Preparation of highly infective Leishmania promastigotes by cultivation on SNB-9 biphasic medium

Protozoan hemoflagellates Leishmania are causative agents of leishmaniases and an important biological model for study of host-pathogen interaction. A wide range of methods of Leishmania cultivation on both biphasic and liquid media is available. Biphasic media are considered to be superior for initial isolation of the parasites and obtaining high promastigote infectivity; however, liquid media are more suitable for large-scale experiments. The aim of the present study was the adaptation and optimization of the cultivation of Leishmania promastigotes on a biphasic SNB-9 (saline-neopeptone-blood 9) medium that was originally developed for Trypanosoma cultivation and combines the advantages of biphasic and liquid media. SNB-9 medium is characterized with a large volume of the liquid phase, which facilitates the manipulation with the culture and provides parasite yields comparable to parasite yields on such liquid medium as Schneider's Insect Medium. We demonstrate that SNB-9 very considerably surpasses Schneider's Insect Medium in in vitro infectivity of the parasites. Additionally, we show that the ratio of apoptotic parasites, which are important for the infectivity of the inoculum, in Leishmania culture in SNB-9 is higher than in Leishmania culture in Schneider's Insect Medium. Thus, we demonstrate that the cultivation of Leishmania on SNB-9 reliably yields highly infective promastigotes suitable for experimental infection.     

The nutritive value of concentrate feedstuffs for ruminant animals: Part II: In situ ruminal degradability of crude protein

The objective of the study was to generate a database of in situ ruminal degradability of crude protein (CP) from different samples of a range of concentrate feedstuffs comprising protein, energy or protein+energy feeds commonly offered to ruminant animals in European countries. The in situ disappearance of CP was calculated using the in situ nylon bag technique where the test feedstuffs were subject to ruminal incubation in four Friesian steers offered grass silage and concentrate. Disappearance of CP from the test feeds from the rumen was measured at 0, 2, 4, 8, 14, 24 and 48 h. The exponential model of Ørskov and McDonald (1979) was used to measure degradation kinetics of CP. Test protein feeds were sunflower meal (SUN), rapeseed meal (RAP), soyabean meal (SBM) and cottonseed meal (CSM). Test energy feeds were palm kernel meal (PK), pollard (PO), barley (BA) and beet pulp (BP). Test protein+energy feeds were maize distillers grains (MDG), maize gluten feed (MGF), copra meal (CO) and malt combings (MC). The effective degradability of CP (EDP) in the protein feed SBM, where outflow rate (k)=0.02 was not influenced (P>0.05) by the sample of feed used. For the energy feeds, the EDP in PO for k=0.05 h⁻¹, 0.06 h⁻¹ and 0.08 h⁻¹ and EDP in BA for k=0.08 h⁻¹ were not significantly affected (P>0.05) by the sample of feed used. The data has shown that for the majority of feeds examined in this study, the different samples of any one feed are not equal in nutritive value and it is necessary to screen feeds for nutritive value before using them in ration formulation systems.     

Physiological and biochemical characterization of a suspensionculture system for sustained exponential growth of Nicotiana silvestris

A strong approach to understanding the regulation of enzymes in metabolic pathways, such as those responsible for amino acid biosynthesis, is to follow enzyme levels throughout the growth curve of higher plant cells in suspension culture. The rise and fall of enzyme levels can be traced as a function of physiological stage of growth Subculturing, as typically carried out by low-factor dilution of stationary phase cells, yields a system suitable for the study of changes in enzyme and metabolite levels that accompany the transition from stationary-phase physiology to exponential-phase physiology. However, the short duration of exponential growth in such subculture protocols is inadequate to avoid carryover effects from previous stationary-phase physiology. Suspension cultures of Nicotiana silvestris Speg, et Comes (2N = 24) were used to demonstrate substantial carryover levels of acid phosphatase, alkaline phosphatase and protease activities. A subculture routine is described for maintaining cell populations in exponential phase indefinitely. About 10 generations of sustained exponential growth is required to approach a true balanced state of exponential growth. Such exponential phase populations consist of cells termed EE cells. EE-cell populations were similar to cells that have been in exponential phase for only a few generations (E cells), with respect to doubling time (about 40 h) and to minimal density of diluted populations able to resume growth (about 500 cells ml^?1). EE cells possess a high content of soluble protein; they are smaller and more aggregated than are E cells. Upon dilution into fresh medium, EE cells resume exponential growth without a lag. In contrast to E cells, EE cells exhibit properties of balanced growth, since proportionate increases in cell number, dry weight, wet weight and packed-cell volume were observed. E cells, sampled at different elapsed times of growth, are likely to differ in metabolite, enzyme and cell properties, whereas EE cells exhibit near-constant properties.     

