A microfluidic competitive immuno-aggregation assay for high sensitivity cell secretome detection

We report a high-sensitivity cell secretome detection method using competitive immuno-aggregation and a micro-Coulter counter. A target cell secretome protein competes with anti-biotin-coated microparticles (MPs) to bind with a biotinylated antibody (Ab), causing decreased aggregation of the functionalized MPs and formation of a mixture of MPs and aggregates. In comparison, without the target cell secretome protein, more microparticles are functionalized, and more aggregates are formed. Thus, a decrease in the average volume of functionalized microparticles/aggregates indicates an increase in cell secretome concentration. This volume change is measured by the micro-Coulter counter, which is used to quantitatively estimate the cell secretome concentration. Vascular endothelial growth factor (VEGF), one of the key cell secretome proteins that regulate angiogenesis and vascular permeabilization, was used as the target protein to demonstrate the sensing principle. A standard calibration curve was generated by testing samples with various VEGF concentrations. A detection range from 0.01 ng/mL to 100.00 ng/mL was achieved. We further demonstrated the quantification of VEGF concentration in exogenous samples collected from the secretome of human mesenchymal stem cells (hMSCs) at different incubation times. The results from the assay agree well with the results of a parallel enzyme-linked immunoabsorbent assay (ELISA) test, indicating the specificity and reliability of the competitive immuno-aggregation assay. With its simple structure and easy sample preparation, this assay not only enables high sensitivity detection of VEGF but also can be readily extended to other types of cell secretome analysis as long as the specific Ab is known.     

Of feathers and fur: Differential pollinator roles of birds and small mammals in the grassland succulent Aloe peglerae

The pollination syndrome of the genus Aloe, with gaudy inflorescences and orange–red flowers, suggests bird pollination. However, the diversity of flowering phenologies and structures suggests that generalizations within the group are currently uninformative because few studies have addressed the role of multiple pollinator guilds, especially mammals. Aloe peglerae, endemic to montane grassland of the Magaliesberg Mountains, South Africa, is primarily bird pollinated, although small mammals are recorded nocturnal visitors. We compared the independent contributions of diurnal and nocturnal visitors, i.e. birds and small mammals respectively, to reproductive success by assigning 12 flowering A. peglerae plants to each of four pollinator selective exclusion treatments: (i) no‐visitors; (ii) nocturnal‐visitors; (iii) diurnal‐visitors; and (iv) all‐visitors (control). Birds alone contributed more to fruit set (1.2–2.6 times), average seed/fruit (1.0–1.8 times) and total seed production (1.1–2.8 times) than both the all visitors (birds and small mammals combined) and the small mammal visitors respectively. The exclusion of all visitors resulted in no fruit set, suggesting that A. peglerae requires pollinators to set seed. Germination trials over six weeks and viability testing with tetrazolium staining identified no significant differences among the three treatments that produced fruit and seed. Germination success was 90–97%, indicating that seed quality of small‐mammal and bird‐pollinated plants are similar. Aloe peglerae inflorescences were visited continuously throughout the day and night, with the Cape rock‐thrush (Monticola rupestris) and dark‐capped bulbul (Pycnonotus tricolor) being the most abundant pollinators, accounting for 68.2% and 13.7% of all visits respectively in the control treatment. Small mammals, i.e. Namaqua rock mouse (Micaelamys namaquensis) and eastern rock sengi (Elephantulus myurus), while not increasing reproductive success in the absence of birds, are suitable alternative pollinators. The presence of diurnal bird and nocturnal small mammal visitors provides a diversity of pollinators in this resilient pollination syndrome.     

Essential sulfhydryl groups of rat liver monoamine oxidase.

The inhibition by some thiol reagents of partly purified mitochondrial monoamine oxidase (MAO) (EC 1.4.3.4) from rat liver was studied, and the molar content of sulfhydryl groups in the enzyme determined. Sodium nitroprusside and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) inhibited the enzyme, apparently reversibly, while sodium arsenite was not inhibitory. Concentrations of the respective inhibitors causing 50% inhibition after 15 min of preincubation with the enzyme at pH 7.0 and 37 degrees C are 5.80 times 10(-4) M and 4.35 times 10(-5) M. The thiol compounds cysteine, dithiothreitol, and 2-mercaptoethanol did not inhibit MAO. The average number of sulfhydryl groups per mole of enzyme, determined by reaction with DTNB, increased from 3.6 +/- 0.2 freely reacting sulfhydryl groups (n = 4) to 18.4 to total sulfhydryl groups (n = 2) on denaturation with 8 M urea.     

An investigation of the specification of unequal cleavages in leech embryos.

Leech embryos develop via stereotyped cell divisions, many of which are unequal. The first division generates identifiable cells, blastomeres AB and CD, which normally follow distinct developmental pathways. When these two cells are dissociated and cultured in isolation, their fates remain distinct and are reminiscent of normal development, but their typical cleavage patterns are disrupted; cell AB undergoes relatively few cell divisions, giving rise to a variable number of macromeres and micromeres, while cell CD cleaves many times, usually forming a poorly organized set of macromeres, embryonic stem cells (teloblasts), and micromeres. We have investigated the hypothesis that the abnormal cleavage pattern of isolated CD blastomeres is due to removal of mechanical constraints normally imposed by cell AB. We find that when cell CD is constrained in vitro to mimic its in vivo shape, it cleaves more normally.     

Improved separation of bacteria with a dual flocculant hydrocol™ system

Summary Polyelectrolyte flocculants are inefficient at flocculating Z. mobilis and B. subtilis in a complex medium, when administered singly or when combined with an oppositely charged polymer. However, when polymer dosing is followed by bentonite addition, particularly when chitosan constitutes the soluble polymer, effective flocculation occurs. High broth clarities can be attributed to chitosan interaction with cells, extracellular polymer and bentonite.     

Spontaneous fibroblast‐derived pericyte recruitment in a human tissue‐engineered angiogenesis model in vitro

Cooperation between endothelial cells and pericytes is essential to the stabilization and maturation of blood microvessels. We developed a unique in vitro tissue‐engineered model to study angiogenesis. The human endothelialized reconstructed connective tissue model promotes the formation of a three‐dimensional branching network of capillary‐like tubes (CLT) with closed lumens. The purpose of this work was to investigate whether pericytes were spontaneously recruited around CLT in the model. We demonstrated that smooth muscle α‐actin (SMA)‐positive cells were found closely associated with PECAM‐1‐positive capillaries in the model. Twelve percent (±2.6) of SMA‐positive cells were detected along with 15% (±1.64) von Willebrand factor‐positive endothelial cells in the culture system after 31 days of in vitro maturation. Conversely, no SMA‐positive cells were detected in reconstructed connective tissues made solely of fibroblasts. Knowing that PDGF is a major factor in the recruitment of pericytes, we showed that blockade of the PDGFB receptor using the inhibitor AG1296 induced an overall 5, 2.6, and 2.4‐fold decrease in the SMA‐positive cells, von Willebrand factor‐positive cells, and number of capillaries, respectively. Using combinations of human GFP‐positive fibroblasts and endothelial cells, we demonstrated that pericytes were recruited from the fibroblast population in the model. In conclusion, our tissue‐engineered culture system promotes the spontaneous formation of a network of capillaries and the recruitment of pericytes derived from fibroblasts. Since pericytes are essential components of the blood microvasculature, this culture system is a powerful model to study angiogenesis and endothelial cell/pericyte interactions in vitro. J. Cell. Physiol. 227: 2130–2137, 2012. © 2011 Wiley Periodicals, Inc.