Using DNA barcoding to assess Caribbean reef fish biodiversity: expanding taxonomic and geographic coverage

This paper represents a DNA barcode data release for 3,400 specimens representing 521 species of fishes from 6 areas across the Caribbean and western central Atlantic regions (FAO Region 31). Merged with our prior published data, the combined efforts result in 3,964 specimens representing 572 species of marine fishes and constitute one of the most comprehensive DNA barcoding "coverages" for a region reported to date. The barcode data are providing new insights into Caribbean shorefish diversity, allowing for more and more accurate DNA-based identifications of larvae, juveniles, and unknown specimens. Examples are given correcting previous work that was erroneous due to database incompleteness.     

The assessment of post-vasectomy pain in mice using behaviour and the Mouse Grimace Scale

Background

Current behaviour-based pain assessments for laboratory rodents have significant limitations. Assessment of facial expression changes, as a novel means of pain scoring, may overcome some of these limitations. The Mouse Grimace Scale appears to offer a means of assessing post-operative pain in mice that is as effective as manual behavioural-based scoring, without the limitations of such schemes. Effective assessment of post-operative pain is not only critical for animal welfare, but also the validity of science using animal models.

Methodology/Principal Findings

This study compared changes in behaviour assessed using both an automated system (“HomeCageScan”) and using manual analysis with changes in facial expressions assessed using the Mouse Grimace Scale (MGS). Mice (n = 6/group) were assessed before and after surgery (scrotal approach vasectomy) and either received saline, meloxicam or bupivacaine. Both the MGS and manual scoring of pain behaviours identified clear differences between the pre and post surgery periods and between those animals receiving analgesia (20 mg/kg meloxicam or 5 mg/kg bupivacaine) or saline post-operatively. Both of these assessments were highly correlated with those showing high MGS scores also exhibiting high frequencies of pain behaviours. Automated behavioural analysis in contrast was only able to detect differences between the pre and post surgery periods.

Conclusions

In conclusion, both the Mouse Grimace Scale and manual scoring of pain behaviours are assessing the presence of post-surgical pain, whereas automated behavioural analysis could be detecting surgical stress and/or post-surgical pain. This study suggests that the Mouse Grimace Scale could prove to be a quick and easy means of assessing post-surgical pain, and the efficacy of analgesic treatment in mice that overcomes some of the limitations of behaviour-based assessment schemes.     

Use of Postmortem Human Dura Mater and Scalp for Deriving Human Fibroblast Cultures

Fibroblasts can be collected from deceased individuals, grown in culture, reprogrammed into induced pluripotent stem cells (iPSCs), and then differentiated into a multitude of cell types, including neurons. Past studies have generated iPSCs from somatic cell biopsies from either animal or human subjects. Previously, fibroblasts have only been successfully cultured from postmortem human skin in two studies. Here we present data on fibroblast cell cultures generated from 146 scalp and/or 53 dura mater samples from 146 postmortem human brain donors. In our overall sample, the odds of successful dural culture was almost two-fold compared with scalp (OR = 1.95, 95% CI: [1.01, 3.9], p = 0.047). Using a paired design within subjects for whom both tissues were available for culture (n = 53), the odds of success for culture in dura was 16-fold as compared to scalp (OR = 16.0, 95% CI: [2.1–120.6], p = 0.0007). Unattended death, tissue donation source, longer postmortem interval (PMI), and higher body mass index (BMI) were associated with unsuccessful culture in scalp (all p<0.05), but not in dura. While scalp cells proliferated more and grew more rapidly than dura cells [F (1, 46) = 12.94, p<0.008], both tissues could be generated and maintained as fibroblast cell lines. Using a random sample of four cases, we found that both postmortem scalp and dura could be successfully reprogrammed into iPSC lines. Our study demonstrates that postmortem dura mater, and to a lesser extent, scalp, are viable sources of living fibroblasts for culture that can be used to generate iPSCs. These tissues may be accessible through existing brain tissue collections, which is critical for studying disorders such as neuropsychiatric diseases.     

Ecotin-like serine peptidase inhibitor ISP1 of Leishmania major plays a role in flagellar pocket dynamics and promastigote differentiation

Leishmania ISPs are ecotin-like natural peptide inhibitors of trypsin-family serine peptidases, enzymes that are absent from the Leishmania genome. This led to the proposal that ISPs inhibit host serine peptidases and we have recently shown that ISP2 inhibits neutrophil elastase, thereby enhancing parasite survival in murine macrophages. In this study we show that ISP1 has less serine peptidase inhibitory activity than ISP2, and in promastigotes both are generally located in the cytosol and along the flagellum. However, in haptomonad promastigotes there is a prominent accumulation of ISP1 and ISP2 in the hemidesmosome and for ISP2 on the cell surface. An L. major mutant deficient in all three ISP genes (Δisp1/2/3) was generated and compared with Δisp2/3 mutants to elucidate the physiological role of ISP1. In in vitro cultures, the Δisp1/2/3 mutant contained more haptomonad, nectomonad and leptomonad promastigotes with elongated flagella and reduced motility compared with Δisp2/3 populations, moreover it was characterized by very high levels of release of exosome-like vesicles from the flagellar pocket. These data suggest that ISP1 has a primary role in flagellar homeostasis, disruption of which affects differentiation and flagellar pocket dynamics.     

