A novel in vivo protein refolding technique

Proteins perform their functions in their native folded states and misfolding of proteins may cause severe diseases, including Alzheimer\u27s disease, Parkinson\u27s disease, prion disease and diabetes. Understanding protein folding is important for us to engineer proteins to treat these diseases. For protein therapeutics, large quantities of properly folded and functional proteins are required. The current technology produces recombinant proteins using either eukaryotic or prokaryotic expression system, both of them have major problems that prevent production of large quantities of properly folded and functional human proteins for protein therapeutics. Although the eukaryotic cells have comprehensive folding machinery that contains chaperones and folding enzymes and a complex quality control (QC) system to ensure that only properly folded proteins will be generated to perform their functions, either intracellular or extracellular, the protein yield is usually very low. Protein production using this system is usually costly. In contrast, prokaryotic cells can be used to produce large quantities of recombinant human proteins at a low cost. However, the produced human proteins using prokaryotic cells usually misfold and are not functional due to the much simpler protein folding machinery and QC system of these prokaryotic cells. To solve this problem, the in vitro protein refolding technique has been developed that either mimics the intracellular redox conditions to promote protein folding at a diluted concentration or uses column chromatography to refold the misfolded recombinant proteins. Although this in vitro protein folding technique has some success for small proteins with simple folds, the refolding efficiency is generally very low. For large proteins of complex folds of multiple domains, this in vitro protein refolding technique is usually not working. To solve these challenges, our lab recently developed an in vivo protein refolding technique that uses the intracellular folding machinery and QC system of the Endoplasmic reticulum (ER) of mammalian cells to refold the misfolded recombinant proteins produced using bacterial expression system. This novel technique uses the QQ-protein delivery technology developed in our lab to directly deliver bacterially expressed proteins into the ER for refolding. We showed that the intracellular folding machinery of mammalian cells had a large capacity to properly refold large quantities of misfolded bacterially expressed proteins and the QC system of the mammalian cells ensured that only properly folded proteins followed the normal intracellular trafficking pathway as their endogenous counterparts. Since the refolded proteins contain an affinity tag, we can purify the properly refolded proteins. This in vivo refolded technique takes the advantage of the high yield prokaryotic expression system and the comprehensive protein folding machinery/QC system of mammalian cells to efficiently produce large quantities of properly folded and biologically functional proteins. I optimized this in vivo protein refolding technique for the beta-propeller/EGF domain I of LDL receptor-related protein 6 (BP1-LRP6) and the ligand-binding domain of apolipoprotein E receptor 2 (LBD-ApoER2). These two proteins contain a large number of cysteines that form intracellular disulfide bonds. The folding of these two proteins is very challenging. I performed optimizations of experimental conditions that allow me to produce large quantities of properly folded and functional BP1-LRP6 and LBD-apoER2. The yield of refolding is about 20-60%, depending on different proteins, allowing me to produce milligram quantity of properly refolded and functional BP1-LRP6 and LBD-apoER2. The Far-UV Circular Dichroism (CD) Spectrum of refolded BP1-LRP6 showed a high percentage of beta-sheet which is consistent with the x-ray crystal structure of the beta-propeller/EGF domain of low-density lipoprotein receptor (LDLR). Refolded LBD-apoER2 showed the biological function of active binding the chaperone receptor-associated protein (RAP) in the ligand-blotting assay. My results suggested that, as a new tool, this protein refolding technique can be used to produce large quantities of properly folded and biologically functional proteins for many applications including protein therapeutics to treat human disease, structural biology and protein folding studies

Velocity distribution and 3D turbulence characteristic analysis for flow over water-worked rough bed

YesTo reproduce the natural flow topography in a laboratory environment, it is crucial to recapture its bed condition in order to ensure the accurate representation. Water-worked bed represents a state-of-the-art experimentally formed bed to imitate the natural-formed channel in most rivers or natural streams. Recently, this technique has been intensively studied through experimental and computational approaches; however, its actual influence towards the near-bed flow as compared to experimentally prepared rough bed in well-packed bedform order are still yet to be investigated deeply. This experimental study systematically investigated and compared the differences in velocity distribution and three-dimensional (3D) turbulence characteristics, including turbulence intensities and Reynolds stresses, between uniform smooth bed, laboratory-prepared rough bed and water-worked bed open channel flows. The flow comparisons were concentrated at near-bed region where clear flow behaviour change can be observed. Through these comparisons, the study inspected the characteristics of water-worked bedform thoroughly, in order to inform future experimental research that tries to reproduce natural stream behaviours.the Major State Basic Research Development Grant No. 2013CB036402 from Tsinghua University. The support from the Major State Basic Research Development Program (973 program) of China is also greatly appreciated. We also acknowledge the National Key Research and Development Project from the Ministry of Science and Technology during the Thirteenth Five-year Plan Period (Grant No. 2017YFC0403600) and the Science and Technology Projects State Grid Corporation of China (Grant No. 52283014000T)

