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

Modeled foraminiferal calcification and strontium partitioning in benthic foraminifera helps reconstruct calcifying fluid composition

Abstract: Foraminifera are unicellular organisms that inhabit the oceans. They play an important role in the global carbon cycle and record valuable paleoclimate information through the uptake of trace elements such as strontium into their calcitic shells. Understanding how foraminifera control their internal fluid composition to make calcite is important for predicting their response to ocean acidification and for reliably interpreting the chemical and isotopic compositions of their shells. Here, we model foraminiferal calcification and strontium partitioning in the benthic foraminifera Cibicides wuellerstorfi and Cibicidoides mundulus based on insights from inorganic calcite experiments. The numerical model reconciles inter-ocean and taxonomic differences in benthic foraminifer strontium partitioning relationships and enables us to reconstruct the composition of the calcifying fluid. We find that strontium partitioning and mineral growth rates of foraminiferal calcite are not strongly affected by changes in external seawater pH (within 7.8–8.1) and dissolved inorganic carbon (DIC, within 2100–2300 μmol/kg) due to a regulated calcite saturation state at the site of shell formation

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

Identifying Vegetation Dynamics and Sensitivities in Response to Water Resources Management in the Heihe River Basin in China

The Heihe River Basin, the second largest inland river basin in China, plays a vital role in the ecological sustainability of the Hexi Corridor. However, the requirements for regional economic development and ecological balance cannot be fully met due to water resource shortage and overexploitation induced by an extremely dry climate and population growth, especially in the middle and lower basins. Thus, environmental conservation projects that reallocate water resources have been planned and implemented step by step since 2001. The aim of this study is to evaluate ecosystem restoration benefits by identifying vegetation dynamics and sensitivities. The MODIS Normalized Difference Vegetation Index (NDVI) and its derivative indices, coupled with Geographic Information System (GIS), are introduced to explore ecosystem evolution at the pixel level, based on the hydrological and meteorological data in the whole region at varying temporal and spatial scales. Results indicate there are slight vegetation restoration trends in the upper, middle, and lower basin; the results of correlation analyses between vegetation and runoff into the lower basin suggest that the impact of a water supplement lasts at most three years, and engineering or nonengineering measures should be maintained for permanent ecosystem recovery