characteristics were analyzed.ResultsWe identified VEGFA-189, VEGFA-165, and VEGFA-121 as predominant isoforms in liver tissue. Anti-angiogenic VEGFA-xxxb variants constituted no more than 5% of all mature VEGFA transcripts detected and their expression was not changed significantly in HCC tissue. We demonstrated for the first time that the least active variant VEGFA-189 is frequently repressed in HCC (p < 0.001), while no uniform changes were detected for potent angiogenesis stimulators VEGFA-165 and VEGFA-121. Isoform balance in HCC shifts from VEGFA-189 towards VEGFA-165 or VEGFA-121 in the majority of cases (p < 0.001). Changes in fractions, but not expression levels, of VEGFA-189 (decrease) and VEGFA-121 (increase) correlated with advanced Tumor-Node-Metastasis (TNM) and Barcelona Clinic Liver Cancer (BCLC) tumor stages (p < 0.05), VEGFA-189 fraction reduction was also associated with poor tumor differentiation (p < 0.05).DiscussionA distinct shift in VEGFA isoform balance towards more pro-angiogenic variants occurs in HCC tissue and may modulate overall impact of VEGFA signaling. We suppose that the ratio between VEGFA isoforms is an important parameter governing HCC angiogenesis that may affect HCC progression and be used for optimizing the strategy of HCC therapy by predicting the response to anti-angiogenic drugs. creator: Mikhail S. Chesnokov creator: Polina A. Khesina creator: Darya A. Shavochkina creator: Inna F. Kustova creator: Leonid M. Dyakov creator: Olga V. Morozova creator: Nikolai S. Mugue creator: Nikolay E. Kudashkin creator: Ekaterina A. Moroz creator: Yuri I. Patyutko creator: Natalia L. Lazarevich uri: https://doi.org/10.7717/peerj.4915 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2018 Chesnokov et al. title: Discovery of digestive enzymes in carnivorous plants with focus on proteases link: https://peerj.com/articles/4914 last-modified: 2018-06-05 description: BackgroundCarnivorous plants have been fascinating researchers with their unique characters and bioinspired applications. These include medicinal trait of some carnivorous plants with potentials for pharmaceutical industry.MethodsThis review will cover recent progress based on current studies on digestive enzymes secreted by different genera of carnivorous plants: Drosera (sundews), Dionaea (Venus flytrap), Nepenthes (tropical pitcher plants), Sarracenia (North American pitcher plants), Cephalotus (Australian pitcher plants), Genlisea (corkscrew plants), and Utricularia (bladderworts).ResultsSince the discovery of secreted protease nepenthesin in Nepenthes pitcher, digestive enzymes from carnivorous plants have been the focus of many studies. Recent genomics approaches have accelerated digestive enzyme discovery. Furthermore, the advancement in recombinant technology and protein purification helped in the identification and characterisation of enzymes in carnivorous plants.DiscussionThese different aspects will be described and discussed in this review with focus on the role of secreted plant proteases and their potential industrial applications. creator: Rishiesvari Ravee creator: Faris ‘Imadi Mohd Salleh creator: Hoe-Han Goh uri: https://doi.org/10.7717/peerj.4914 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2018 Ravee et al. title: Hindering the illegal trade in dog and cat furs through a DNA-based protocol for species identification link: https://peerj.com/articles/4902 last-modified: 2018-06-05 description: In Western countries dogs and cats are the most popular pets, and people are increasingly opposed to their rearing for the fur industry. In 2007, a Regulation of the European Union (EU) banned the use and trade of dog and cat furs, but an official analytical protocol to identify them as source species was not provided, and violations of law are still frequent in all Member States. In this paper we report on the development and validation of a simple and affordable DNA method for species detection in furs to use as an effective tool to combat illegal trade in fur products. A set of mitochondrial primers was designed for amplification of partial cytochrome b, control region and ND1 gene in highly degraded samples, like furs and pelts. Our amplification workflow involved the use of a non-specific primer pair to perform a first test to identify the species through sequencing, then the application of species-specific primer pairs to use in singleplex end-point PCRs as confirmation tests. The advantage of this two-step procedure is twofold: on the one hand it minimises the possibility of negative test results from degraded samples, since failure of amplification with a first set of primers can be offset by successful amplification of the second, and on the other it adds confidence and reliability to final authentication of species. All designed primers were validated on a reference collection of tissue samples, obtaining solid results in terms of specificity, sensitivity, repeatability and reproducibility. Application of the protocol on real caseworks from seized furs yielded successful results also from old and dyed furs, suggesting that age and chemical staining do not necessarily affect positive amplifications. Major pros of this approach are: (1) sensitive and informative primer sets for detection of species; (2) short PCR amplicons for the analysis of poor quality DNA; (3) binding primers that avoid contamination from human DNA; (4) user-friendly protocol for any laboratory equipped for analysis of low-copy-number DNA. Our molecular procedure proved to be a good starting point for enforcing the EU Regulation against dog and cat fur trade in forensic