Noninvasive and safe cell viability assay for Euglena gracilis using natural food pigment
Noninvasive and safe cell viability assay is required in many fields such as regenerative medicine, genetic engineering, single-cell analysis, and microbial food culture. In this case, a safe and inexpensive method which is a small load on cells and the environment is preferable without requiring expensive and space-consuming equipment and a technician to operate. We examined eight typical natural food pigments to find Monascus pigment (MP) or anthocyanin pigment (AP) works as a good viability indicator of dye exclusion test (DET) for Euglena gracilis which is an edible photosynthetic green microalga. This is the first report using natural food pigments as cell viability assay. Euglena gracilis stained by MP or AP can be visually judged with a bright field microscope. This was spectrally confirmed by scan-free, non-invasive absorbance spectral imaging A(x, y, λ) microscopy of single live cells and principal component analysis (PCA). To confirm the ability of staining dead cells and examine the load on the cells, these two natural pigments were compared with trypan blue (TB) and methylene blue (MP), which are synthetic dyes conventionally used for DET. As a result, MP and AP had as good ability of staining dead cells treated with microwave as TB and MB and showed faster and more uniform staining for dead cells in benzalkonium chloride than them. The growth curve and the ratio of dead cells in the culture showed that the synthetic dyes inhibit the growth of E. gracilis, but the natural pigments do not. As the cell density increased, however, AP increased the ratio of stained cells, which was prevented by the addition of glucose. MP can stain dead cells in a shorter time than AP, while AP is more stable in color against long-term irradiation of intense light than MP. Due to the low toxicity of these pigments, viability of cells in culture can be monitored with them over a long period.
Yamashita et al. (2019) Noninvasive and safe cell viability assay for Euglena gracilis using natural food pigment. PeerJ 7:e6636 https://doi.org/10.7717/peerj.6636
Photo-hydrogen and lipid production from lactate, acetate, butyrate, and sugar manufacturing wastewater with an alternative nitrogen source by Rhodobacter sp. KKU-PS1
Photo-hydrogen and lipid production from individual synthetic volatile fatty acids (VFAs) and sugar manufacturing wastewater (SMW) by Rhodobacter sp. KKU-PS1 with sodium glutamate or Aji-L (i.e., waste from the process of crystallizing monosodium glutamate) as a nitrogen source was investigated. Using individual synthetic VFAs, the maximum hydrogen production was achieved with Aji-L as a nitrogen source rather than sodium glutamate. The maximum hydrogen production was 1,727, 754 and 1,353 mL H2/L, respectively, using 25 mM of lactate, 40 mM of acetate and 15mM of butyrate as substrates. Under these conditions, lipid was produced in the range of 10.6–16.9% (w/w). Subsequently, photo-hydrogen and lipid production from SMW using Aji-L as nitrogen source was conducted. Maximal hydrogen production and hydrogen yields of 1,672 mL H2/L and 1.92 mol H2/mol substrate, respectively, were obtained. Additionally, lipid content and lipid production of 21.3% (w/w) and 475 mg lipid/L were achieved. The analysis of the lipid and fatty acid components revealed that triacyglycerol (TAG) and C18:1 methyl ester were the main lipid and fatty acid components, respectively, found in Rhodobacter sp. KKU-PS1 cells.