A new regime for highly robust gamma oscillation with co-exist of accurate and weak synchronization in excitatory–inhibitory networks

A great number of biological experiments show that gamma oscillation occurs in many brain areas after the presentation of stimulus. The neural systems in these brain areas are highly heterogeneous. Specifically, the neurons and synapses in these neural systems are diversified; the external inputs and parameters of these neurons and synapses are heterogeneous. How the gamma oscillation generated in such highly heterogeneous networks remains a challenging problem. Aiming at this problem, a highly heterogeneous complex network model that takes account of many aspects of real neural circuits was constructed. The network model consists of excitatory neurons and fast spiking interneurons, has three types of synapses (GABA_A, AMPA, and NMDA), and has highly heterogeneous external drive currents. We found a new regime for robust gamma oscillation, i.e. the oscillation in inhibitory neurons is rather accurate but the oscillation in excitatory neurons is weak, in such highly heterogeneous neural networks. We also found that the mechanism of the oscillation is a mixture of interneuron gamma (ING) and pyramidal-interneuron gamma (PING). We explained the mixture ING and PING mechanism in a consistent-way by a compound post-synaptic current, which has a slowly rising-excitatory stage and a sharp decreasing-inhibitory stage.     

Inhibition of Cancer Cell Adhesion,Migration and Proliferation by a Bispecific Antibody that Targets two Distinct Epitopes on αv Integrins

Members of the αv family of integrins regulate activation of transforming growth factor beta (TGFβ) and are directly involved in pro-tumorigenic phenotypes. Thus, αv integrins may be therapeutic targets for fibrosis and cancer, yet the isolation of selective inhibitors is currently a challenge. We generated synthetic antibodies selective for αv integrins by phage display selections on cell lines that displayed integrin heterodimers. We identified antibodies that targeted two distinct epitopes on cell-surface αv integrins and partially inhibited cell adhesion mediated by interactions between integrins and the latency-associated peptide, part of the pro-form of TGFβ. Using the isolated antibody paratope sequences we engineered a bispecific antibody capable of binding to both epitopes simultaneously; this antibody potently and completely inhibited cell adhesion mediated by integrins αvβ1, αvβ3 and αvβ5. In addition, the bispecific antibody inhibited proliferation and migration of lung carcinoma lines, where the highest and lowest potencies observed correlated with integrin-αv cell surface expression levels. Taken together, our results demonstrate that phage display selections with live cells can yield high quality anti-integrin antibodies, which we used as biparatopic building blocks to construct a bispecific antibody that strongly inhibited integrin function and may be a therapeutic candidate for cancer and fibrosis.     

