Virus removal from semen with a pinched flow fractionation microfluidic chip.

Nowadays pigs are bred with artificial insemination to reduce costs and transportation. To prevent the spread of diseases, it is important to test semen samples for viruses. Screening techniques applied are enzyme-linked immunosorbent assays and/or polymerase chain reaction, which are labor-intensive and expensive methods. In contrast to the current used screening techniques, it is possible to remove viruses physically from semen. However, existing methods for virus removal techniques have a low yield of spermatozoa. Therefore, we have developed a microfluidic chip that performs size-based separation of viruses and spermatozoa in boar semen samples, thereby having the potential to reduce the risk of disease spreading in the context of artificial insemination in the veterinary industry. As the head of a spermatozoon is at least twenty times larger than a virus particle, the particle size can be used to achieve separation, resulting in a semen sample with lower viral load and of higher quality. To achieve the size separation, our microfluidic device is based on pinched-flow fractionation. A model virus, cowpea chlorotic mottle virus, was used and spiked to porcine semen samples. With the proposed microfluidic chip and the optimized flow parameters, at least 84 ± 4% of the model viruses were removed from the semen. The remaining virus contamination is caused by the model virus adhering to spermatozoa instead of the separation technique. The spermatozoa recovery was 86 ± 6%, which is an enormous improvement in yield compared to existing virus removal techniques.

Polymorphic assembly of virus-capsid proteins around DNA and the cellular uptake of the resulting particles.

Virus-like particles (VLPs), i.e. molecular assemblies that resemble the geometry and organization of viruses, are promising platforms for therapeutics and imaging. Understanding the assembly and cellular uptake pathways of VLPs can contribute to the development of new antiviral drugs and new virus-based materials for the delivery of drugs or nucleic acid-based therapies. Here we report the assembly of capsid proteins of the cowpea chlorotic mottle virus (CCMV) around DNA into defined structures at neutral pH. Depending on the type of DNA used, we are able to create spherical structures of various diameters and rods of various lengths. In order to determine the shape dependency, the cellular uptake routes and intracellular positioning of these formed polymorphic VLPs in RAW264.7, HeLa and HEK 293 cells are evaluated using flow cytometry analysis with specific chemical inhibitors for different uptake routes. We observed particular uptake routes for the various CCMV-based nanostructures, but the experiments point to clathrin-mediated endocytosis as the major route for cell entry for the studied VLPs. Confocal microscopy reveals that the formed VLPs enter the cells, with clear colocalization in the endosomes. The obtained results provide insight in the cargo dependent VLP morphology and increase the understanding of shape dependent uptake into cells, which is relevant in the design of new virus-based structures with applications in drug and gene delivery.

Effects of calcium-fortified ice cream on markers of bone health.

UNLABELLED: Premenopausal women with low calcium intakes consumed calcium-fortified ice cream daily for 28 days. Bone markers, NTX, CTX and PTH decreased significantly by 7 days, with some evidence of a calcium dose-dependent effect. Bone marker responses were observed within 1 h of consuming ice cream. Body weight remained constant over 28 days. INTRODUCTION: Dietary calcium is important for lifelong bone health. Milk is a good source of bioavailable calcium, but consumption has declined among young adults. The aims were to determine whether calcium-fortified ice cream, a palatable source of calcium, produces significant, sustainable changes in bone turnover markers and parathyroid hormone (PTH) in premenopausal women with calcium intake below recommended UK levels. METHODS: Eighty women, ages 20-39 years (calcium intake <750 mg/day) were randomised to consume lower saturated fat/sugar ice cream containing 96, 244, 459 or 676 mg calcium daily for 28 days. Urinary NTX/Cr, serum CTX, PINP, 1,25D and PTH were measured (baseline, days 1, 7 and 28). Acute changes in CTX and PTH were measured over 5 h (n = 29 women). RESULTS: There were significant mean decreases by 7 days in NTX/Cr, CTX, PTH and 1,25D and increases in PINP (one sample t tests), with a significant dose-dependent effect on CTX analysis of covariance. Only CTX remained suppressed at 28 days. Serum CTX and PTH decreased within 1 h. Body weight did not change significantly between baseline and 28 days. CONCLUSIONS: Daily consumption of calcium-fortified ice cream by premenopausal women may significantly reduce levels of the bone resorption marker serum CTX, without stimulating weight gain. The ice cream could be incorporated into the diet to replace low-calcium snacks and thus help individuals with habitually low calcium intakes to meet recommended intakes. The 244 mg calcium preparation would provide more than a quarter of the UK daily recommended nutrient intake for premenopausal women.

Inhibition of nitric oxide synthase by nasal decongestants.

The nasal decongestants oxymetazoline and xylometazoline are frequently used in the topical treatment of rhinitis and sinusitis. As nitric oxide (NO) is thought to play a role in inflammation of the upper respiratory tract, the aim of this study was to examine the in vitro effects of these compounds on the activity and the expression of NO producing enzymes, including the inducible form of NO synthase (iNOS) and the constitutive isoform of NO synthase (cNOS). Experiments concerning the effects of both compounds on enzymatic activity and enzyme induction of iNOS were performed in a lipopolysaccharide (LPS) induced rat alveolar macrophage cell line (NR8383) using the Griess assay and the 3H-citrulline assay respectively. The effects on cNOS were examined in fresh rat synaptosomes using the 3H-citrulline assay. The direct scavenging properties of both compounds were investigated using a amperometric NO sensor. Oxymetazoline and xylometazoline were shown to have a dose dependent inhibitory effect on total iNOS activity indicated by nitrite/nitrate formation in the Griess assay. This effect was found to be due to an inhibition of induction of the enzyme rather than inhibition of the enzyme activity, as was investigated in two separate experiments using the 3H-citrulline assay. Inhibition of cNOS was moderate and in the same order of magnitude as the inhibition of enzymatic iNOS activity. Direct scavenging of NO could not be detected. As constitutive nitric oxide synthase activity is thought to serve beneficial physiological functions, and exaggerated inducible nitric oxide synthase activity may cause exacerbation of the inflammatory process, pharmacological treatment influencing the nitric oxide generating system should focus on inhibition of inducible nitric oxide synthase alone. The specific characteristics of these decongestants in vitro suggests suitability for this application and may indicate an additional beneficial effect in the treatment of upper respiratory tract inflammation.