Non-viral adeno-associated virus-based platform for stable expression of antibody combination therapeutics: Non-viral adeno-associated virus-based platform for stable expression of antibody combination therapeutics

Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures.     

Water temperature and upstream migration of glass eels in New Zealand: implications of climate change

Glass eels migrating upstream in a New Zealand river showed a clear preference for water temperatures between 12 and 20°C, with an optimum of 16.5°C. Water temperatures <12°C and >22°C almost completely inhibited migration, which implies that warmer temperatures associated with global climate change might have a detrimental impact on glass eel recruitment in their current ranges. We established this by trapping glass eels of shortfin, Anguilla australis, and longfin, A. dieffenbachii, eels nightly from September to November. Eels caught in 2001 (50,287) outnumbered those caught in 2002 (19,954); shortfin glass eels dominated catches in both years, comprising 91–93% of the catch. Longfins were larger than shortfins, and size and pigmentation in both species increased as the seasons progressed. Temperatures within the migratory season in 2001 showed ∼14-day intervals between maxima that appeared to be associated with the new and full moons.     

The calcium‐dependent protein kinase CPK7 acts on root hydraulic conductivity

The hydraulic conductivity of plant roots (Lp_r) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post‐translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 (PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lp_r of knockout Arabidopsis plants for four Ca²⁺‐dependent protein kinases. cpk7 plants showed a 30% increase in Lp_r because of a higher aquaporin activity. A quantitative proteomic analysis of wild‐type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lp_r of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7‐dependent stability of specific membrane proteins.     

Anatomical and chemical characteristics of the seed coat of <i>Medicago sativa</i> L. (alfalfa) cv. Baralfa 85 seeds and their association with seed dormancy

Seeds of alfalfa (Medicago sativa L.) can exhibit seedcoat imposed dormancy, which produces hard seeds within a seed lot. These seeds do not germinate because they do not imbibe water due to a barrier to water entry in the seed coat. The aim of this work was to analyze the anatomical and chemical characteristics of the testa of alfalfa seeds with respect to water permeability levels. The anatomy of seeds of the cv. Baralfa 85 was studied and structural substances, polyphenols, tannins and cutin present in the testa of seeds of different water permeability levels were determined. The anatomical characteristics of the seed coat and the proportions of components were found to determine the permeability level of the seed coat, an aspect that is associated with the physical seed dormancy level. Anatomically, increased thickness of the testa was associated with a lower permeability level. The difference may be attributed to the variation in cuticle thickness, length of macrosclereids and thickness of the cell wall, and presence and development of osteosclereids. From the physiological and chemical points of view, the mechanism of physical dormancy of the testa is explained by a greater amount of components that repel water and cement the cell wall, such as polyphenols, lignins, condensed tannins, pectic substances, and a lower proportion of cellulose and hemicellulose.     

Roles of AtpI and Two YidC-Type Proteins from Alkaliphilic Bacillus pseudofirmus OF4 in ATP Synthase Assembly and Nonfermentative Growth

AtpI, a membrane protein encoded by many bacterial atp operons, is reported to be necessary for c-ring oligomer formation during assembly of some ATP synthase complexes. We investigated chaperone functions of AtpI and compared them to those of AtpZ, a protein encoded by a gene upstream of atpI that has a role in magnesium acquisition at near-neutral pH, and of SpoIIIJ and YqjG, two YidC/OxaI/Alb3 family proteins, in alkaliphilic Bacillus pseudofirmus OF4. A strain with a chromosomal deletion of atpI grew nonfermentatively, and its purified ATP synthase had a c-ring of normal size, indicating that AtpI is not absolutely required for ATP synthase function. However, deletion of atpI, but not atpZ, led to reduced stability of the ATP synthase rotor, reduced membrane association of the F₁ domain, reduced ATPase activity, and modestly reduced nonfermentative growth on malate at both pH 7.5 and 10.5. Both spoIIIJ and yqjG, but not atpI or atpZ, complemented a YidC-depleted Escherichia coli strain. Consistent with such overlapping functions, single deletions of spoIIIJ or yqjG in the alkaliphile did not affect membrane ATP synthase levels or activities, but functional specialization was indicated by YqjG and SpoIIIJ showing respectively greater roles in malate growth at pH 7.5 and 10.5. Expression of yqjG was elevated at pH 7.5 relative to that at pH 10.5 and in ΔspoIIIJ strains, but it was lower than constitutive spoIIIJ expression. Deletion of atpZ caused the largest increase among the mutants in magnesium concentrations needed for pH 7.5 growth. The basis for this phenotype is not yet resolved.     

