Magnetic separation of algae genetically modified for increased intracellular iron uptake

a b s t r a c t Algae were investigated in the past as a potential source of biofuel and other useful chemical derivatives. Magnetic separation of algae by iron oxide nanoparticle binding to cells has been proposed by others for dewatering of cellular mass prior to lipid extraction. We have investigated feasibility of magnetic separation based on the presence of natural iron stores in the cell, such as the ferritin in Auxenochlorella protothecoides (A. protothecoides) strains. The A. protothecoides cell constructs were tested for inserted genes and for increased intracellular iron concentration by inductively coupled plasma atomic absorption (ICP-AA). They were grown in Sueoka's modified high salt media with added vitamin B1 and increasing concentration of soluble iron compound (FeCl 3 EDTA, from 1 Â to 8 Â compared to baseline). The cell magnetic separation conditions were tested using a thin rectangular flow channel pressed against interpolar gaps of a permanent magnet forming a separation system of a well-defined fluid flow and magnetic fringing field geometry (up to 2.2 T and 1000 T/m) dubbed "magnetic deposition microscopy", or MDM. The presence of magnetic cells in suspension was detected by formation of characteristic deposition bands at the edges of the magnet interpolar gaps, amenable to optical scanning and microscopic examination. The results demonstrated increasing cellular Fe uptake with increasing Fe concentration in the culture media in wild type strain and in selected genetically-modified constructs, leading to magnetic separation without magnetic particle binding. The throughput in this study is not sufficient for an economical scale harvest

Substance P induces gastric mucosal protection at supraspinal level via increasing the level of endomorphin-2 in rats.

The aim of the present study was to analyze the potential role of substance P (SP) in gastric mucosal defense and to clarify the receptors and mechanisms that may be involved in it. Gastric ulceration was induced by oral administration of acidified ethanol in male Wistar rats. Mucosal levels of calcitonin gene-related peptide (CGRP) and somatostatin were determined by radioimmunoassay. For analysis of gastric motor activity the rubber balloon method was used. We found that central (intracerebroventricular) injection of SP (9.3-74pmol) dose-dependently inhibited the formation of ethanol-induced ulcers, while intravenously injected SP (0.37-7.4nmol/kg) had no effect. The mucosal protective effect of SP was inhibited by pretreatment with neurokinin 1-, neurokinin 2-, neurokinin 3- and mu-opioid receptor antagonists, while delta- and kappa-opioid receptor antagonists had no effect. Endomorphin-2 antiserum also antagonized the SP-induced mucosal protection. In the gastroprotective dose range SP failed to influence the gastric motor activity. Inhibition of muscarinic cholinergic receptors, or the synthesis of nitric oxide or prostaglandins significantly reduced the effect of SP. In addition, centrally injected SP reversed the ethanol-induced reduction of gastric mucosal CGRP content. It can be concluded, that SP may induce gastric mucosal protection initiated centrally. Its protective effect is likely to be mediated by endomorphin-2, and vagal nerve may convey the centrally initiated protection to the periphery, where both prostaglandins, nitric oxide and CGRP are involved in mediating this effect

Clinical analysis of germline copy number variation in DMD using a non-conjugate hierarchical Bayesian model

Abstract Background Detection of copy number variants (CNVs) is an important aspect of clinical testing for several disorders, including Duchenne muscular dystrophy, and is often performed using multiplex ligation-dependent probe amplification (MLPA). However, since many genetic carrier screens depend instead on next-generation sequencing (NGS) for wider discovery of small variants, they often do not include CNV analysis. Moreover, most computational techniques developed to detect CNVs from exome sequencing data are not suitable for carrier screening, as they require matched normals, very large cohorts, or extensive gene panels. Methods We present a computational software package, geneCNV (http://github.com/vkozareva/geneCNV), which can identify exon-level CNVs using exome sequencing data from only a few genes. The tool relies on a hierarchical parametric model trained on a small cohort of reference samples. Results Using geneCNV, we accurately inferred heterozygous CNVs in the DMD gene across a cohort of 15 test subjects. These results were validated against MLPA, the current standard for clinical CNV analysis in DMD. We also benchmarked the tool’s performance against other computational techniques and found comparable or improved CNV detection in DMD using data from panels ranging from 4,000 genes to as few as 8 genes. Conclusions geneCNV allows for the creation of cost-effective screening panels by allowing NGS sequencing approaches to generate results equivalent to bespoke genotyping assays like MLPA. By using a parametric model to detect CNVs, it also fulfills regulatory requirements to define a reference range for a genetic test. It is freely available and can be incorporated into any Illumina sequencing pipeline to create clinical assays for detection of exon duplications and deletions

