Recombinant diphtheria toxin derivatives: Perspectives of application

Diphtheria toxin (DT) is a unique bacterial protein which consists of three domains with various biological functions. Using genetic engineering for the creation of various recombinant constructions of DT with definite features, it is possible to create unique tools for cellular biology and toxins with efficient and selective action on certain populations of cells. The review highlights the structural and functional aspects of the DT molecule, its fragments and domains, as well as the major areas of application of its recombinant derivatives. In particular, the perspectives for practical use of recombinant DT derivatives are discussed for creating immunobiological preparations, cytotoxins, blockers of the heparin-binding epidermal growth factor-like growth factor (HB-EGF), protein constructions for direct delivery of substances into the cell, and also the possibility to use DT recombinant derivatives for therapy and prevention of a number of diseases.     

Overview of Cell Models: From Organs Cultured in a Petri Dish to Organs-on-Chips

In this review, we tried to elucidate the origin and development of different animal and human cell culture methodologies used to evaluate the effects of various factors and substances in vitro. Organ cultures and conventional two-dimensional cultures of dissociated cells of various types, such as primary, tumor, induced pluripotent, stem, etc., have their advantages and drawbacks but usually do not represent accurate models for studying biological processes that take place in living organisms. Nowadays, high-throughput cell assays on the basis of various methods of signal detection (optical utilizing colorimetric, luminescent and fluorescent methods of detection, and electrochemical) are widely used at early stages of drug development for selection of the most active compounds and evaluation of their cytotoxic effects. The use of animals as models for drug testing is being criticized because of the lack of correlation between the results obtained in studies on them and on humans, and also because of the high cost and ethical issues. Therefore, much effort is put to create models based on human cells. This is how cultures emerged that utilize a three-dimensional network to simulate the architecture of tissues in vivo, and then so-called organs-on-chips—microfluidic microfabricated devices combining several types of cells—that replicate physical and chemical parameters of the microenvironment of cells in living organisms. In summary, experimental cell models have come a long way from the whole organs cultivated in a growth medium to almost complete reconstruction of organs in vitro based on the cutting-edge engineering approach with the use of different cell types. This currently enables one to replicate complex biological processes and study the influence of different substances and factors on them more successfully.     

动物细胞代谢工程进展

动物细胞被越来越广泛地用于工业生产,一些现代化企业已采用分子生物学技术,将一些比较重要的基因导入动物细胞,生产具有医用价值的药物。但该技术并未成熟,主要是因为体外培养的细胞,其生长代谢及生理模式都比较复杂,而且细胞的应答机制还受外界因素的影响。因此,采用细胞代谢工程手段,提高体外培养细胞的生长率、产品产率及有效性,成为人们追求的新目标。我们从细胞代谢中心途径、抑制细胞调亡的因素、细胞生长周期的控制及其相关代谢、多基因共表达代谢工程及糖基化代谢工程等方面对代谢工程进行阐述,为动物细胞的培养提供新的思路。     

Metabolic engineering of animal cells

Animal cells are widely used in industrial processes as sophisticated cell factories to obtain a high number of complex proteins with correct post-translation modifications and biological activity, with many applications in diagnostic and therapeutic uses. However, from the bioprocess point of view these are still sub-optimal processes, mainly due to the complex requirements for the in vitro growth of the cells, their metabolic and physiological patterns, and the response of mechanisms developed for in vivo growth to the external conditions found in culture in vitro. Metabolic engineering, combined with the corresponding redesign of the process itself, offers the possibility to the enhance the performance of animal cells grown in in vitro systems, targeting how to redesign the cells themselves to make them more robust, efficient, and productive. This paper reviews efforts made in this direction, and how the metabolic engineering of animal cells has been approached to reshape their profiles in various key aspects, namely: central metabolism, protection of apoptosis, regulation of cell cycle, and finally, the combined engineering of different aspects.     

