Multifactorial Optimization of Contrast-Enhanced Nanofocus Computed Tomography for Quantitative Analysis of Neo-Tissue Formation in Tissue Engineering Constructs

To progress the fields of tissue engineering (TE) and regenerative medicine, development of quantitative methods for non-invasive three dimensional characterization of engineered constructs (i.e. cells/tissue combined with scaffolds) becomes essential. In this study, we have defined the most optimal staining conditions for contrast-enhanced nanofocus computed tomography for three dimensional visualization and quantitative analysis of in vitro engineered neo-tissue (i.e. extracellular matrix containing cells) in perfusion bioreactor-developed Ti6Al4V constructs. A fractional factorial ‘design of experiments’ approach was used to elucidate the influence of the staining time and concentration of two contrast agents (Hexabrix and phosphotungstic acid) and the neo-tissue volume on the image contrast and dataset quality. Additionally, the neo-tissue shrinkage that was induced by phosphotungstic acid staining was quantified to determine the operating window within which this contrast agent can be accurately applied. For Hexabrix the staining concentration was the main parameter influencing image contrast and dataset quality. Using phosphotungstic acid the staining concentration had a significant influence on the image contrast while both staining concentration and neo-tissue volume had an influence on the dataset quality. The use of high concentrations of phosphotungstic acid did however introduce significant shrinkage of the neo-tissue indicating that, despite sub-optimal image contrast, low concentrations of this staining agent should be used to enable quantitative analysis. To conclude, design of experiments allowed us to define the most optimal staining conditions for contrast-enhanced nanofocus computed tomography to be used as a routine screening tool of neo-tissue formation in Ti6Al4V constructs, transforming it into a robust three dimensional quality control methodology.     

Hyperosmolaric contrast agents in cartilage tomography may expose cartilage to overload-induced cell death

In clinical arthrographic examination, strong hypertonic contrast agents are injected directly into the joint space. This may reduce the stiffness of articular cartilage, which is further hypothesized to lead to overload-induced cell death. We investigated the cell death in articular cartilage while the tissue was compressed in situ in physiological saline solution and in full strength hypertonic X-ray contrast agent Hexabrix^TM. Samples were prepared from bovine patellae and stored in Dulbecco’s Modified Eagle’s Medium overnight. Further, impact tests with or without creep were conducted for the samples with contact stresses and creep times changing from 1 MPa to 10 MPa and from 0 min to 15 min, respectively. Finally, depth-dependent cell viability was assessed with a confocal microscope. In order to characterize changes in the biomechanical properties of cartilage as a result of the use of Hexabrix?, stress-relaxation tests were conducted for the samples immersed in Hexabrix? and phosphate buffered saline (PBS). Both dynamic and equilibrium modulus of the samples immersed in Hexabrix? were significantly (p<0.05) lower than those of the samples immersed in PBS. Cartilage samples immersed in physiological saline solution showed load-induced cell death primarily in the superficial and middle zones. However, under high 8–10 MPa contact stresses, the samples immersed in full strength Hexabrix? showed significantly (p<0.05) higher number of dead cells than the samples compressed in physiological saline, especially in the deep zone of cartilage. In conclusion, excessive loading stresses followed by tissue creep might increase the risk for chondrocyte death in articular cartilage when immersed in hypertonic X-ray contrast agent, especially in the deep zone of cartilage.     

Two-dimensional dose finding in discrete dose space

The objective of a Phase I trial with two agents is to find a set of maximum-tolerated dose combinations that yield a prespecified toxicity rate. In this article, we consider the case where several doses of one agent are fixed and the goal is to find the maximum-tolerated dose of the other agent to be used in combination with each of the doses of agent one. We propose a Bayesian design that uses a parsimonious working model for the dose-toxicity relationship. We show that the new design is more effective in identifying the maximum-tolerated combinations than one-dimensional designs applied at each dose level of one of the agents.     

