DNA barcoding of nematodes using the MinION

Many nematode species are parasitic and threaten the health of plants and animals, including humans, on a global scale. Advances in DNA sequencing techniques have allowed for the rapid and accurate identification of many organisms including nematodes. However, the steps taken from sample collection in the field to molecular analysis and identification can take many days and depend on access to both immovable equipment and a specialized laboratory. Here, we present a protocol to genetically identify nematodes using 18S SSU rRNA sequencing using the MinION, a portable third generation sequencer, and proof that it is possible to perform all the molecular preparations on a fully portable molecular biology lab – the Bentolab. We show that both parasitic and free-living nematode species (Anisakis simplex, Panagrellus redivivus, Turbatrix aceti, and Caenorhabditis elegans) can be identified with a 96–100% accuracy compared to Sanger sequencing, requiring only 10–15 min of sequencing. This protocol is an essential first step toward genetically identifying nematodes in the field from complex natural environments (such as feces, soil, or marine sediments). This increased accessibility could in turn improve global information of nematode presence and distribution, aiding near-real-time global biomonitoring

The Application of PCR and STR DNA Profiling for the Identification of Haematoxylin Eosin Histological Slides in a Case of Sample Mix-Up Involving Synonymous Patients

A good laboratory practice ensures that biopsy material is correctly identified and associated with a given patient. Nevertheless, there are cases where the proof of origin of a tissue sample may be questioned. In this case study we have identified the source of cervical cancer glass slide sections stained with H/E, (hematoxylin eosin), after the request of a patient of Northern Greek origin who suspected sample mix-up when she coincidentally found out that a synonymous patient was examined for cervical cancer at the same time period in the same hospital in Greece. The patient was prepared to legally challenge the administrators of the downstream chemotherapeutic regimen. A combination of organic gradient clean up and silica membrane method was used for DNA isolation. Powerplex-16® system (Promega U.S.A) was used to generate complete DNA profiles from histological slides and the reference blood sample collected from the patient. Histochemical slides often yield inadequate STR profiles for successful DNA typing. Complete profiling in this case could be attributed to the adequate removal of stain and fixatives inhibitors and the isolation of good quality DNA for PCR or STR, protocols. Matching of histochemical slide DNA with patient blood DNA prevented legal action

Mortality and Effect on Growth of Artemia franciscana Exposed to Two Common Organic Pollutants

Acute toxicity and inhibition on growth of Artemia franciscana nauplii (Instar I-II) after exposure to the reference toxicants bisphenol a (BPA) and sodium dodecyl sulfate (SDS) were studied. LC50 values were calculated and differences in body growth were recorded after 24, 48, and 72 h of exposure to the toxicants. The results indicated that BPA had lower toxicity than SDS. Development of the nauplii was clearly influenced by duration of exposure. Growth inhibition was detected for both toxicants. Abnormal growth of the central eye of several Artemia nauplii after 72 h of exposure to BPA was also detected. Our results indicate that growth inhibition could be used as a valid endpoint for toxicity studies

The Inclusion of a Matrix Metalloproteinase-9 Responsive Sequence in Self-assembled Peptide-based Brain-Targeting Nanoparticles Improves the Efficiency of Nanoparticles Crossing the Blood-Brain Barrier at Elevated MMP-9 Levels

This study investigated whether the inclusion of a matrix metalloproteinase-9 (MMP-9) responsive sequence in self-assembled peptide-based brain-targeting nanoparticles (NPs) would enhance the blood-brain barrier (BBB) penetration when MMP-9 levels are elevated both in the brain and blood circulation. Brain-targeting peptides were conjugated at the N-terminus to MMP-9-responsive peptides, and these were conjugated at the N-terminus to lipid moiety (cholesteryl chloroformate or palmitic acid). Two constructs did not have MMP-9-responsive peptides. NPs were characterised for size, charge, critical micelle concentration, toxicity, blood compatibility, neural cell uptake, release profiles, and in vitro BBB permeability simulating normal or elevated MMP-9 levels. The inclusion of MMP-9-sensitive sequences did not improve the release of a model drug in the presence of active MMP-9 from NPs compared to distilled water. 19F NMR studies suggested the burial of MMP-9-sensitive sequences inside the NPs making them inaccessible to MMP-9. Only cholesterol-GGGCKAPETALC (responsive to MMP-9) NPs showed <5% haemolysis, <1 pg/mL release of IL-1β at 500 μg/mL from THP1 cells, with 70.75 ± 5.78% of NPs crossing the BBB at 24 h in presence of active MMP-9. In conclusion, brain-targeting NPs showed higher transport across the BBB model when MMP-9 levels were elevated and the brain-targeting ligand was responsive to MMP-9