Process monitoring of virus-like particle reassembly by diafiltration with UV/Vis spectroscopy and light scattering

Virus-like particles (VLPs) have shown great potential as biopharmaceuticals in the market and in clinics. Nonenveloped, in vivo assembled VLPs are typically disassembled and reassembled in vitro to improve particle stability, homogeneity, and immunogenicity. At the industrial scale, cross-flow filtration (CFF) is the method of choice for performing reassembly by diafiltration. Here, we developed an experimental CFF setup with an on-line measurement loop for the implementation of process analytical technology (PAT). The measurement loop included an ultraviolet and visible (UV/Vis) spectrometer as well as a light scattering photometer. These sensors allowed for monitoring protein concentration, protein tertiary structure, and protein quaternary structure. The experimental setup was tested with three Hepatitis B core Antigen (HBcAg) variants. With each variant, three reassembly processes were performed at different transmembrane pressures (TMPs). While light scattering provided information on the assembly progress, UV/Vis allowed for monitoring the protein concentration and the rate of VLP assembly based on the microenvironment of Tyrosine-132. VLP formation was verified by off-line dynamic light scattering (DLS) and transmission electron microscopy (TEM). Furthermore, the experimental results provided evidence of aggregate-related assembly inhibition and showed that off-line size-exclusion chromatography does not provide a complete picture of the particle content. Finally, a Partial-Least Squares (PLS) model was calibrated to predict VLP concentrations in the process solution. values of 0.947–0.984 were reached for the three HBcAg variants. In summary, the proposed experimental setup provides a powerful platform for developing and monitoring VLP reassembly steps by CFF.     

Mechanism of selective stabilization of extrinsic polypeptides in PSII particles by glycinebetaine

Ｔｈｒｅｅｅｘｔｒｉｎｓｉｃｐｏｌｙｐｅｐｔｉｄｅｓ,ｗｈｉｃｈａｒｅｌｏｃａｔｅｄｏｎｔｈｅｉｎｎｅｒｓｕｒｆａｃｅｏｆｔｈｙｌａｋｏｉｄｍｅｍｂｒａｎｅ,ｐｌａｙｅｓｓｅｎｔｉａｌｒｏｌｅｓｉｎｍａｉｎｔａｉｎｉｎｇｔｈｅｐｈｏｔｏｓｙｎｔｈｅｔｉｃｅｖｏｌｕｔｉｏｎｏｆｏｘｙｇｅｎ.Ｔｈｅｙａｒｅｇｅｎｅｒａｌｌｙｒｅｃｏｇｎｉｚｅｄａｓｌｉａｂｌｅｃｏｍｐｏｎｅｎｔｓｏｆｔｈｅｐｈｏｔｏｓｙｎｔｈｅｔｉｃａｐｐａｒａｔｕｓ,ａｎｄｃａｎｂｅｒｅｌｅａｓｅｄｂｙａｖａｒｉｅｔｙｏｆｐｈｙｓｉｃ…     

Mechanism of proteasome gate modulation by assembly chaperones Pba1 and Pba2

The active sites of the proteasome are housed within its central core particle (CP), a barrel-shaped chamber of four stacked heptameric rings, and access of substrates to the CP interior is mediated by gates at either axial end. These gates are constitutively closed and may be opened by the regulatory particle (RP), which binds the CP and facilitates substrate degradation. We recently showed that the heterodimeric CP assembly chaperones Pba1/2 also mediate gate opening through an unexpected structural arrangement that facilitates the insertion of the N terminus of Pba1 into the CP interior; however, the full mechanism of Pba1/2-mediated gate opening is unclear. Here, we report a detailed analysis of CP gate modulation by Pba1/2. The clustering of key residues at the interface between neighboring α-subunits is a critical feature of RP-mediated gate opening, and we find that Pba1/2 recapitulate this strategy. Unlike RP, which inserts at six α-subunit interfaces, Pba1/2 insert at only two α-subunit interfaces. Nevertheless, Pba1/2 are able to regulate six of the seven interfacial clusters, largely through direct interactions. The N terminus of Pba1 also physically interacts with the center of the gate, disrupting the intersubunit contacts that maintain the closed state. This novel mechanism of gate modulation appears to be unique to Pba1/2 and therefore likely occurs only during proteasome assembly. Our data suggest that release of Pba1/2 at the conclusion of assembly is what allows the nascent CP to assume its mature gate conformation, which is primarily closed, until activated by RP.     

灵井许昌人遗址第5层细石核工艺

本文研究的细石核出自灵井"许昌人"遗址第5层,该层为桔黄色粉细砂,2008-2013年发掘,出土细石核82件,其他还有与之相关的材料。该层碳十四年龄为13402±406BP。细石核素材一般为燧石质的石片、小砾石等。根据其毛坯形状、细石叶剥离进度等的差异,形成了角锥形、柱形、圆锥形等各种形状。在剥离石叶过程中,曾运用过台面修理、工作面上端(细石叶头部)修正、台面更新、台面转移等调整手法。灵井石器工业的细石叶工艺是一种以角锥形(型)细石核为主的技术。通过对比,灵井与华北各地的同类细石核大小尺寸接近,与本省临近的大岗、李家沟等细石器遗址应属相同或相近技术传统的细石叶工艺。     

University Multi-User Facility Survey��2010

Multi-user facilities serve as a resource for many universities. In 2010, a survey was conducted investigating possible changes and successful characteristics of multi-user facilities, as well as identifying problems in facilities. Over 300 surveys were e-mailed to persons identified from university websites as being involved with multi-user facilities. Complete responses were received from 36 facilities with an average of 20 years of operation. Facilities were associated with specific departments (22%), colleges (22%), and university research centers (8.3%) or were not affiliated with any department or college within the university (47%). The five most important factors to succeed as a multi-user facility were: 1) maintaining an experienced, professional staff in an open atmosphere; 2) university-level support providing partial funding; 3) broad client base; 4) instrument training programs; and 5) an effective leader and engaged strategic advisory group. The most significant problems were: 1) inadequate university financial support and commitment; 2) problems recovering full service costs from university subsidies and user fees; 3) availability of funds to repair and upgrade equipment; 4) inability to retain highly qualified staff; and 5) unqualified users dirtying/damaging equipment. Further information related to these issues and to fee structure was solicited. Overall, there appeared to be a decline in university support for facilities and more emphasis on securing income by serving clients outside of the institution and by obtaining grants from entities outside of the university.     