Impact of mitochondriotropic quercetin derivatives on mitochondria

Mitochondria-targeted polyphenols are being developed with the intent to intervene on the levels of reactive oxygen species (ROS) in mitochondria. Polyphenols being more than just anti-oxidants, the interaction of these derivatives with the organelles needs to be characterised. We have studied the effects of two quercetin derivatives, 3-(4-O-triphenylphosphoniumbutyl)quercetin iodide (Q3BTPI) and its tetracetylated analogue (QTA3BTPI), on the inner membrane aspecific permeability, transmembrane voltage difference and respiration of isolated rat liver mitochondria. While the effects of low concentrations were too small to be reliably defined, when used in the 5-20 μM range these compounds acted as inducers of the mitochondrial permeability transition (MPT), an effect due to pro-oxidant activity. Furthermore, Q3BTPI behaved as an uncoupler of isolated mitochondria, causing depolarisation and stimulating oxygen consumption. When applied to tetramethylrhodamine methyl ester (TMRM)-loaded HepG2 or Jurkat cells uptake of the compounds was predictably associated with a loss of TMRM fluorescence, but there was no indication of MPT induction. A production of superoxide could be detected in some cells upon prolonged incubation of MitoSOX®-loaded cells with QTA3BTPI. The overall effects of these model mitochondriotropic polyphenols may thus differ considerably depending on whether their hydroxyls are protected or not and on the experimental system. In vivo assays will be needed for a definitive assessment of their bioactivities.     

4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.     

Hydrophobic matching controls the tilt and stability of the dimeric platelet-derived growth factor receptor (PDGFR) β transmembrane segment

The platelet-derived growth factor receptor β is a member of the cell surface receptor tyrosine kinase family and dimerizes upon activation. We determined the structure of the transmembrane segment in dodecylphosphocholine micelles by liquid-state NMR and found that it forms a stable left-handed helical dimer. Solid-state NMR and oriented circular dichroism were used to measure the tilt angle of the helical segments in macroscopically aligned model membranes with different acyl chain lengths. Both methods showed that decreasing bilayer thickness (DEPC-POPC-DMPC) led to an increase in the helix tilt angle from 10° to 30° with respect to the bilayer normal. At the same time, reconstitution of the comparatively long hydrophobic segment became less effective, eventually resulting in complete protein aggregation in the short-chain lipid DLPC. Unrestrained molecular dynamics simulations of the dimer were carried out in explicit lipid bilayers (DEPC, POPC, DMPC, sphingomyelin), confirming the observed dependence of the helix tilt angle on bilayer thickness. Notably, molecular dynamics revealed that the left-handed dimer gets tilted en bloc, whereas conformational transitions to alternative (e.g. right-handed dimeric) states were not supported. The experimental data along with the simulation results demonstrate a pronounced interplay between the platelet-directed growth factor receptor β transmembrane segment and the bilayer thickness. The effect of hydrophobic mismatch might play a key role in the redistribution and activation of the receptor within different lipid microdomains of the plasma membrane in vivo.     

平衡超滤技术对小儿先心病术后炎症因子、凝血功能及肺功能的影响

目的:探讨平衡超滤技术对小儿先心病术后炎症因子、凝血功能及肺功能的影响。方法:选择2014年9月至2017年9月我院接诊的100例先天性心脏病患儿进行研究,通过随机数表法分为观察组55例和对照组45例,两组均于体外循环下实施心内直视手术,观察组在体外循环启动后患儿复温时开始平衡超滤,并于体外循环结束后即刻进行改良超滤,对照组仅在体外循环结束后即刻进行改良超滤。比较两组不同时间点炎症因子、凝血功能及肺功能的变化、术后恢复情况及并发症。结果:于体外循环结束后(术后)20 min(T1)、术后2 h(T2)、术后6 h(T3)各时点,观察组血清肿瘤坏死因子(TNF)-α、白介素(IL)-6、IL-10均明显比对照组低(P<0.05);观察组各时点活化部分凝血活酶时间(APTT)、凝血酶时间(TT)、纤维蛋白原(FIB)均明显低于对照组(P<0.05);观察组各时点肺静脉顺应性(Cstat)、氧合指数(OI)在各时点均明显高于对照组,肺泡-动脉氧分压梯度(AaDO2)明显低于对照组(P<0.05);观察组血管活性药使用时间、呼吸机使用时间和ICU住院时间均明显比对照组短(P<0.05),观察组感染、弥散性血管性凝血、肺功能损伤等发生率明显低于对照组(P<0.05)。结论:在改良超滤技术上,联合平衡超滤更有助于减轻小儿先心病术后炎症因子的释放,具有较好的凝血功能、肺功能保护作用,可有效促进术后恢复,减少围术期并发症。