Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter

Immunocamouflaged red blood cells (RBC) are produced by cell surface derivatization with methoxypolyethylene glycol (mPEG). These immunologically attenuated cells may reduce the risk of allosensitization in chronically transfused patients. To characterize the effects of differing linker chemistries and polymer lengths, RBC were modified with cyanuric chloride activated mPEG (C-mPEG 5 kDa), benzotriazole carbonate methoxyPEG (BTC-mPEG; 5 or 20 kDa) or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPA-mPEG; 2, 5 or 20 kDa). Biophysical methods including particle electrophoresis and aqueous two-phase polymer partitioning were employed to compare the PEG derivatives. While C-mPEG was faster reacting, both BTC-mPEG and SPA-mPEG gave comparable findings after 1 h. Both PEG surface density and molecular mass had a large effect on RBC surface properties. Proportional changes in electrophoretic mobility and preferential phase partitioning were achieved by increasing either the quantity of surface PEG or the PEG molecular mass. In addition, two-phase partitioning may provide a means for efficiently removing unmodified or lightly modified (hence potentially immunogenic) RBC in the clinical setting. Furthermore, mPEG modification significantly inhibits cell-cell interaction as evidenced by loss of Rouleaux formation and, consequently, sedimentation rate. Importantly, BTC-mPEG 20 kDa RBC showed normal in vivo survival in mice at immunoprotective concentrations (up to 2 mM).     

Immobilization and characterization of horseradish peroxidase into chitosan and chitosan/PEG nanoparticles: A comparative study

Chitosan (CS) is considered a suitable biomaterial for enzyme immobilization. CS combination with polyethylene glycol (PEG) can improve the biocompatibility and the properties of the immobilized system. Thus, the present work investigated the effect of the PEG in the horseradish peroxidase (HRP) immobilization into chitosan nanoparticles from the morphological, physicochemical, and biochemical perspectives. CS and CS/PEG nanoparticles were obtained by ionotropic gelation and provided immobilization efficiencies (IE) of 65.8 % and 51.7 % and activity recovery (AR) of 76.4 % and 60.4 %, respectively. The particles were characterized by DLS, ZP, SEM, FTIR, TGA and DSC analysis. Chitosan nanoparticles showed size around 135 nm and increased to 229 nm after PEG addition and HRP immobilization. All particles showed positive surface charges (20−28 mV). Characterizations suggest nanoparticles formation and effective immobilization process. Similar values for optimum temperature and pH for immobilized HRP into both nanoparticles were found (45 °C, 7.0). V_max value decreased by 5.07 to 3.82 and 4.11 mM/min and K_M increased by 17.78 to 18.28 and 19.92 mM for free and immobilized HRP into chitosan and chitosan/PEG nanoparticles, respectively. Another biochemical parameters (K_cat, K_e, and K_α) evaluated showed a slight reduction for the immobilized enzyme in both nanoparticles compared to the free enzyme.     

大蜡螟和黄粉虫肠道菌中聚乙烯地膜降解细菌的筛选及其降解性能

【背景】在我国农业生产中大量使用的聚乙烯(polyethylene,PE)地膜难以降解,在土壤中长期累积影响农作物生长并破坏生态环境,发掘微生物资源,寻求聚乙烯生物降解途径对治理“白色污染”具有重要意义。【目的】以不同来源的啮食塑料昆虫大蜡螟、黄粉虫为材料,从肠道菌群中分离筛选出对PE具有降解能力的细菌菌株,研究其降解农用地膜的效能。【方法】饲喂PE膜片驯化大蜡螟、黄粉虫幼虫,采集肠道液富集培养、共代谢驯化、选择培养基筛选等方法从肠道细菌中分离出以PE为唯一碳源的细菌菌株。将菌株接种到以PE膜片为唯一碳源的培养基中共培养,通过测定菌体生长量,定期检测膜片失重率,结合高分辨场发射扫描电子显微镜观察、红外扫描分析和膜片力学性能测定,评价菌株对聚乙烯地膜的降解效果。对筛选出的降解性能良好的菌株通过16S rRNA基因扩增和序列分析进行菌株鉴定。【结果】从新疆蜜蜂蜂巢中的土著大蜡螟肠道分离获得的聚乙烯降解菌菌株最多,其聚乙烯的降解效率高于其他来源的分离菌株。从中筛选出具有较高降解能力的3个菌株XJDLM-3、XJDLM-8和XJDLM-12,它们能利用PE膜片生长,扫描电镜观察经过30 d降解的PE膜片表面出现明显的侵蚀孔洞和裂痕,红外扫描图谱发生改变,拉伸强度、断裂伸长率和弹性模量等力学性能显著下降,膜片失重率分别达到了8.06%、5.66%和5.39%。从新疆蜜蜂蜂巢中的土著大蜡螟肠道分离出降解效果较好的细菌菌株,经鉴定XJDLM-8和XJDLM-12为Bacillus cereus,XJDLM-3为Enterobacter bugandensis。【结论】证明了新疆蜜蜂蜂巢中的土著大蜡螟肠道存在对PE具有较高降解能力的菌株,丰富了PE降解菌的菌种资源,在PE地膜降解中具有开发应用的潜力。     

Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging flow cytometry for the study of nuclear reprogramming

Fusion of somatic cells to embryonic stem cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell over the differentiated nucleus. However, fusion only occurs in a small fraction of the cells exposed to fusogenic conditions, hence the need for a protocol that produces high fusion rate with minimal cell damage, coupled with a method capable of identifying and selecting these rare events. Here, we describe a protocol to induce formation of bi-species mouse pluripotent/bovine somatic heterokaryons, as well as same-species homokaryons, using polyethylene glycol (PEG). To identify bi-species fusion products, heterokaryons were labeled using cell type-specific fluorescent antibodies and selected using imaging (Amnis ImageStream Mark II) and traditional (BD FACSAria I) flow cytometry. Heterokaryons selected with this method produced ES cell-like colonies in vitro. This procedure can be combined with downstream applications such as nucleic acid isolation for RT-PCR and RNA-Seq, and used as a tool to study somatic cell nuclear reprogramming.     