Selective cyclo-oxygenase-2 inhibitors and their influence on the protective effect of a mild irritant in the rat stomach

1. The effects of the non-selective cyclo-oxygenase (COX) inhibitor indomethacin and the selective COX-2 inhibitors, N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulphonamide (NS-398), 5-methanesulphonamido-6-(2,4-difluorothio-phenyl)-1-indanone (L-745,337) and 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furanone (DFU), on the protection induced by the mild irritant 20% ethanol were investigated in the rat stomach. 2. Instillation of 20% ethanol (1 ml, p.o.) effectively protected against gastric mucosal injury induced by subsequent instillation of 70% or 96% ethanol (1 ml, p.o.). 3. Oral administration of indomethacin (1.25–20 mg kg(−1)) dose-dependently counteracted the protective effect of 20% ethanol (ID(50): 3.5 mg kg(−1)). 4. Likewise, NS-398 (0.1–1 mg kg(−1)), L-745,337 (0.2–2 mg kg(−1)) and DFU (0.02–0.2 mg kg(−1)) inhibited the protective effect of 20% ethanol in a dose-dependent manner with ID(50) values of 0.3 mg kg(−1), 0.4 mg kg(−1) and 0.06 mg kg(−1), respectively. 5. Inhibition of mild irritant-induced protection was also found when NS-398 (1 mg kg(−1)) was administered s.c. or when 96% ethanol was used to damage the mucosa. 6. Pretreatment with 16,16-dimethyl-prostaglandin (PG)E(2) at 4 ng kg(−1), a dose that did not protect against ethanol (70%)-induced mucosal damage when given alone, completely reversed the effect of the selective COX-2 inhibitors on the mild irritant-induced protection. 7. Pretreatment with dexamethasone (3 mg kg(−1), 24 and 2 h before instillation of 20% ethanol) did not affect the protective activity of the mild irritant, indicating that enzyme induction is not involved. 8. Indomethacin (20 mg kg(−1), p.o.) did not prevent the protection conferred by sodium salicylate (100 mg kg(−1)), dimercaprol (30 μg kg(−1)), iodoacetamide (50 mg kg(−1)) and lithium (20 mg kg(−1)). Likewise, the protective effect of these agents was not counteracted by NS-398 (1 mg kg(−1), p.o.). 9. Whereas indomethacin (20 mg kg(−1), p.o.) near-maximally inhibited gastric mucosal formation of PGE(2), 6-keto-PGF(1α) and thromboxane (TX) B(2) as well as platelet TXB2 release, the selective COX-2 inhibitors were ineffective. 10. The findings show that selective COX-2 inhibitors, although lacking in ulcerogenic activity, prevent the protection conferred by a mild irritant. Prostaglandis generated by a constitutive COX-2 could thus contribute to physiological functions involved in gastric homeostasis, although at present a non-COX-2-related mechanism underlying the effect of the selective COX-2 inhibitors tested on mild irritant-induced protection cannot be completely excluded

Role of endogenous gastrin in gastroprotection

Gastrin has a potent influence on gastric acid secretion and mucosal growth but its role in mucosal integrity has been little studied. This study investigated in rats whether gastrin protects the gastric mucosa against the damage by 100% ethanol and what are the possible mechanisms of this protection. Exogenous gastrin-17 (0.6–5.0 pmol/kg) injected subcutaneously (s.c.) reduced dose dependently ethanol-induced mucosal damage and the dose decreasing the ethanol lesions by 50% was about 1.8 pmol/kg. The protection afforded by gastrin-17 was accompanied by a dose-dependent increase in gastric blood flow and these effects were almost completely abolished by the pretreatment with specific CCK B (L-365,260) but not CCK A receptor antagonist (loxiglumide). Endogenous gastrin released by intragastric (i.g.) peptone meal or s.c. injection of gastrin-releasing peptide prevented the formation of acute ethanol-induced lesions and these effects were also abolished by the pretreatment with L-365,260 but not by loxiglumide. The inhibition of nitric oxide (NO) synthase, by N G-nitro- l-arginine methyl ester almost completely eliminated both the protective and hyperemic effects of gastrin-17 and the addition of l-arginine (but not d-arginine) to N G-nitro- l-arginine-methyl ester restored, in part, these effects of gastrin-17. Deactivation of sensory nerves with capsaicin did not influence the protective or hyperemic effects of gastrin-17. We conclude that both exogenous and endogenous gastrin exert its protective activity against ethanol damage of gastric mucosa and this effect is mediated through the interaction with specific CCK B receptors and arginine-NO pathway, but does not involve sensory nerves