Assawamongkholsiri et al. (2019) Photo-hydrogen and lipid production from lactate, acetate, butyrate, and sugar manufacturing wastewater with an alternative nitrogen source by Rhodobacter sp. KKU-PS1. PeerJ 7:e6653 https://doi.org/10.7717/peerj.6653
Hematological parameters in largemouth bass (Micropterus salmoides) with formalin-preservation: comparison between wild tournament-caught and captive-raised fish
Background Largemouth bass (Micropterus salmoides) are an economically important freshwater fish species that have been investigated for both the short and long-term effects of stress, secondary to angling. Limited data has been published on the hematological parameters of this species and blood sample stability is a notable limitation of hematologic field studies. A relatively novel technique using 10% neutral buffered formalin preserves heparinized whole blood and maintains blood cell stability beyond one month in striped bass. The objective of this study was to evaluate the differences in hematological parameters between tournament-caught and captive-raised largemouth bass using whole blood preservation with neutral buffered formalin. Methods Two populations of largemouth bass (n = 26 wild; n = 29 captive) underwent coccygeal venipuncture to collect heparinized whole blood for packed cell volume, total solids, and manual differential. Formalin preservation of heparinized whole blood facilitated manual hemocytometer analysis. Results were compared between the populations (tournament-caught, and captive-raised) with Wilcoxon rank sum test, a Hotelling’s T2 test, and Bonferroni simultaneous 95% confidence intervals to determine significance. Results The mean packed cell volume (44.9 ± 5.4%) and total solids (7.2 ± 1.1 g/dL) were significantly higher, while the total leukocyte count (7.08 ± 1.86 × 103/µL) was significantly lower in the wild tournament-caught population of largemouth bass, as compared to the captive-raised counterparts (PCV 34.4 ± 7.2%; TS 5.2 ± 1.0 g/dL; WBC 16.43 ± 8.37 × 103/µL). The wild population demonstrated a significantly distinct leukogram characterized by a neutropenia (24.1 ± 12.7%), lymphocytosis (67.7 ± 13.0%), and monocytopenia (8.3 ± 2.9%), while the erythrocyte and thrombocyte counts were not significantly different between populations. Discussion Numerous factors have been demonstrated to influence hematologic parameters in fish including age, size, sex, temperature, environmental oxygen level, population density, and infection. The wild population endured stress during angling capture, live-well hypoxia, transport, and extended air exposures at weigh in, which may have caused a stress leukopenia as well as osmoregulatory dysfunction and subsequent hemoconcentration. Further evaluation of seasonal impact as well as increased sample size is warranted to enhance our understanding of largemouth bass hematology. Conclusion This study concluded that wild largemouth bass captured via tournament angling have higher packed cell volume and total solids, and lower total leukocyte counts, compared to captive-reared individuals. Through the completion of this study, we demonstrated the successful use of 10% neutral buffered formalin to preserve heparinized whole blood for precise hemocytometer cell counts in a new teleost species, the largemouth bass.
Whitehead et al. (2019) Hematological parameters in largemouth bass (Micropterus salmoides) with formalin-preservation: comparison between wild tournament-caught and captive-raised fish. PeerJ 7:e6669 https://doi.org/10.7717/peerj.6669
Genome-wide characterization of the auxin response factor (ARF) gene family of litchi (Litchi chinensis Sonn.): phylogenetic analysis, miRNA regulation and expression changes during fruit abscission
Auxin response factors (ARFs) play fundamental roles in modulating various biological processes including fruit development and abscission via regulating the expression of auxin response genes. Currently, little is known about roles of ARFs in litchi (Litchi chinensis Sonn.), an economically important subtropical fruit tree whose production is suffering from fruit abscission. In this study, a genome-wide analysis of ARFs was conducted for litchi, 39 ARF genes (LcARFs) were identified. Conserved domain analysis showed that all the LcARFs identified have the signature B3 DNA-binding (B3) and ARF (Aux_rep) domains, with only 23 members having the dimerization domain (Aux_IAA). The number of exons in LcARF genes ranges from 2 to 16, suggesting a large variation for the gene structure of LcARFs. Phylogenetic analysis showed that the 39 LcARFs could be divided into three main groups: class I, II, and III. In total, 23 LcARFs were found to be potential targets of small RNAs, with three conserved and one novel miRNA-ARF (miRN43-ARF9) regulatory pathways discovered in litchi. Expression patterns were used to evaluate candidate LcARFs involved in various developmental processes, especially in flower formation and organ abscission. The results revealed that most ARF genes likely acted as repressors in litchi fruit abscission, that is, ARF2D/2E, 7A/7B, 9A/9B, 16A/16B, while a few LcARFs, such as LcARF5A/B, might be positively involved in this process. These findings provide useful information and resources for further studies on the roles of ARF genes in litchi growth and development, especially in the process of fruit abscission.
Zhang et al. (2019) Genome-wide characterization of the auxin response factor (ARF) gene family of litchi (Litchi chinensis Sonn.): phylogenetic analysis, miRNA regulation and expression changes during fruit abscission. PeerJ 7:e6677 https://doi.org/10.7717/peerj.6677
Association and diagnostic value of serum SPINK4 in colorectal cancer
The role of serum serine peptidase inhibitor, Kazal type 4 (SPINK4), in colorectal cancer (CRC) is largely unknown. This study aimed to explore the association and diagnostic value of serum SPINK4 in CRC. A total of 70 preoperative CRC patients, 30 postoperative CRC patients, 30 gastric cancer patients, and 30 healthy controls were enrolled. Using enzyme-linked immunosorbent assays, we found that the serum SPINK4 level was significantly increased in preoperative CRC compared with postoperative CRC patients, gastric cancer patients, and healthy controls (p < 0.05). The serum SPINK4 level was remarkably elevated in colon cancer compared with rectal cancer and was enhanced in the M1 stage compared with the M0 stage (p < 0.05). The area under the receiver operating characteristic curve of serum SPINK4 level in the diagnosis of CRC was 0.9186, with a sensitivity and specificity of 0.886 and 0.900, respectively, and a cut-off value of 2.065. There was no significant difference between high and low expression of serum SPINK4 regarding the overall survival time and disease-free survival (p > 0.05). This study demonstrated that the serum SPINK4 level increased in CRC and was associated with the location and distant metastasis of CRC. It had a high diagnostic value in CRC but was not associated with the survival of CRC patients.