New Concept in Stroke Diagnosis

Stroke is a life-threatening event that is expected to more than double over the next 40 years. Approximately 85% of strokes are ischemic in nature and result from thromboembolic occlusion of a major cerebral artery or its branches. One of the diagnostic methods for detection of the cerebral ischemia is the gadolinium-enhanced MRI imaging. It is mainly used in patients to detect brain tissue damaged by an ischemic stroke and brain hemorrhage. These techniques are expensive, require sophisticated machines and are time consuming. A recent study in acute stroke patients showed gadolinium leakage into ocular structures (GLOS) during MRI imaging with gadolinium administration. The results indicate that at 2 hours after administration of the contrast agent, GLOS was more common in the aqueous chamber alone, compared to the vitreous chamber with increasing amount in 24 hours after the administration of the contrast agent. This could be due to disruption of blood-ocular barrier similar to the disruption of blood-brain barrier in acute stroke. A new approach to diagnosis of acute stroke and transient ischemic attack (TIA) is through the detection of sodium fluorescein contrast agent in the eye by i.v. injection. The agent is safe and is used routinely in eye fluorescein angiography. Fluorescein fluorescence occurs at the visible wavelengths and can be detected by fluorescein angiography camera. The fluorescein angiography camera prices are affordable for any medical clinic. The innovation of this method is to leverage the eye as the window to the brain. The method can potentially detect acute stroke and TIA without MRI. This can have a far-reaching impact on the healthcare system. The eventual feature of the device will be portability and simplicity of operation that can be used by a medical technician in medical office, emergency outfits and even in ambulances given the portability.     

MR contrast agent composed of cholesterol and peptide nucleic acids: Design,synthesis and cellular uptake

A new mRNA targeting contrast agent consisting of three main functional domains, (i) gadolinium based magnetic resonance reporter part, (ii) antisense peptide nucleic acids targeted to mRNA, and (iii) cholesterol as the delivery vector, was developed and synthesized. The new contrast agent showed efficient cellular uptake and significant contrast enhancement at very low labeling concentrations (0.5 μM). However, after uptake into cells the agent was located predominantly in endosomes like a similar cell penetrating peptide conjugated probe. Our results indicate that this newly developed contrast agent could be used for the labeling of cells for optical as well as magnetic resonance imaging.     

A spin label method for measuring internal volumes in liposomes or cells, applied to Ca-dependent fusion of negatively charged vesicles

A new spin label - broadening agent system for measuring trapped volumes of vesicles or cells is described. The method seems to be more advantageous than existing procedures when volumes of highly negatively charged vesicles are to be determined. The membrane permeable spin label is TEMPONE (2,2,6,6-tetramethyl piperidone-N-oxyl), and the nonpermeable broadening agent is chromium oxalate (K3Cr(C2O4)3). Absolute values for the trapped volumes down to 0.1% in 0.1 ml can be measured with an accuracy of about +/- (1-10%). The method is used to study the final volume of fused phosphatidylserine vesicles as a function of the temperature at which the Ca-induced fusion takes place.     

Proteins induced by DNA-damaging agents in cultured Drosophila cells

In Drosophila cultured cells, the effects of several DNA-damaging agents on the expression of proteins were investigated. Poly(A+) RNA prepared from both untreated cells and cells treated with DNA-damaging agents was translated in vitro. The translation products were analyzed by two-dimensional electrophoresis. Methyl methanesulfonate, the most potent agent used, induced about 25 proteins, some new and some enhanced pre-existing proteins. Angelicin plus near UV irradiation, 4-nitroquinoline N-oxide and ethyl methanesulfonate were efficient inducers. Mitomycin C, UV irradiation and hydrogen peroxide were poor inducers, inducing only a few proteins at low levels. A tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, and a DNA gyrase inhibitor, nalidixic acid, also were used. In this system they were weak inducers of new proteins. Several of the new or enhanced proteins were common to several agents, but others were agent specific. The distribution of mutagen-induced proteins was compared with that of proteins induced in cells heated at 37 degrees C. Some of the proteins induced by DNA-damaging agents were found to overlap heat-shock proteins. These results suggest that there are sets of induced genes that are regulated differently.     

Identification of genes involved in terbinafine resistance in Aspergillus nidulans

AIMS: To determine the pattern and the genetic basis of resistance to terbinafine, a drug extensively used for the treatment of fungal infections in humans. METHODS AND RESULTS: Four resistant mutants from Aspergillus nidulans isolated after irradiation with ultraviolet light were crossed with the master strain F (MSF). Genetic analysis revealed that a single gene, located on chromosome IV, is responsible for resistance to terbinafine and that the alleles responsible for this resistance in these mutants are of a codominant or dominant nature at high terbinafine concentrations. Furthermore, the interaction of this mutation with another one identified on chromosome II causes the double mutant to be highly resistant. CONCLUSIONS: Periodic surveillance of antimycotic susceptibility would be an important measure in detecting the emergence and spread of resistance. Mutation in a single gene could be responsible for resistance to terbinafine and a genic interaction may be responsible for a higher level of antimycotic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of the mechanisms that lead to changes in the sensitivity of a fungus to a given antifungal agent is important both in order to define strategies for the use of such agent and to guide the development of new antifungal agents.     