供体细胞与哺乳动物体细胞核移植

哺乳动物体细胞核移植(克隆)技术在转基因动物生产、珍稀动物资源复原与保护、生物学基础研究等方面业已显示出重要的应用价值,而目前该技术还与诱导多能干细胞技术一同被认为是创制患者特异性多能干细胞,为再生医学临床"细胞治疗"提供素材的最佳手段。但是,体细胞克隆的效率仍不理想,关键机制还不清楚,严重制约了该技术的推广。因此,如何提高克隆效率已成为人们普遍关心的首要问题。在体细胞克隆技术所涉及的各环节中,供体细胞是影响克隆效率的最关键因素之一。该文从供体细胞的生物学因素和技术因素两方面进行了回顾,旨在为进一步探寻建立物种或供体细胞个性化准备方案,为提高动物克隆效率提供参考。     

Cell and molecular biology of intervertebral disc degeneration: current understanding and implications for potential therapeutic strategies

Intervertebral disc degeneration (IDD) is a chronic, complex process associated with low back pain; mechanisms of its occurrence have not yet been fully elucidated. Its process is not only accompanied by morphological changes, but also by systematic changes in its histological and biochemical properties. Many cellular and molecular mechanisms have been reported to be related with IDD and to reverse degenerative trends, abnormal conditions of the living cells and altered cell phenotypes would need to be restored. Promising biological therapeutic strategies still rely on injection of active substances, gene therapy and cell transplantation. With advanced study of tissue engineering protocols based on cell therapy, combined use of seeding cells, bio‐active substances and bio‐compatible materials, are promising for IDD regeneration. Recently reported progenitor cells within discs themselves also hold prospects for future IDD studies. This article describes the background of IDD, current understanding and implications of potential therapeutic strategies.     

Drug delivery and nanoparticles:applications and hazards

The use of nanotechnology in medicine and more specifically drug delivery is set to spread rapidly. Currently many substances are under investigation for drug delivery and more specifically for cancer therapy. Interestingly pharmaceutical sciences are using nanoparticles to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient. The kind of hazards that are introduced by using nanoparticles for drug delivery are beyond that posed by conventional hazards imposed by chemicals in classical delivery matrices. For nanoparticles the knowledge on particle toxicity as obtained in inhalation toxicity shows the way how to investigate the potential hazards of nanoparticles. The toxicology of particulate matter differs from toxicology of substances as the composing chemical(s) may or may not be soluble in biological matrices, thus influencing greatly the potential exposure of various internal organs. This may vary from a rather high local exposure in the lungs and a low or neglectable exposure for other organ systems after inhalation. However, absorbed species may also influence the potential toxicity of the inhaled particles. For nanoparticles the situation is different as their size opens the potential for crossing the various biological barriers within the body. From a positive viewpoint, especially the potential to cross the blood brain barrier may open new ways for drug delivery into the brain. In addition, the nanosize also allows for access into the cell and various cellular compartments including the nucleus. A multitude of substances are currently under investigation for the preparation of nanoparticles for drug delivery, varying from biological substances like albumin, gelatine and phospholipids for liposomes, and more substances of a chemical nature like various polymers and solid metal containing nanoparticles. It is obvious that the potential interaction with tissues and cells, and the potential toxicity, greatly depends on the actual composition of the nanoparticle formulation. This paper provides an overview on some of the currently used systems for drug delivery. Besides the potential beneficial use also attention is drawn to the questions how we should proceed with the safety evaluation of the nanoparticle formulations for drug delivery. For such testing the lessons learned from particle toxicity as applied in inhalation toxicology may be of use. Although for pharmaceutical use the current requirements seem to be adequate to detect most of the adverse effects of nanoparticle formulations, it can not be expected that all aspects of nanoparticle toxicology will be detected. So, probably additional more specific testing would be needed.     

