Generation of Inducible CRISPRi and CRISPRa Human Stromal/Stem Cell Lines for Controlled Target Gene Transcription during Lineage Differentiation

Background. Human bone marrow stromal/stem cells (hMSCs, also known as the skeletal stem cells or mesenchymal stem cells) are being employed to study lineage fate determination to osteoblasts, adipocytes, and chondrocytes. However, mechanistic studies employing hMSC have been hampered by the difficulty of deriving genetically modified cell lines due to the low and unstable transfection efficiency. Methods. We infected hMSC with a CRISPR/Cas9 lentivirus system, with specific inducible dCas9-coupled transcription activator or repressor: dCas9-KRAB or dCas9-VP64, respectively, and established two hMSC lines (hMSC-CRISPRi and hMSC-CRISPRa) that can inhibit or activate gene expression, respectively. The two cell lines showed similar cell morphology, cell growth kinetics, and similar lineage differentiation potentials as the parental hMSC line. The expression of KRAB-dCas9 or VP64-dCas9 was controlled by the presence or absence of doxycycline (Dox) in the cell culturing medium. To demonstrate the functionality of the dCas9-effector hMSC system, we tested controlled expression of alkaline phosphatase (ALP) gene through transfection with the same single ALP sgRNA. Results. In the presence of Dox, the expression of ALP showed 60-90% inhibition in hMSC-CRISPRi while ALP showed more than 20-fold increased expression in hMSC-CRISPRa. As expected, the ALP was functionally active and the cells showed evidence for inhibition or enhancement of in vitro osteoblast differentiation, respectively. Conclusion. hMSC-CRISPRi and hMSC-CRISPRa are useful resources to study genes and genetic pathways regulating lineage-specific differentiation of hMSC

Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Keywords: Actin cytoskeleton, Actin depolymerizing factors, Adipocyte differentiation, Human stromal stem cell

Effect of suction chamber baffles on pressure fluctuations in a low specific speed centrifugal pump

In order to study the effect of suction chamber baffles on hydraulic performance and unsteady characteristics in a low specific speed centrifugal pump, a model pump was design with enlarging flow mothed and four schemes of suction baffle, including no baffle (scheme 0), only one baffle in the suction (scheme 1), two baffles (scheme 2) and three baffles in the suction (scheme 3), were considered. Commercial code FLUENT was applied to simulate the flow of the pump. RNG k-ε turbulence model was adopted to handle with the turbulent flows in the pump. The sliding mesh technique was applied to take into account the impeller-volute interaction. Based on the simulation results, the hydraulic performance and pressure fluctuations were obtained and analyzed in detail. The head value of no baffle in the suction (scheme 0) is lower than that with baffles in the suction (scheme 1, scheme 2, scheme 3) at each condition point. Hump point in scheme 0 is at φ= 0.00596 (1.2 times Qo). The hump point in scheme 1, scheme 2, scheme 3 is at 0.8Qo, 1.0Qo, 1.0Qo, respectively. The ε value of scheme 1 is the smallest and that of scheme 0 is the largest in the four schemes. Six wave troughs are observed clearly at each monitoring point as the impeller rotates in a circle. Each time the impeller is turned 10 degrees, there are six obvious troughs around the impeller. With the rotation of the impeller, peak value of pressure fluctuations at blade passing frequency (BPF) is gradually decrease. At low flow (ϕ= 0.002383), the main frequency of pressure fluctuation at P36 and P1 under scheme 0, scheme 2 and scheme 3 is 295 Hz, which is corresponding to BPF. The pressure fluctuation levels are decreased by –2.72 %, –2.13 %, and –2.21 % respectively when the number of baffle in the suction is one, two, three, respectively. And decrease rate of pressure fluctuation (∆Cp) on scheme 1 is maximum. It indicates that Adding baffles to the suction chamber is beneficial to reduce the amplitude of pressure pulsation at BPF in the volute. The best number of baffles in the suction is one. Based on scheme 1 simulation results, the prototype was manufactured and the performance experiments were carried out. A good agreement of the head and efficiency between numerical results and experimental results are observed

Multiagent-Based Reactive Power Sharing and Control Model for Islanded Microgrids

In islanded microgrids (MGs), the reactive power cannot be shared proportionally among distributed generators (DGs) with conventional droop control, due to the mismatch in feeder impedances. For the purpose of proportional reactive power sharing, a multiagent system (MAS)-based distributed control model for droop-controlled MGs is proposed. The proposed control model consists of two layers, where the bottom layer is the electrical distribution MG, while the top layer is a communication network composed of agents. Moreover, agents on the communication network exchange the information acquired from DGs with neighbors, and calculate set points for DGs they connect to, according to the control laws. Furthermore, a theorem is demonstrated, which yields a systematic method to derive the control laws from a given communication network. Finally, three cases are carried out to test the performance of the control model, in which the uncertainty of intermittent DGs, variations in load demands, as well as impacts of time delays are considered. The simulation results demonstrate the effectiveness of the control model in proportional reactive power sharing, and the plug and play capability of the control model is also verified.Feixiong Chen, Minyou Chen, Qiang Li, Kaikai Meng, Josep M. Guerrero, Fellow and Derek Abbot

Pronounced ductility in CuZrAl ternary bulk metallic glass composites with optimized microstructure through melt adjustment

Microstructures and mechanical properties of as-cast Cu47.5Zr47.5Al5 bulk metallic glass composites are optimized by appropriate remelting treatment of master alloys. With increasing remelting time, the alloys exhibit homogenized size and distribution of in situ formed B2 CuZr crystals. Pronounced tensile ductility of ∼13.6% and work-hardening ability are obtained for the composite with optimized microstructure. The effect of remelting treatment is attributed to the suppressed heterogeneous nucleation and growth of the crystalline phase from undercooled liquid, which may originate from the dissolution of oxides and nitrides as well as from the micro-scale homogenization of the melt

Cost-Based Droop Schemes for Economic Dispatch in Islanded Microgrids

In this paper, cost-based droop schemes are proposed, to minimize the total active power generation cost in an islanded microgrid (MG), while the simplicity and decentralized nature of the droop control are retained. In cost-based droop schemes, the incremental costs of distributed generators (DGs) are embedded into the droop schemes, where the incremental cost is a derivative of the DG cost function with respect to output power. In the steady state, DGs share a single common frequency, and cost-based droop schemes equate incremental costs of DGs, thus minimizing the total active power generation cost, in terms of the equal incremental cost principle. Finally, simulation results in an islanded MG with high a penetration of intermittent renewable energy sources are presented, to demonstrate the effectiveness, as well as plug and play capability of the cost-based droop schemes.Feixiong Chen, Minyou Chen, Qiang Li, Kaikai Meng, Yongwei Zheng, Josep M. Guerrero, Derek Abbot