Inhibition of monoamine oxidase by metaiodobenzylguanidine, a new radiodiagnostic agent

Metaiodobenzylguanidine, which is used in the diagnosis and treatment of pheochromocytoma, produced 50% inhibition of monoamine oxidase at a concentration of 8 X 10(-6) M in vitro. These results suggest that a careful study of the structure of a new radiodiagnostic agent should be done to anticipate and prepare for possible unwanted biological effects when administering the preparation to patients.     

4-羟基异亮氨酸的研究进展

4-羟基异亮氨酸是一种新型胰岛素分泌促进剂,可用于治疗Ⅱ型糖尿病。本文综述了近年来4羟基异亮氨酸的研究进展,包括它的存在形式、活性、提取分离方法和4羟基异亮氨酸及内酯的部分立体异构体的合成方法。     

1-O-methyl-rac-glycerol: A new agent for the cryopreservation of mononuclear cells

1-O-methyl-rac-glycerol (1-O-MG), also known as 3-methoxy-1,2-propanediol is a lipophilic derivative of glycerol, and has been studied as a new cryoprotective agent (CPA) for mononuclear blood cells (MNC), a well-established experimental model in cryopreservation. The chemical modification of the glycerol molecule results in improved cryobiological properties, such as membrane permeability, thus allowing easier handling in the freeze/thaw process. The optimum preincubation period for MNC and 1-O-MG before freezing is 5 min at 4 degree C, resulting in 86% recovery of viable cells, whereas optimal recovery of glycerol-frozen MNC is only guaranteed after 30 min of preincubation at room temperature (74% viable recovery). The optimal concentration of 1-O-MG is 10% (v/v). Although this new agent offers no improvement in cryoprotective properties over dimethyl sulfoxide (Me2SO) there may be possible pharmacological advantages when used in humans. It is, however, obviously superior to glycerol with regard to its permeation kinetics. 1-O-MG might therefore also be of interest in the cryoprotection of other hematopoietic cells and biological tissues.     

Separation of epidermal cells by density gradient centrifugation on a continuous colloidal silica (Percoll) gradient

A new method designed for the specific isolation and characterization of ligand-receptor complexes using a heterobifunctional crosslinking agent and immunoprecipitation is described. The complexes are first covalently crosslinked by photoactivation of the crosslinking agent. After lysis of the cells, the crosslinked complexes are immunoprecipitated using an antiserum directed against the crosslinking agent. With this method, ligand-receptor complexes formed in only minute amounts become available for further investigation. By using this anticrosslinker antiserum, different receptor systems can be investigated without raising new receptor- or ligand-specific antibodies for each system. As a test system, a radioiodinated lectin was used as ligand molecule and erythrocyte membranes acted as receptor carriers.     

连续光照转暗培养联合药物处理实现衣藻细胞的高水平同步化

单细胞衣藻(Chlamydomonas)是光合作用和植物细胞周期等生物学过程研究的一个重要模式系统, 同步化培养是进行相关研究的必要手段。该研究探索了连续光照转暗培养联合细胞周期阻断剂实现莱茵衣藻(Chlamydomonas reinhardtii)细胞高水平同步化的新方法, 并利用流式细胞术对同步化程度进行了精确的分析。结果表明, 连续光照转暗培养或联合S期阻断剂可以使衣藻细胞同步化到G1期或G1/S期边界; 连续光照转暗培养联合M期阻断剂或者在“加入-释放”S期阻断剂后再加入M期阻断剂可以使衣藻细胞同步化到M期, 同步化水平可达80%。具体的同步化培养步骤要根据研究对象(特别是某些衣藻突变株系)的特性和研究目的确定。     

Effect of curcumin-nanoemulsion associated with photodynamic therapy in breast adenocarcinoma cell line