Some new methodological aspects of the hen's egg test for micronucleus induction (HET-MN)

In a previous publication we introduced the hen's egg test for micronucleus induction (HET-MN) as an extremely simple, inexpensive and rapid animal free genotoxicity assay which is positioned between pure in vitro and in vivo assays, strictly in line with animal protection regulations and ethical aspects. The HET-MN combines the use of the commonly accepted genetic endpoint "formation of micronuclei" with the well characterized and complex model of the chick embryo. The high metabolic competency provided by this model enables metabolic activation, elimination and excretion of xenobiotics including mutagens and promutagens.In this paper we present some new methodological aspects, which are important for improving the experimental protocol. We used cyclophosphamide (CP) and 7,12-dimethyl-benz[a]anthracene (DMBA) as model substances. Dose-response-relationship for both chemicals and cytotoxic effects for CP are described. In addition to the standard proliferation marker PCE/NCE-ratio we found an increased frequency of primitive erythrocytes (E I) and the appearance of proerythroblasts and erythroblasts as further alerting signals for cytotoxic or erythrosuppressive effects. From the total cell population we could further qualify the group of target cells. We found that all definite erythrocytes (E II), observed at day 11 (d11), are relevant target cells, independent from their stage of maturity (polychromatic as well as normochromatic definite erythrocytes). E I cells do not belong to the group of target cells, however. An additional important methodological aspect is the optimal time frame. We found the time period from d8 of incubation (administration of the test substance) up to d11 (time point of blood sampling) as most favorable. In this way an exposure period of up to 72h is covered. Further results indicate that the air cell route provides a higher response to the test substances than the albumen route. The consideration of the described methodological aspects will contribute to the improvement of the experimental protocol of the HET-MN.     

My pigs are ok,why change? – animal welfare accounts of pig farmers

Intensive pig production systems are a source of stress, which is linked to reduced animal welfare and increased antimicrobial use. As the gatekeepers of the welfare of the animals under their care, farmers are seen as the stakeholder responsible for improving animal welfare. The aim of this study was to explore the knowledge and attitudes of pig farmers towards pig welfare and the impact of such attitudes on farmers' selection of management strategies on the farm. We conducted in-depth semi-structured interviews with 44 pig farmers in one of the main pig producing regions of Brazil. Interviews covered knowledge and attitudes towards pig sentience and behaviour and welfare-related issues commonly observed in intensive pig farms (belly-nosing, fights, tail-biting, diarrhoea and castration without pain control) and farmers' conception and attitudes towards pig welfare. We identified many management and animal-based indicators of poor welfare, such as the use of painful and stressful management practices and use of environments that limit the expression of natural behaviours. However, most farmers were satisfied with animal welfare standards at their farms. Farmers' perceptions are aligned with their understanding of animal welfare. Although they identified all the dimensions that impact the welfare of a pig on a farm (affect, biological functioning and naturalness), their social reality, industry demands and available advice pushed them to perceive their range of action limited to biological and environmental aspects of the animals that do not necessarily benefit affective state. This precluded farmers from making associations between good health and the animal's ability to express a full behavioural repertoire, as well as from viewing abnormal behaviours as problems. The negative consequences for the welfare of the animals were commonly alleviated by routines that relied on constant use of medication, including high dependence on antibiotics. Expressions of estrangement from the production chain were common voices among the participants. This suggests that farmers may not be sufficiently informed or engaged in responding to consumers' expectations and commitments made by companies, which can pose a severe economic risk for farmers. The findings of this study indicate that economic, technical and social factors restrict farmers' autonomy and their ability to perform their role as stewards of animal welfare. (Re)connecting different human, animal and environmental interests may be a step to changing this scenario.     

Structured modelling of animal cells

Recent advances in computer technology have promoted the design and use of detailed, computer-based models for biological systems. For many non-biological systems, the complexity of such simulations may be considered inappropriate and unwieldy, but in biological systems, and more specifically in animal cell culture, this level of complexity simply mimics what is only beginning to be understood about metabolic processs. With this in mind, we contend that complex, structured models are vital tools in the investigation of fundamental biological processes. An example of such a simulation, which describes the commercial production of therapeutic proteins by animal cell cultures, is considered.     