Xie et al. (2019) Association and diagnostic value of serum SPINK4 in colorectal cancer. PeerJ 7:e6679 https://doi.org/10.7717/peerj.6679
Multi-gene phylogeny and divergence estimations for Evaniidae (Hymenoptera)
Ensign wasps (Hymenoptera: Evaniidae) develop as predators of cockroach eggs (Blattodea), have a wide distribution and exhibit numerous interesting biological phenomena. The taxonomy of this lineage has been the subject of several recent, intensive efforts, but the lineage lacked a robust phylogeny. In this paper we present a new phylogeny, based on increased taxonomic sampling and data from six molecular markers (mitochondrial 16S and COI, and nuclear markers 28S, RPS23, CAD, and AM2), the latter used for the first time in phylogenetic reconstruction. Our intent is to provide a robust phylogeny that will stabilize and facilitate revision of the higher-level classification. We also show the continued utility of molecular motifs, especially the presence of an intron in the RPS23 fragments of certain taxa, to diagnose evaniid clades and assist with taxonomic classification. Furthermore, we estimate divergence times among evaniid lineages for the first time, using multiple fossil calibrations. Evaniidae radiated primarily in the Early Cretaceous (134.1–141.1 Mya), with and most extant genera diverging near the K-T boundary. The estimated phylogeny reveals a more robust topology than previous efforts, with the recovery of more monophyletic taxa and better higher-level resolution. The results facilitate a change in ensign wasp taxonomy, with Parevania, and Papatuka, syn. nov. becoming junior synonyms of Zeuxevania, and Acanthinevania, syn. nov. being designated as junior synonym of Szepligetella. We transfer 30 species to Zeuxevania, either reestablishing past combinations or as new combinations. We also transfer 20 species from Acanthinevania to Szepligetella as new combinations.
Sharanowski et al. (2019) Multi-gene phylogeny and divergence estimations for Evaniidae (Hymenoptera). PeerJ 7:e6689 https://doi.org/10.7717/peerj.6689
Spiders did not repeatedly gain, but repeatedly lost, foraging webs
Much genomic-scale, especially transcriptomic, data on spider phylogeny has accumulated in the last few years. These data have recently been used to investigate the diverse architectures and the origin of spider webs, concluding that the ancestral spider spun no foraging web, that spider webs evolved de novo 10–14 times, and that the orb web evolved at least three times. These findings in fact result from a particular phylogenetic character coding strategy, specifically coding the absence of webs as logically equivalent, and homologous to, 10 other observable (i.e., not absent) web architectures. “Absence” of webs should be regarded as inapplicable data. To be analyzed properly by character optimization algorithms, it should be coded as “?” because these codes—or their equivalent—are handled differently by such algorithms. Additional problems include critical misspellings of taxon names from one analysis to the next (misspellings cause some optimization algorithms to drop terminals, which affects taxon sampling and results), and mistakes in spider natural history. In sum, the method causes character optimization algorithms to produce counter-intuitive results, and does not distinguish absence from secondary loss. Proper treatment of missing entries and corrected data instead imply that foraging webs are primitive for spiders and that webs have been lost ∼5–7 times, not gained 10–14 times. The orb web, specifically, may be homologous (originated only once) although lost 2–6 times.
