Capillarity Guided Patterning of Microliquids.

Soft lithography and other techniques have been developed to investigate biological and chemical phenomena as an alternative to photolithography-based patterning methods that have compatibility problems. Here, a simple approach for nonlithographic patterning of liquids and gels inside microchannels is described. Using a design that incorporates strategically placed microstructures inside the channel, microliquids or gels can be spontaneously trapped and patterned when the channel is drained. The ability to form microscale patterns inside microfluidic channels using simple fluid drain motion offers many advantages. This method is geometrically analyzed based on hydrodynamics and verified with simulation and experiments. Various materials (i.e., water, hydrogels, and other liquids) are successfully patterned with complex shapes that are isolated from each other. Multiple cell types are patterned within the gels. Capillarity guided patterning (CGP) is fast, simple, and robust. It is not limited by pattern shape, size, cell type, and material. In a simple three-step process, a 3D cancer model that mimics cell-cell and cell-extracellular matrix interactions is engineered. The simplicity and robustness of the CGP will be attractive for developing novel in vitro models of organ-on-a-chip and other biological experimental platforms amenable to long-term observation of dynamic events using advanced imaging and analytical techniques.

Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the "biological glue." We take advantage of hydroxyproline (Hyp)-based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm(2) . Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable.

Predicting motor gains with home-based telerehabilitation after stroke.

INTRODUCTION: Telerehabilitation (TR) may be useful for rehabilitation therapy after stroke. However, stroke is a heterogeneous condition, and not all patients can be expected to derive the same benefit from TR, underscoring the need to identify predictors of response to TR. METHODS: A prior trial provided patients with 6 weeks of intensive rehabilitation therapy targeting arm movement, randomly assigned to be provided in the home via TR (current focus) or in clinic. Eligible patients had moderate arm motor deficits and were in the subacute-chronic stage post stroke. Behavioral gains were measured as change in the arm motor Fugl-Meyer score from baseline to 30 days post therapy. To delineate predictors of TR response, multivariable linear regression was performed, advancing the most significant predictor from each of eight categories: patient demographics, stroke characteristics, medical history, rehabilitation therapy outside of study procedures, motivation, sensorimotor impairment, cognitive/affective deficits, and functional status. RESULTS: The primary focus was on patients starting TR >90 days post stroke onset (n = 44), among whom female sex, less spasticity, and less visual field defects predicted greater motor gains. This model explained 39.3% of the variance in treatment-related gains. In secondary analysis that also included TR patients enrolled ≤90 days post stroke (total n = 59), only female sex was a predictor of treatment gains. A separate secondary analysis examined patients >90 days post stroke (n = 34) randomized to in-clinic therapy, among whom starting therapy earlier post stroke and less ataxia predicted greater motor gains. DISCUSSION: Response to TR varies across patients, emphasizing the need to identify characteristics that predict treatment-related behavioral gain. The current study highlights factors that might be important to patient selection for home-based TR after stroke.

Regulation of yeast Yak1 kinase by PKA and autophosphorylation-dependent 14-3-3 binding.

Yak1 is a member of an evolutionarily conserved family of Ser/Thr protein kinases known as dual-specificity Tyr phosphorylation-regulated kinases (DYRKs). Yak1 was originally identified as a growth antagonist, which functions downstream of Ras/PKA signalling pathway. It has been known that Yak1 is phosphorylated by PKA in vitro and is translocated to the nucleus upon nutrient deprivation. However, the regulatory mechanisms for Yak1 activity and localization are largely unknown. In the present study, we investigated the role of PKA and Bmh1, a yeast 14-3-3 protein, in regulation of Yak1. We demonstrate that PKA-dependent phosphorylation of Yak1 on Ser295 and two minor sites inhibits nuclear localization of Yak1. We also show that intramolecular autophosphorylation on at least four Ser/Thr residues in the non-catalytic N-terminal domain is required for full kinase activity of Yak1. The most potent autophosphorylation site, Thr335, plays an essential role for Bmh1 binding in collaboration with a yet unidentified second binding site in the N-terminal domain. Bmh1 binding decreases the catalytic activity of Yak1 without affecting its subcellular localization. Since the binding of 14-3-3 proteins to Yak1 coincides with PKA activity, such regulatory mechanisms might allow cytoplasmic retention of an inactive form of Yak1 under high glucose conditions.

Overproduction of recombinant E. coli malate synthase enhances Chlamydomonas reinhardtii biomass by upregulating heterotrophic metabolism.

High uptake of malate and efficient distribution of intracellular malate to organelles contributed to biomass increase, reducing maintenance energy. In this study, transgenic Chlamydomonas reinhardtii was developed that stably expresses malate synthase in the chloroplast. The strains under glyoxylate treatment showed 19% more increase in microalgal biomass than wild-type. By RNA analysis, transcript levels of malate dehydrogenase (MDH4) and acetyl-CoA synthetase (ACS3), isocitrate lyase (ICL1) and malate synthase (MAS1), were significantly more expressed (17%, 42%, 24%, and 18% respectively), which was consistent with reported heterotrophic metabolism flux analysis with the objective function maximizing biomass. Photosynthetic Fv/Fm was slightly reduced. A more meticulous analysis is necessary, but, in the transgenic microalgae with malate synthase overexpression, the metabolism is likely to more rely on heterotrophic energy production via TCA cycle and glyoxylate shunt than on photosynthesis, resulting in the increase in microalgal biomass.

Vibration-induced stress priming during seed culture increases microalgal biomass in high shear field-cultivation.