The contribution of reduction in evaporative cooling to higher surface air temperatures during drought

Higher temperatures are usually reported during meteorological drought and there are two prevailing interpretations for this observation. The first is that the increase in temperature (T) causes an increase in evaporation (E) that dries the environment. The second states that the decline in precipitation (P) during drought reduces the available water thereby decreasing E, and in turn the consequent reduction in evaporative cooling causes higher T. To test which of these interpretations is correct, we use climatic data (T, P) and a recently released database (CERES) that includes incoming and outgoing shortwave and longwave surface radiative fluxes to study meteorological drought at four sites (parts of Australia, US, and Brazil), using the Budyko approximation to calculate E. The results support the second interpretation at arid sites. The analysis also showed that increases in T due to drought have a different radiative signature from increases in T due to elevated CO₂.This research was supported by the Australian Research Council (CE11E0098), the National Natural Science Foundation of China (91125018), and the China Scholarship Council (201306210089)

The Milky Way's rotation curve out to 100 kpc and its constraint on the Galactic mass distribution

The rotation curve (RC) of the Milky Way out to $\sim$ 100 kpc has been constructed using $\sim$ 16,000 primary red clump giants (PRCGs) in the outer disk selected from the LSS-GAC and the SDSS-III/APOGEE survey, combined with $\sim$ 5700 halo K giants (HKGs) selected from the SDSS/SEGUE survey. To derive the RC, the PRCG sample of the warm disc population and the HKG sample of halo stellar population are respectively analyzed using a kinematical model allowing for the asymmetric drift corrections and re-analyzed using the spherical Jeans equation along with measurements of the anisotropic parameter $\beta$ currently available. The typical uncertainties of RC derived from the PRCG and HKG samples are respectively 5-7 km/s and several tens km/s. We determine a circular velocity at the solar position, $V_c (R_0)$ = 240 $\pm$ 6 km/s and an azimuthal peculiar speed of the Sun, $V_{\odot}$ = 12.1 $\pm$ 7.6 km/s, both in good agreement with the previous determinations. The newly constructed RC has a generally flat value of 240 km/s within a Galactocentric distance $r$ of 25 kpc and then decreases steadily to 150 km/s at $r$ $\sim$ 100 kpc. On top of this overall trend, the RC exhibits two prominent localized dips, one at $r$ $\sim$ 11 kpc and another at $r$ $\sim$ 19 kpc. From the newly constructed RC, combined with other constraints, we have built a parametrized mass model for the Galaxy, yielding a virial mass of the Milky Way's dark matter halo of $0.90^{+0.07}_{-0.08} \times 10^{12}$ ${\rm M}_{\odot}$ and a local dark matter density, $\rho_{\rm \odot, dm} = 0.32^{+0.02}_{-0.02}$ GeV cm$^{-3}$.Comment: MNRAS accepted, 18 pages, 15 figures, 4 table

SWE-SPHysics Simulation of Dam Break Flows at South-Gate Gorges Reservoir

This paper applied a Smoothed Particle Hydrodynamics (SPH) approach to solve Shallow Water Equations (SWEs) to study practical dam-break flows. The computational program is based on the open source code SWE-SPHysics, where a Monotone Upstream-centered Scheme for Conservation Laws (MUSCL) reconstruction method is used to improve the Riemann solution with Lax-Friedrichs flux. A virtual boundary particle method is applied to treat the solid boundary. The model is first tested on two benchmark collapses of water columns with the existence of downstream obstacle. Subsequently the model is applied to forecast a prototype dam-break flood, which might occur in South-Gate Gorges Reservoir area of Qinghai Province, China. It shows that the SWE-SPH modeling approach could provide a promising simulation tool for practical dam-break flows in engineering scale

SPHysics Simulation of Experimental Spillway Hydraulics

In this paper, we use the parallel open source code parallelSPHysics based on the weakly compressible Smoothed Particle Hydrodynamics (WCSPH) approach to study a spillway flow over stepped stairs. SPH is a robust mesh-free particle modelling technique and has great potential in treating the free surfaces in spillway hydraulics. A laboratory experiment is carried out for the different flow discharges and spillway step geometries. The physical model is constructed from a prototype reservoir dam in the practical field. During the experiment, flow discharge over the weir crest, free surface, velocity and pressure profiles along the spillway are measured. In the present SPH study, a straightforward push-paddle model is used to generate the steady inflow discharge in front of the weir. The parallelSPHysics model is first validated by a documented benchmark case of skimming flow over a stepped spillway. Subsequently, it is used to reproduce a laboratory experiment based on a prototype hydraulic dam project located in Qinghai Province, China. The detailed comparisons are made on the pressure profiles on the steps between the SPH results and experimental data. The energy dissipation features of the flows under different flow conditions are also discussed. It is shown that the pressure on the horizontal face of the steps demonstrates an S-shape, while on the vertical face it is negative on the upper part and positive on the lower part. The energy dissipation efficiency of the spillway could reach nearly 80%