Structural and functional domains of cellobiohydrolase I from trichoderma reesei

Limited proteolysis (papain) of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei led to the seperation of two functional domains: a core protein (55 kDa) containing the active site, and a C-terminal glycopeptide (10 kDa) implicated in binding to the insoluble matrix (cellulose). The quaternary structures of the intact CBH I and its core in solution are now compared by small angle X-ray scattering (SAXS) measurements. The molecular parameters derived for the core (Rg=2.09 nm, D_max=6.5 nm) and for the intact enzyme (Rg=4.27 nm, D_max=18 nm) indicate very different shapes. The resulting models show a tadpole-like structure for the intact enzyme where the isotropic part coincides with the core protein and the flexible tail part should be identified with the C-terminal glycopeptide. Thus in this enzyme, functional differentiation is reflected in structural peculiarities.Abbreviations SAXS small angle X-ray scattering - SDS-PAGE SDS-polyacrylamide gel electrophoresis - IEF-PAG polyacrylamide gel isoelectric focusing; cellobiohydrolase (CBH, 1,4--glucan cellobio hydrolase (E.C.3.2.1.91)) - D_max maximum diameter - Rg radius of gyration     

De novo design of the hydrophobic core of ubiquitin.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.     

A tetracopper(II) complex with N,N′-bis(picolinoyl)hydrazine

Synthesis, physical properties and X-ray structure of a hydrated tetranuclear copper(II) complex [Cu₄(μ-diph)₂(μ-H₂O)₂(O₂CCH₃)₄(H₂O)₂]·4H₂O with N,N′-bis(picolinoyl)hydrazine (H₂diph) are reported. The centrosymmetric complex has two types of copper(II) centres with distorted square-pyramidal N₂O₃ coordination spheres. The dinucleating trans planar diph²⁻ ligands are parallel to each other and act as N₂O-donor to one metal centre and N₂-donor to the other metal centre. The complex has a rectangular {Cu₄(μ-N-N)₂(μ-OH₂)₂} core with Cu···Cu distances as 4.834(1) and 3.762(1) Å. Solid state as well as solution electronic spectra show several transitions in the wavelength range 700-280 nm. The room temperature (298 K) solid state magnetic moment is 3.55 μ_B. The powder EPR spectra at 298 and 130 K are very similar and axial (g_∥ = 2.25 and g_⊥ = 2.08) in character.     

Strain‐specific proportion of the two isoforms of the dopamine D2 receptor in the mouse striatum: associated neural and behavioral phenotypes

Genetic variability in the proportion of the two alternative dopamine D2 receptor (D2R) mRNA splice variants, D2R‐long (D2L) and D2R‐short (D2S), influence corticostriatal functioning and could be implicated in liability to psychopathology. This study compared mesostriatal D2L/D2S ratios and associated neural and behavioral phenotypes in mice of the DBA/2J and C57BL/6J‐inbred strains, which differ for schizophrenia‐ and addiction‐like phenotypes. Results showed that DBA/2J mice lack the striatal predominance of D2L that has been reported in the rat and in C57BL/6J mice and confirmed in the latter strain by this study. Only C57BL/6J mice showed enhanced striatal c‐Fos expression under D1R and D2/3R co‐stimulation, indicating synergistic interaction between the subtypes of DA receptors. Instead, DBA/2J mice were characterized by opposing effects of D2/3R and D1R stimulation on striatal c‐Fos expression, in line with a more pronounced influence of D2S isoform, and did not express stereotyped climbing under D1R and D2/3R co‐stimulation, as reported for D2L?/? mice. Finally, strain‐specific modulation of c‐Fos expression by D1R and D2/3R co‐stimulation was selectively observed in striatal compartments receiving inputs from the prefrontal cortex and involved in the control of motivated behaviors. These results show differences in tissue‐specific D2R splicing in mice with intact genotypes and support a role for this phenotype in individual variability of corticostriatal functioning and in liability to psychopathology.     

Encapsulation and Permeability Characteristics of Plasma Polymerized Hollow Particles

In this protocol, core-shell nanostructures are synthesized by plasma enhanced chemical vapor deposition. We produce an amorphous barrier by plasma polymerization of isopropanol on various solid substrates, including silica and potassium chloride. This versatile technique is used to treat nanoparticles and nanopowders with sizes ranging from 37 nm to 1 micron, by depositing films whose thickness can be anywhere from 1 nm to upwards of 100 nm. Dissolution of the core allows us to study the rate of permeation through the film. In these experiments, we determine the diffusion coefficient of KCl through the barrier film by coating KCL nanocrystals and subsequently monitoring the ionic conductivity of the coated particles suspended in water. The primary interest in this process is the encapsulation and delayed release of solutes. The thickness of the shell is one of the independent variables by which we control the rate of release. It has a strong effect on the rate of release, which increases from a six-hour release (shell thickness is 20 nm) to a long-term release over 30 days (shell thickness is 95 nm). The release profile shows a characteristic behavior: a fast release (35% of the final materials) during the first five minutes after the beginning of the dissolution, and a slower release till all of the core materials come out.