Multidirectional Time-Dependent Effect of Sinigrin and Allyl Isothiocyanate on Metabolic Parameters in Rats

Sinigrin (SIN) and allyl isothiocyanate (AITC) are compounds found in high concentrations in Brassica family vegetables, especially in Brussels sprouts. Recently, they have been used as a nutrition supplement for their preventive and medicinal effect on some types of cancer and other diseases. In this research, nutritional significance of parent glucosinolate sinigrin 50 μmol/kg b. w./day and its degradation product allyl isothiocyanate 25 μmol/kg b. w./day and 50 μmol/kg b. w./day was studied by the evaluation of their influence on some parameters of carbohydrate and lipid metabolism in an animal rat model in vivo after their single (4 h) and 2 weeks oral administration. Additionally, the aim of this trial was to evaluate the direct action of AITC on basal and epinephrine-induced lipolysis in isolated rat adipocytes at concentration 1 μM, 10 μM and 100 μM in vitro. Sole AITC after 4 h of its ingestion caused liver triacylglycerols increment at both doses and glycaemia only at the higher dose. Multiple SIN treatment showed its putative bioconversion into AITC. It was found that SIN and AITC multiple administration in the same way strongly disturbed lipid and carbohydrate homeostasis, increasing esterified and total cholesterol, free fatty acids and lowering tracylglycerols in the blood serum. Additionally, AITC at both doses elevated insulinaemia and liver glycogen enhancement. The in vitro experiment revealed that AITC potentiated basal lipolysis process at 10 μM, and had stimulatory effect on epinephrine action at 1 μM and 10 μM. The results of this study demonstrated that the effect of SIN and AITC is multidirectional, indicating its impact on many organs like liver as well as pancreas, intestine in vivo action and rat adipocytes in vitro. Whilst consumption of cruciferous vegetables at levels currently considered “normal” seems to be beneficial to human health, this data suggest that any large increase in intake could conceivably lead to undesirable effect. This effect is potentiated with time of action of the examined compounds, whose influence is rather adverse for the majority of metabolic pathways (liver steatosis at short duration and insulinaemia, cholesterolaemia at long time treatment). Beneficial action of AITC concerned intensified hydrolysis of TG in the blood serum with a simultaneous lipolysis in adipocytes

Microfluidics with fluid walls

Microfluidics has great potential, but the complexity of fabricating and operating devices has limited its use. Here we describe a method - Freestyle Fluidics - that overcomes many key limitations. In this method, liquids are confined by fluid (not solid) walls. Aqueous circuits with any 2D shape are printed in seconds on plastic or glass Petri dishes; then, interfacial forces pin liquids to substrates, and overlaying an immiscible liquid prevents evaporation. Confining fluid walls are pliant and resilient; they self-heal when liquids are pipetted through them. We drive flow through a wide range of circuits passively by manipulating surface tension and hydrostatic pressure, and actively using external pumps. Finally, we validate the technology with two challenging applications - triggering an inflammatory response in human cells and chemotaxis in bacterial biofilms. This approach provides a powerful and versatile alternative to traditional microfluidics.The complexity of fabricating and operating microfluidic devices limits their use. Walsh et al. describe a method in which circuits are printed as quickly and simply as writing with a pen, and liquids in them are confined by fluid instead of solid walls

Single cell–resolution western blotting

This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine

Real-time size modulation and synchronization of a microfluidic dropmaker with pulsed surface acoustic waves (SAW)

We show that a microfluidic flow focusing drop maker can be synchronized to a surface acoustic waves (SAW) triggered by an external electric signal. In this way droplet rate and volume can be controlled over a wide range of values in real time. Using SAW, the drop formation rate of a regularly operating water in oil drop maker without SAW can be increased by acoustically enforcing the drop pinch-off and thereby reducing the volume. Drop makers of square cross-sections (w = h = 30 µm, with width w and height h) that produce large drops of length l = 10 w can be triggered to produce drops as short as l ~ 2w, approaching the geometical limit l = w without changing the flow rates. Unlike devices that adjust drop size by changing the flow rates the acoustic dropmaker has very short transients allowing to adjust the size of every single drop. This allows us to produce custom made emulsions with a defined size distribution as demonstrated here not only for a monodisperse emulsion but also for binary emulsions with drops of alternating size. Moreover, we show that the robustness and monodispersity of our devices is enhanced compared to purely flow driven drop makers in the absence of acoustic synchronization