contexts where source attribution is essential to the assignment of responsibilities. creator: Luisa Garofalo creator: Alessia Mariacher creator: Rita Fanelli creator: Rosario Fico creator: Rita Lorenzini uri: https://doi.org/10.7717/peerj.4902 license: http://creativecommons.org/licenses/by/4.0/ rights: © 2018 Garofalo et al. title: In quest of contact: phylogeography of helmeted terrapins (Pelomedusa galeata, P. subrufa sensu stricto) link: https://peerj.com/articles/4901 last-modified: 2018-06-05 description: Based on rangewide sampling and three mitochondrial and two nuclear markers (together up to 1,850 bp and 1,840 bp, respectively), we examine the phylogeography of two helmeted terrapin species (Pelomedusa galeata and P. subrufa sensu stricto) and infer shifts of climatically suitable spaces since the Last Glacial Maximum using a modeling approach. Whilst P. galeata displays significant phylogeographic structuring across its range and consists of two deeply divergent lineages that could represent distinct species, P. subrufa shows no obvious phylogeographic differentiation. This seems to be related to historically stable or fluctuating ranges. One of the lineages within P. galeata appears to be confined to the westernmost, winter-rainfall region of South Africa and deserves special conservational attention due to the scarcity of surface water. The other lineage is distributed further east and is differentiated in three weakly supported subclades with parapatric distribution; one occurring inland, and two along the south and east coasts, respectively. As far as is known, P. subrufa occurs in South Africa only in the northeast of the country (Limpopo, Mpumalanga) and we report the species for the first time from the Lapalala Wilderness Area in the Waterberg region (Limpopo), approximately 350 km further west than previously recorded. We confirmed the occurrence of P. galeata only 80 km south of Lapalala. Thus, a sympatric occurrence of P. galeata and P. subrufa is possible. Another putative contact zone, for the two lineages within P. galeata, must be located in the Western Cape region, and further contact zones are likely for the eastern subclades within P. galeata. The nuclear loci provided no evidence for gene flow across taxa or genetic clusters within taxa. Future investigations should use denser sampling from putative contact zones and more nuclear markers to re-examine this situation. Despite few phylogeographic studies published for southern African biota, it seems likely that differentiation follows general rules, and that climate and physiographic barriers (e.g., the Great Escarpment) have shaped phylogeographic patterns. creator: Melita Vamberger creator: Margaretha D. Hofmeyr creator: Flora Ihlow creator: Uwe Fritz uri: https://doi.org/10.7717/peerj.4901 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2018 Vamberger et al. title: Growth, biochemical response and liver health of juvenile barramundi (Lates calcarifer) fed fermented and non-fermented tuna hydrolysate as fishmeal protein replacement ingredients link: https://peerj.com/articles/4870 last-modified: 2018-06-05 description: Conventional aquaculture feed materials available in Australia are expensive, which has prompted the search for alternatives that would be cost-effective and locally available. The present study was undertaken in order to maximize the use of a tuna hydrolysate (TH), which was produced locally from the tuna-processing discards. The growth performance, biochemical status, antioxidant capacity and liver health of juvenile barramundi (Lates calcarifer) were assessed. Two series of isonitrogenous and isocaloric diets labelled as TH50, TH75 (non-fermented tuna hydrolysate) and FTH50, FTH75 (fermented tuna hydrolysate) were formulated to replace FM at 50% and 75%, respectively. A basal diet without the TH supplementation was used as a control. The experimental diets were fed to the triplicate groups of fish three times a day for 56 days. The results of the experiment revealed that fish fed on both fermented and non-fermented TH-containing diets significantly reduced (p < 0.05) the final body weight, weight gain and specific growth rate compared to the control. The highest apparent digestibility coefficients for dry matter, protein and lipid were obtained in the control group, and decreased with the increasing level of TH in the diets. However, the whole-body proximate compositions and the blood biochemical indices of fish were not affected by the TH inclusion in the diets. The fish fed on TH diets of TH50, FTH50 and TH75 exhibited reduced (p < 0.05) glutathione peroxidase (GPx) activity compared to the control; whereas the FTH75 exhibited no difference with the control. The excessive inclusion of TH in the diets of TH75 and FTH75 resulted in cytoplasmic vacuolization, with an increased amount of lipid accumulation, and necrosis in the liver tissue. These results indicated that the replacement of the FM protein with TH at 50% and 75% inclusion levels negatively affected the growth performance, feed utilization, and digestibility in juvenile barramundi; and it also increased the potential risk of hepatic failure in the fish. Further investigation is, therefore, required in order to optimize the TH levels in the fish diets which would be suitable for the growth of fish, as well as for maintaining the enhanced biochemical response in juvenile barramundi. creator: Muhammad A.B. Siddik creator: Janet Howieson creator: Ilham Ilham creator: Ravi Fotedar uri: https://doi.org/10.7717/peerj.4870 license: http://creativecommons.org/licenses/by/4.0/ rights: ©2018 Siddik et al.