Vibrational wave treatment has been used to increase proliferation of microalgae. When directly applied at large scale, however, turbulence can offset positive effects of vibration on microalgae proliferation. Moreover, severe hydrodynamic shear fields in the bioreactor decrease cell viability that detrimentally influence maximum yieldable biomass. In this study, vibration pretreatment (between 10-30 Hz and 0.15-0.45 G) was used to prime the cells for enhanced biomass. When exposed to 10 Hz at 0.15 G for 72 h and inoculated in baffled flasks of large shear fields (0.292 Pa for the average wall shear force (aveWSF) and 184 s-1 for the average shear strain rate (aveSSR)), microalgae showed 27% increase in biomass as well as 39% increase in corresponding amount of heterologous protein (i.e. GFP-3HA). Our results show that stress primed microalgae with vibrations can lead to improved proliferation that results in increased biomass production at industrial scale bioprocesses.

Microstructure guided multi-scale liquid patterning on an open surface.

Liquid patterning is a quintessential aspect in cell-based screening. While there are a variety of methods to handle microliquids utilizing surface treatments, complex microfluidic systems, and automated dispensing, most of the stated methods are both expensive and difficult to implement. Here, we present a fast multi-scale microliquid-patterning method on an open surface using embossed microstructures without surface modification. Arrays of micropillars can trap microliquids when a bulk drop is swept by an elastic sweeper on polystyrene (PS) substrates. The patterning mechanism on a basic form of a 2 × 2 rectangular array of circular pillars is analyzed theoretically and verified with experiments. Nanoliter-to-microliter volumes of liquids are patterned into various shapes by arranging the pillars based on the analysis. Furthermore, an array of geometrically modified pillars can capture approximately 8000 droplets on a large substrate (55 mm × 55 mm) in one step. Given the simplistic method of wipe patterning, the proposed platform can be utilized in both manual benchtop and automated settings. We will provide proof of concept experiments of single colony isolation using nanoliter-scale liquid patterning and of human angiogenic vessel formation using sequential patterning of microliter-scale liquids.

Microfluidic perfusion bioreactor for optimization of microalgal lipid productivity.

Nutrient deprivation in a batch process induces microbes to produce secondary metabolites while drastically constraining cellular growth. A microfluidic continuous perfusion system was designed and tested to culture microalgae, Chlamydomonas reinhardtii, under constant nutrient concentration slightly lower than normal condition. When cultured in 7.5%/7.5% of NH4+/PO42-, C. reinhardtii showed a 2.4-fold increase in TAG production with a 3.5-fold increase in biomass compared to level obtained under an only NH4+ depleted condition. The microfluidic continuous perfusion bioreactor with steady continuous nutrient flow can be used to optimize conditions for enhancing secondary metabolite production and increasing microbial biomass.

Rapid large area fabrication of multiscale through-hole membranes.

There are many proposed mechanisms by which single cells can be trapped; among them is the through-hole membrane for the characterization of individual microorganisms. Due to the small scale of the fabricated pores, the construction of through-hole membranes on a large scale and with relatively large areas faces many difficulties. This paper describes novel fabrication methods for a large-area, freestanding micro/nano through-hole membrane constructed from versatile membrane materials using through-hole membranes on a microfluidic chip (THMMC). This process can rapidly (<20 min) fabricate membranes with high fidelity multiscale hole size without residual layers. The through-hole site was easily customizable from the micro to the nanoscale, with a low or high aspect ratio giving rise to reliable membranes. Also, the rigidity and biocompatibility of the through-hole membrane are easily tunable by simple injection of versatile membrane materials to obtain a large area (up to 3600 mm2). Membranes produced in this manner were then applied as a proof of concept for the isolation, cultivation, and quantification of individual micro-algal cells for selection with respect to the growth rate, while controlling the quorum sensing mediated metabolic and proliferative changes.

Effect of topography of an electrospun nanofiber on modulation of activity of primary rat astrocytes.

Several biomaterials for neural tissue engineering have recently been proposed for regeneration of damaged tissue and promotion of axonal guidance following CNS injury. When implanted into damaged nerve tissue, biomaterials should favorably induce cell infiltration and axonal guiding while suppressing inflammation. Nanofiber scaffolds are regarded as adequate materials to meet the above requirements; however, most studies of these materials conducted to date have targeted neuronal cells, not glial cells, despite their important function in the injured CNS. In this study, an electrospun nanofibrous scaffold of polycaprolactone (PCL) was investigated with respect to its topographic effects on astrocyte behavior and expression of GFAP. The results revealed that the PCL nanofiber topograghy promoted adhesion, but GFAP expression was down-regulated, leading to reduced astrocytes activity. Taken together, these results indicate that the topographic structure of electrospun nanofibers provides a scaffold that is favorable to neural regeneration via alleviation of astrogliosis.

Rim15-dependent activation of Hsf1 and Msn2/4 transcription factors by direct phosphorylation in Saccharomyces cerevisiae.

Rim15 kinase, a downstream effector of PKA and TORC1 signaling pathways, initiates the quiescent program upon nutrient starvation via induction of genes whose expression depends on transcription factors Msn2, Msn4, and Gis1. Here, we demonstrate that Rim15 also induces expression of Hsf1 target genes upon glucose depletion by both transcriptional activation and stabilization of the transcripts. Rim15 phosphorylates Hsf1 in vitro, suggesting that Rim15 might directly activate Hsf1. In addition, Igo1 and Igo2, Rim15 substrate proteins involved in mRNA stabilization, regulate mRNA levels of Hsf1 target genes. We also show that Rim15 can phosphorylate Msn2, but not Gis1, in vitro, implying different mechanisms for the activation of these transcription factors.