Determining the effect of the incorporation of unnatural amino acids into antimicrobial peptides on the interactions with zwitterionic and anionic membrane model systems

Circular Dichroism (CD), isothermal calorimetry (ITC) and calcein fluorescence leakage experiments were conducted to provide insight into the mechanisms of binding of a series of antimicrobial peptides containing unnatural amino acids (Ac-XF-Tic-Oic-XK-Tic-Oic-XF-Tic-Oic-XK-Tic-KKKK-CONH₂) to zwitterionic and anionic micelles, SUVs and LUVs; where X (Spacer# 1) is either Gly, β-Ala, Gaba or 6-aminohexanoic acid. It is the intent of this investigation to correlate these interactions with the observed potency and selectivity against several different strains of bacteria. The CD spectra of these compounds in the presence of zwitterionic DPC micelles and anionic SDS micelles are very different indicating that these compounds adopt different conformations on binding to the surface of anionic and zwitterionic membrane models. These compounds also exhibited very different CD spectra in the presence of zwitterionic POPC and anionic mixed 4:1 POPC/POPG SUVs and LUVs, indicating the formation of different conformations on interaction with the two membrane types. This observation is also supported by ITC and calcein leakage data. ITC data suggested these peptides interact primarily with the surface of zwitterionic LUVs and was further supported by fluorescence experiments where the interactions do not appear to be concentration dependent. In the presence of anionic membranes, the interactions appear more complex and the calorimetric and fluorescence data both imply pore formation is dependent on peptide concentration. Furthermore, evidence suggests that as the length of Spacer# 1 increases the mechanism of pore formation also changes. Based on the observed differences in the mechanisms of interactions with zwitterionic and anionic LUVs these AMPs are potential candidates for further drug development.     

原核表达载体pOPE101-8E5的改构及单链抗体的表达鉴定

本研究利用基因重组技术将链亲和素(core-streptavidin)cDNA插入原核表达载体pOPE101-8E5的3′端,并用单链抗体scFv-C4的重链和轻链可变区cDNA取代其scFv-8E5,构建重组表达载体pOPE101-C4::core-streptavidin。将该表达载体转化入在大肠杆菌中进行诱导表达,用聚丙烯酰胺凝胶电泳和免疫印迹法分析表达产物的表达量和产物活性。结果提示我们成功地获得一个分子量约为45kDa的scFv-C4::core-streptavidin的融合蛋白,它可结合KG1a细胞裂解物中分子量约为60kDa、45kDa的蛋白带,且其结合功能可以通过融合蛋白中的链亲和素基因直接测定。     

Does hatchery-reared Siniperca chuatsi (Actinopterygii,Perciformes) compete significantly with two wild Siniperca populations for diets in a shallow lake?

Greenhouse gas emissions of Lake Neusiedl, the westernmost European shallow steppe lake, were analysed to identify differences between the seasons of the years and between different locations in the pelagic zone and reed belt. Emissions of CO₂, CH₄ and N₂O were measured in gas samples that had been recovered from the gas space of floating chambers operated as closed systems. Sampling periods covered all seasons except winter. Scaled up to the whole lake area (320 km²), the diffusive emissions of spring, summer and autumn totalled to about 79,500 t CO₂e, disregarding bubble emissions, winter emissions and plant-mediated emissions. The emission sum consisted of about 57,000 t CO₂, 760 t CH₄, and 12 t N₂O. Approximately one-third of the methane and carbon dioxide emissions originated in the pelagic zone and two-thirds in the reed belt (without plant emissions) whereas nitrous oxide emissions were similar in these two zones. An estimate of ebullitive emissions resulted in additional 1,765 t CH₄ that predominantly originated in or near the reed belt from spring to autumn.     

Interplay of isoprenoid and peptide substrate specificity in protein farnesyltransferase

Protein farnesyltransferase (FTase) catalyzes the post-translational modification of many important cellular proteins, and is a potential anticancer drug target. Crystal structures of the FTase ternary complex illustrate an unusual feature of this enzyme, the fact that the isoprenoid substrate farnesyl diphosphate (FPP) forms part of the binding site for the peptide substrate. This implies that changing the structure of FPP could alter the specificity of the FPP-FTase complex for peptide substrates. We have found that this is the case; a newly synthesized FPP analogue, 3-MeBFPP, is a substrate with three peptide cosubstrates, but is not an effective substrate with a fourth peptide (dansyl-GCKVL). Addition of this analogue also inhibits farnesylation of dansyl-GCKVL by FPP. Surprisingly, the differential substrate abilities of these four peptides with FPP-FTase and 3-MeBFPP-FTase complexes do not correlate with their binding affinities for these isoprenoid-enzyme complexes. The possible mechanistic rationales for this observation, along with its potential utility for the study of protein prenylation, are discussed.