Kinesin-1 prevents capture of the oocyte meiotic spindle by the sperm aster

Centrioles are lost during oogenesis and inherited from the sperm at fertilization. In the zygote, the centrioles recruit pericentriolar proteins from the egg to form a mature centrosome that nucleates a sperm aster. The sperm aster then captures the female pronucleus to join the maternal and paternal genomes. Because fertilization occurs before completion of female meiosis, some mechanism must prevent capture of the meiotic spindle by the sperm aster. Here we show that in wild-type Caenorhabditis elegans zygotes, maternal pericentriolar proteins are not recruited to the sperm centrioles until after completion of meiosis. Depletion of kinesin-1 heavy chain or its binding partner resulted in premature centrosome maturation during meiosis and growth of a sperm aster that could capture the oocyte meiotic spindle. Kinesin prevents recruitment of pericentriolar proteins by coating the sperm DNA and centrioles and thus prevents triploidy by a nonmotor mechanism.     

Formaldehyde-induced genome instability is suppressed by an XPF-dependent pathway

Formaldehyde is a reactive chemical that is commonly used in the production of industrial, laboratory, household, and cosmetic products. The causal association between formaldehyde exposure and increased incidence of cancer led the International Agency for Research on Cancer to classify formaldehyde as a carcinogen. Formaldehyde-induced DNA-protein crosslinks (DPCs) elicit responses involving nucleotide excision repair (NER) and homologous recombination (HR) repair pathways; however, little is known about the cellular and genetic changes that subsequently lead to formaldehyde-induced genotoxic and cytotoxic effects. Herein, investigations of genes that modulate the cytotoxic effects of formaldehyde exposure revealed that of five NER-deficient Chinese Hamster Ovary (CHO) cell lines tested, XPF- and ERCC1-deficient cells were most sensitive to formaldehyde treatment as compared to wild-type cells. Cell cycle analyses revealed that formaldehyde-treated XPF-deficient cells exhibited an immediate G2/M arrest that was associated with altered cell ploidy and apoptosis. Additionally, an elevated number of DNA double-strand breaks (DSBs), chromosomal breaks and radial formation were also observed in XPF-deficient cells following formaldehyde treatment. Formaldehyde-induced DSBs occurred in a replication-dependent, but an XPF-independent manner. However, delayed DSB repair was observed in the absence of XPF function. Collectively, our findings highlight the role of an XPF-dependent pathway in mitigating the sensitivity to formaldehyde-induced DNA damage as evidenced by the increased genomic instability and reduced cell viability in an XPF-deficient background. In addition, centrosome and microtubule abnormalities, as well as enlarged nuclei, caused by formaldehyde exposure are demonstrated in a repair-proficient cell line.     

Rosaceae conserved orthologous sequences marker polymorphism in sweet cherry germplasm and construction of a SNP-based map

The Rosaceae Conserved Orthologous Set (RosCOS) provides a gene-based genome-wide set of markers that have been used in comparative analyses of peach (Prunus persica), apple (Malus × domestica), and strawberry (Fragaria spp.). In order to extend the use of these RosCOS to sweet cherry (Prunus avium L.), we identified markers that are polymorphic in breeding germplasm. Ninety-five percent (595/627) of previously designed RosCOS primer pairs amplified a product in six sweet cherry cultivars predicted to represent the range of genetic diversity in breeding germplasm. A total of 45% (282/627) RosCOS were polymorphic among the six cultivars, and allele number ranged from 2 to 6, with a genome-wide mean of 2.35. A subset of 92 genome-wide single nucleotide polymorphisms (SNPs) corresponding to 76 RosCOS was analyzed in 36 founder accessions and progeny. The expected and observed heterozygosity suggested that 83% of the RosCOS were in Hardy–Weinberg equilibrium, implying that most RosCOS behave as neutral markers. Principal coordinate analysis (PCO) identified one wild accession and two Spanish landraces that clustered differently from the other accessions. The relatively high number of unique alleles found in the three differentially clustered selections suggested that their use as parents has potential to increase the genetic diversity in future US-bred cultivars. Of the 92 RosCOS SNPs, 81 SNPs that represented 68 genome-wide RosCOS segregated in four mapping populations. These RosCOS were mapped in four F₁ populations, thereby greatly improving the genetic linkage map of sweet cherry.     

Galanthamine production by Leucojum aestivum L. shoot culture in a modified bubble column bioreactor with internal sections

Shoot culture of summer snowflake (Leucojum aestivum L.) was successfully cultivated in an advanced modified glass‐column bioreactor with internal sections for production of Amaryllidaceae alkaloids. The highest amounts of dry biomass (20.8 g/L) and galanthamine (1.7 mg/L) were achieved when shoots were cultured at 22°C and 18 L/(L·h) flow rate of inlet air. At these conditions, the L. aestivum shoot culture possessed mixotrophic‐type nutrition, synthesizing the highest amounts of chlorophyll (0.24 mg/g DW (dry weight) chlorophyll A and 0.13 mg/g DW chlorophyll B). The alkaloids extract of shoot biomass showed high acetylcholinesterase inhibitory activity (IC₅₀ = 4.6 mg). The gas chromatography–mass spectrometry (GC/MS) profiling of biosynthesized alkaloids revealed that galanthamine and related compounds were presented in higher extracellular proportions while lycorine and hemanthamine‐type compounds had higher intracellular proportions. The developed modified bubble‐column bioreactor with internal sections provided conditions ensuring the growth and galanthamine production by L. aestivum shoot culture.