Metal ion separations in polyethylene glycol-based aqueous biphasic systems: correlation of partitioning behavior with available thermodynamic hydration data

Solvent extraction, utilizing an oil-water mixture (e.g, chloroform-water) and a suitable complexant, is a proven technology for the selective removal and recovery of metal ions from aqueous solutions. Aqueous biphasic systems (ABS), formed by mixing certain inorganic salts and water-soluble polymers, or by mixing two dissimilar water-soluble polymers, have been studied for more than 40 years for the gentle, non-denaturing separation of fragile biomolecules, yet ABS have been virtually ignored as a possible extraction technology for metal ions. In this report we review our metal ion partitioning work and discuss the three major types of partitioning: (1) those rare instances that the metal ion species present in a given solution partitions to the PEG-rich phase without an extractant; (2) the use of halide salts which produce a metal anion complex that partitions to the PEG-rich phase; and (3) the use of a water-soluble extractant which distributes to the PEG-rich phase. In addition, we correlate the partitioning behavior we observed with available thermodynamic data for metal ions and their complexes.     

Chemical modification of proteases for wool cuticle scale removal

Over the last few decades several enzymatic processes to improve properties of wool fabrics like felting tendency, shrink resistance, dyeing ability and handling characteristics have been described. Previous investigations into the use of proteases to hydrolyse the cuticles at the surface of wool fibres, resulted in high strength and weight losses. Therefore restriction of the enzyme activity to the wool surface or control of enzyme diffusion to the cortex cells is required.

To change the diffusion behaviour of proteases in wool fibres, the soluble polymer PEG was covalently attached to a protease from Bacillus lentus. Modified enzymes with different molecular weights were compared. These modified enzymes retained up to 80% of their activity in the standard assay while hydrolysis of wool fibres was successfully restricted to cuticles, resulting in a 90% decrease in weight losses compared to non-modified enzymes.     

Pore size of swelling-activated channels for organic osmolytes in Jurkat lymphocytes, probed by differential polymer exclusion

The present study explores the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200-1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions. To this end, we analyzed the cell volume responses to PEG-substituted solutions of different osmolalities (100-300 mOsm) using video microscopy. In parallel experiments, the osmotically induced changes in the membrane capacitance and cytosolic conductivity were measured by electrorotation (ROT). Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane remained impermeable to PEG300-1500, which allowed the cells to accomplish regulatory volume decrease (RVD). During RVD, lymphocytes released intracellular electrolytes through the swelling-activated pathways, as proved by a decrease of the cytosolic conductivity measured by electrorotation. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600-1500, whereas 100 mOsm solutions of PEG300-400 inhibited RVD in Jurkat cells. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300-400, but not to PEG600-1500. The swelling-activated channels conducting PEG300-400 were inserted into the plasma membrane from cytosolic vesicles via swelling-mediated exocytosis, as suggested by an increase of the whole cell capacitance. Using the hydrodynamic radii R_h of PEGs (determined by viscosimetry), the observed size-selectivity of membrane permeation yielded an estimate of ∼ 0.74 nm for the cut-off radius of the swelling-activated channel for organic osmolytes. Unlike PEG300-1500, the smallest PEG (PEG200, R_h = 0.5 nm) permeated the lymphocyte membrane under isotonic conditions thus leading to a continuous isotonic swelling. The results are of interest for biotechnology and biomedicine, where PEGs are widely used for cryopreservation of cells and tissues.     

Substrate preferences of scyphozoan <Emphasis Type="Italic">Aurelia labiata</Emphasis> polyps among common dock-building materials

New habitat on proliferating marine construction may increase jellyfish polyp populations, and thereby increase jellyfish populations worldwide. In this investigation, we examined planula settlement and polyp immigration rates of the scyphozoan Aurelia labiata Chamisso & Eysenhardt, 1821 on six common dock-building materials. The planulae and polyps preferred plastics (expanded polystyrenes, low and high density polyethylene) to rubber and treated wood when choosing habitat on man-made surfaces. Substrate surface texture and the presence/absence of anti-fouling chemicals are discussed as possible causes for these substrate preferences. This study illustrates the potential effects of different man-made structures on jellyfish populations, and provides useful information to coastal managers and port authorities for reduction of biofouling and jellyfish bloom effects. Guest editors: K. A. Pitt & J. E. Purcell Jellyfish Blooms: Causes, Consequences, and Recent Advances     

Effect of Surfactants on Activity and Stability of Native and Chemically Modified Lipases A and B from Candida Rugosa

The effect of sodium dodecyl sulfate (SDS) and Triton X-100 on the hydrolytic activity of lipases A and B from Candida rugosa has been studied. Lipase B is significantly more affected than lipase A by the presence of both surfactants; Triton X-100 produces a more deleterious effect than SDS with both isoenzymes. In addition, the stability of lipases A and B in the presence of different concentrations of SDS was investigated; lipase A was more stable than isoform B. Both isoenzymes were chemically modified by reaction of their amino groups with octanoyl chloride or activated polyethylene glycol (PEG, mol. wt. 5000). In all cases the modification produced a protective effect against denaturation by SDS. In particular, PEG₅₀₀₀-liPases A and B were significantly more stable (stabilization factor: 3-4) than the native enzymes at the surfactant concentrations tested.