A poor imitation of a natural process: A call to reconsider the iPSC engineering technique

Reprogramming somatic cells into a pluripotent state is expected to initiate a new era in medicine. Because the precise underlying mechanism of reprogramming remains unclear, many efforts have been made to optimize induced pluripotent stem cell (iPSC) engineering. However, satisfactory results have not yet been attained. In this review, we focus on recent roadblocks in iPSC reprogramming engineering, such as the inefficiency of the process, tumorigenicity and heterogeneity of the generation. We conclude that cell reprogramming is a naturally occurring phenomenon rather than a biological technique. We will only be able to mimic the natural process of reprogramming when we fully understand its underlying mechanism. Finally, we highlight the alternative method of direct conversion, which avoids the use of iPSCs to generate cell materials for patient-specific cell therapy.     

A poor imitation of a natural process

Reprogramming somatic cells into a pluripotent state is expected to initiate a new era in medicine. Because the precise underlying mechanism of reprogramming remains unclear, many efforts have been made to optimize induced pluripotent stem cell (iPSC) engineering. However, satisfactory results have not yet been attained. In this review, we focus on recent roadblocks in iPSC reprogramming engineering, such as the inefficiency of the process, tumorigenicity and heterogeneity of the generation. We conclude that cell reprogramming is a naturally occurring phenomenon rather than a biological technique. We will only be able to mimic the natural process of reprogramming when we fully understand its underlying mechanism. Finally, we highlight the alternative method of direct conversion, which avoids the use of iPSCs to generate cell materials for patient-specific cell therapy.     

The prospects of modifying the antimicrobial properties of milk

Milk contains a variety of substances, which inhibit the infection of pathogens. This is of benefit to the mother, safeguarding the integrity of the lactating mammary gland, but also of huge importance for protection of the suckling offspring. The antimicrobial substances in milk can be classified into two categories. First, nonspecific, broad-spectrum defense substances, which have evolved over long periods of time, and secondly, substances like antibodies, which are specifically directed against particular pathogens and have developed during the mother's lifetime. Substances in both categories may be targets for biological intervention and manipulation with the goal of improving the antimicrobial properties of milk. These alterations of milk composition have applications in human as well as in animal health.     

Primary cell culture from the nose of a marine organism, the banded houndshark, Triakis scyllium

Animal cells have been widely and continuously studied due to their usefulness in biological researches and production of pharmacological agents as well as food additives. Nevertheless, there are several problems such as the existence of viruses which introduce the possibility of mammalian-infection. In this reason, recently, animal cells derived from marine organisms have emerged to overcome these problems by many researches. However, marine animal-derived cells have not yet been well developed. The banded hound shark occupies an important position in an evolutionary perspective and currently a few biological substances have been used for medicines or food additives such as shark squalene and cartilage extract. In this study, primary cells were cultivated from a banded hound shark nose containing many extracellular matrix (ECM) materials. After successful isolation of one type of primary nasal cell, we optimized culture conditions including coating materials, media composition, serum concentrations and pH. Additionally, these cells demonstrated proliferation ability in vitro, generating secretions of collagen and sulfated polysaccharides. The cultivated primary cells are useful in the study of cellular biology and may be used to create a variety of ECM-originated bioactive substances.     

Microfluidics meet cell biology: bridging the gap by validation and application of microscale techniques for cell biological assays

Microscale techniques have been applied to biological assays for nearly two decades, but haven't been widely integrated as common tools in biological laboratories. The significant differences between several physical phenomena at the microscale versus the macroscale have been exploited to provide a variety of new types of assays (such as gradient production or spatial cell patterning). However, the use of these devices by biologists seems to be limited by issues regarding biological validation, ease of use, and the limited available readouts for assays done using microtechnology. Critical validation work has been done recently that highlights the current challenges for microfluidic methods and suggest ways in which future devices might be improved to better integrate with biological assays. With more validation and improved designs, microscale techniques hold immense promise as a platform to study aspects of cell biology that are not possible using current macroscale techniques.     