Coddington et al. (2019) Spiders did not repeatedly gain, but repeatedly lost, foraging webs. PeerJ 7:e6703 https://doi.org/10.7717/peerj.6703
Prehemodialysis arteriovenous access creation is associated with better cardiovascular outcomes in patients receiving hemodialysis: a population-based cohort study
Background Cardiovascular (CV) disease contributes to nearly half of the mortalities in patients with end-stage renal disease. Patients who received prehemodialysis arteriovenous access (pre-HD AVA) creation had divergent CV outcomes. Methods We conducted a population-based cohort study by recruiting incident patients receiving HD from 2001 to 2012 from the Taiwan National Health Insurance Research Database. Patients’ characteristics, comorbidities, and medicines were analyzed. The primary outcome of interest was major adverse cardiovascular events (MACEs), defined as hospitalization due to acute myocardial infarction, stroke, or congestive heart failure (CHF) occurring within the first year of HD. Secondary outcomes included MACE-related mortality and all-cause mortality in the same follow-up period. Results The patients in the pre-HD AVA group were younger, had a lower burden of underlying diseases, were more likely to use erythropoiesis-stimulating agents but less likely to use renin–angiotensin–aldosterone system blockers. The patients with pre-HD AVA creation had a marginally lower rate of MACEs but a significant 35% lower rate of CHF hospitalization than those without creation (adjusted hazard ratio (HR) 0.65, 95% confidence interval (CI) [0.48–0.88]). In addition, the pre-HD AVA group exhibited an insignificantly lower rate of MACE-related mortality but a significantly 52% lower rate of all-cause mortality than the non-pre-HD AVA group (adjusted HR 0.48, 95% CI [0.39–0.59]). Sensitivity analyses obtained consistent results. Conclusions Pre-HD AVA creation is associated with a lower rate of CHF hospitalization and overall death in the first year of dialysis.
Yen et al. (2019) Prehemodialysis arteriovenous access creation is associated with better cardiovascular outcomes in patients receiving hemodialysis: a population-based cohort study. PeerJ 7:e6680 https://doi.org/10.7717/peerj.6680
A longitudinal study of the faecal microbiome and metabolome of periparturient mares
Background Periparturient mares are at increased risk of colic including large colon volvulus, which has a high mortality rate. Alterations in colonic microbiota related to either physiological or management changes, or both, that occur at this time have been suggested as potential causes for increased colic risk in this population of horses. Although the effect of management changes on the horse faecal microbiota has been investigated, limited work has been conducted to investigate changes in faecal microbiota structure and function in the periparturient period. The objectives of the current study were to investigate temporal stability of the faecal microbiota and volatile organic compounds (VOCs) of the faecal metabolome in periparturient mares. Methods Faecal samples were collected weekly from five pregnant mares from 3 weeks pre-foaling to 7 weeks post-foaling. The microbiome data was generated by PCR amplification and sequencing of the V1–V2 regions of the bacterial 16S rRNA genes, while the VOC profile was characterised using headspace solid phase microextraction gas chromatography mass spectrometry. Results The mare faecal microbiota was relatively stable over the periparturient period and most variation was associated with individual mares. A small number of operational taxonomic units were found to be significantly differentially abundant between samples collected before and after foaling. A total of 98 VOCs were identified. The total number of VOCs did not vary significantly between individual mares, weeks of sample collection and feeds available to the mares. Three VOCs (decane, 2-pentylfuran, and oct-2-ene) showed significant increase overtime on linear mixed effects modelling analysis. These results suggest that the mare faecal microbiota is structurally and functionally stable during the periparturient period. The findings also suggest that if changes in the gut microbiota are related to development of colic postpartum, altered risk may be due to inherent differences between individual mares. VOCs offer a cost-effective means of looking at the functional changes in the microbiome and warrant further investigation in mares at risk of colic.
Salem et al. (2019) A longitudinal study of the faecal microbiome and metabolome of periparturient mares. PeerJ 7:e6687 https://doi.org/10.7717/peerj.6687
Density of intertidal barnacles along their full elevational range of distribution conforms to the abundant-centre hypothesis
The abundant-centre hypothesis (ACH) predicts that the density of a species should peak at its distribution centre and decrease similarly towards distribution margins. The ACH has been deduced from a theory that postulates that environmental conditions should be most favourable for a species at the centre of its distribution. This idealised density pattern, however, has been supported by limited field studies, as natural patterns are often more complex. It is thus of interest to examine under what conditions compliance with the ACH could be favoured. Such conditions could be smooth environmental gradients with limited habitat patchiness throughout the distribution range of a species. Thus, we tested the ACH by measuring the density of an intertidal barnacle (Semibalanus balanoides) across its full vertical distribution range (from low to high elevations) on a rocky shore with similar substrate properties across elevations. To do a reliable test, we surveyed eight elevation zones applying an equal sampling effort in each zone. Average barnacle density conformed to the ACH, as it peaked at the middle of the vertical distribution range of this species. The same underlying theory predicts a similar unimodal pattern for maximum body size, but this trait was decoupled from density, as maximum barnacle size increased from low to high elevations. Overall, although the ACH is not a universal predictive tool as once envisioned, it may predict some cases well, as shown by this study. Therefore, the ACH should not be discarded completely, and its domain of application should be further evaluated.
Scrosati et al. (2019) Density of intertidal barnacles along their full elevational range of distribution conforms to the abundant-centre hypothesis. PeerJ 7:e6719 https://doi.org/10.7717/peerj.6719