国际前沿

高伟坚博士,英籍华人,1986年出生于英国。18岁以全优成绩考取曼彻斯特大学生命科学院。2008年以优等生荣誉毕业,获学士学位。2012年,获得该校博士学位。2004～2012年,先后获得“默沙东杰出学术成就奖”、英联邦帕金森氏病学会的“杰出研究和展示奖”、“曼彻斯特领袖项目”金奖、双重博士奖学金。他从小热爱生物医学,经过系统求学过程及严格的科研训练,在科研课题设计和科技论文撰写方面,表现出不菲的成绩。毕业后仅仅两年,便发表有影响的科技论文4篇。并参与Springer 组织的‘L-DOPA-induced dyskinesia in Parkinson ’ s disease ’一书的编撰工作。同时,还组织申请国际合作课题两项,参研课题四项。高博士现供职于法国波尔多第二大学,在华开展联合研发工作期间,多次表达了“自己作为华人,愿意为祖国生物医学发展贡献自己力量的想法”。也深知华人科学家因为语言障碍,而在科技论文发表和科研课题申请上遇到的重重困难。经我刊编委推荐及编辑部与部分专家讨论商议,特聘请高博士为两刊特约通讯员,开设专栏,希望高博士从信息获取、论文撰写、课题设计等方面开展工作,并定期向我刊介绍业内国际前沿动态,为我刊读者扩大视角。 本期推出神经科学研究中的动物选择和模型制作,欢迎读者就相关内容展开互动。     

Kinetic models of hemopoietic stem cell populations

Summary Recent techniques enabled a series of quantitative studies to be made with various aspects of the stem cell functions. Results from several laboratories appear to agree on certain main points: the existence of a pluripotential stem cell in the small rodent, capable of forming visible colonies of hemopoietic foci in the spleen and the existence of some undifferentiated but committed precursor cells from which the differentiating and maturing cell populations originate. There is evidence that the primary stem cell is in a low turnover state in the normal animal, although on demand it is capable of fast and prolonged proliferative activity. The committed undifferentiated precursor cells differ greatly, depending on which cell line they represent. Some of these are in high turnover state in the normal animal (e.g., erythropoietin-responsive cells), others do not appear to be capable of proliferation (e.g., focus forming cells). Perturbation of the steady states by irradiation of the animal, or by treatment with cytotoxic drugs results in recovery patterns which yield valuable kinetic information.     

Biological matrices and bionanotechnology

A crucial step towards the goal of tissue engineering a heart valve will be the choice of scaffold onto which an appropriate cell phenotype can be seeded. Successful scaffold materials should be amenable to modification, have a controlled degradation, be compatible with the cells, lack cytotoxicity and not elicit an immune or inflammatory response. In addition, the scaffold should induce appropriate responses from the cells seeded onto it, such as cell attachment, proliferation and remodelling capacity, all of which should promote the formation of a tissue construct that can mimic the structure and function of the native valve. This paper discusses the various biological scaffolds that have been considered and are being studied for use in tissue engineering a heart valve. Also, strategies to enhance the biological communication between the scaffold and the cells seeded onto it as well as the use of bionanotechnology in the manufacture of scaffolds possessing the desired properties will be discussed.     

电场刺激技术在微生物工程中的应用

电场刺激是利用电解法将惰性电极插入电解液中,形成一种电解池系统,电解液对细胞培养物会产生不同影响,并导致生物过程发生改变。电场刺激对微生物会产生促进和杀灭两种不同作用,并在微生物工程中已有所应用,如促进微生物生长和代谢、强化废水处理、进行生物修复和用于杀菌消毒等。研究表明电场刺激在微生物工程和环境生物工程等方面有广泛的应用前景。