Curcumin, a natural compound has several antineoplastic activities and is a promising natural photosensitizer used in photodynamic therapy. However, its low solubility in physiological medium limit the clinical use of curcumin. This study aimed to analyze the action of curcumin-nanoemulsion, a new and well-designed Drug Delivery System (DDS+) molecule, used as a photosensitizing agent in photodynamic therapy in an in vitro breast cancer model, MCF-7 cells. The empty nanoemulsion fulfils all necessary requirements to be an excellent DDS. Furthermore, the use of curcumin-nanoemulsion in photodynamic therapy resulted in a high phototoxic effect after activation at 440?nm, decreasing to <10% viable tumor cells after two irradiations and increasing the reactive oxygen species (ROS) production. The use of curcumin-nanoemulsion associated with photodynamic therapy resulted in an increase in the levels of caspase 3/7 activity for the studied MCF-7 cell model, indicating that this therapy triggers a cascade of events that lead to cell death, such as cellular apoptosis. In conclusion, curcumin-nanoemulsion proved to be efficient as a photosensitizing agent, had phototoxic effects, significantly decreased the proliferation of MCF-7 cells and stimulating the ROS production in combination with photodynamic therapy, so, this formulation has a great potential for use in treatment of breast cancer.     

Development of a conjugate of (99m)Tc-EC with aminomethylenediphosphonate in the search for a bone tracer with fast clearance from soft tissue.

For the currently used (99m)Tc-labeled diphosphonates such as (99m)Tc-MDP and (99m)Tc-HDP, the required interval of 2.5 to 3 h between injection and the scintigraphic bone imaging is an inconvenience. The present study was set up in an attempt to develop a technetium-99m-labeled diphosphonate with efficient bone uptake and more rapid clearance from blood and soft tissue by renal extraction and excretion so that it would be possible to start imaging as early as 1 h after injection. A conjugate of the new renal tracer agent (99m)Tc-ethylene dicysteine ((99m)Tc-L,L-EC), covalently bound via one of its carboxylates with aminomethylenediphosphonic acid (AMDP), was synthesized in seven steps. EC-AMDP could be labeled easily and efficiently with (99m)Tc at pH > or = 12 and room temperature. Analysis using ion pair reversed phase high performance liquid chromatography showed the formation of a mixture of two main compounds with reproducible relative ratios, which were stable as a function of time. In a baboon, the scintigraphic images obtained with the new agent showed good quality bone scans, with clear visualization of the skeleton and low soft tissue activity at respectively 1 and 2 h after injection.     

Transmissible modification induced in Mycobacterium tuberculosis and Mycobacterium bovis in vitro]

A biologically active material (fraction "S") is isolated from cultures of scotochromogenic mycobacteria. Mycobacterium tuberculosis, or Mycobacterium bovis by disrupting the cells, sedimentation through 2.2 M sucrose, and ultrafiltration. The fraction "S" induces the modification of tubercle bacilli into non acid-fast bacteria forming smooth colonies on nutritive glycerol agar within 24-36 h of incubation. Three new phenotypes are thus obtained; two proved to be stable upon subculturing. Frequently the phenomenon occurs with a very large part of the Koch's bacillus population exposed to the inducing agent effect. It can be reproduced with crude preparations of DNA obtained from the fraction "S." It is inhibited by concanavalin A. The observed modification does not correspond to a transfer of characteristics of the inducing agent from the donor mycobacteria; furthermore it can be manifested even in the strain used for the preparation of the fraction "S."     

The growth-stimulating action of the preparation Balis-2 on sympathetic ganglia in culture

The effect of Balis-2 drug on the growth and differentiation of nervous tissue was studied on organotypic culture of the sympathetic ganglia. It was established that this agent is able to stimulate fiber outgrowth from explant and increase mean value of the maximal magnitude of the zone of the growth by concentration 0.001% and 0.0001%. It was found that Balis-2 drug is able to increase the intensity of reaction by 1.8-2 times. It is suggested that Balis-2 drug can be used as a new neurotropic agent.     

Fluorine-containing aryloxyethyl thiocyanate derivatives are potent inhibitors of Trypanosoma cruzi and Toxoplasma gondii proliferation

As a part of our project aimed at developing new safe chemotherapeutic and chemoprophylactic agents against tropical diseases, fluorine-containing drugs structurally related to 4-phenoxyphenoxyethyl thiocyanate (1) were designed, synthesized, and evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible of American trypanosomiasis (Chagas' disease), and Toxoplasma gondii, the etiological agent of toxoplasmosis. This thiocyanate derivative had previously proven to be an effective agent against T. cruzi proliferation. Fluorine-containing thiocyanate derivatives 2 and 3 were threefold more potent than our lead drug 1 against intracellular T. cruzi. The biological evaluation against T. gondii was also very promising. The IC(50) values corresponding to 2 and 3 were at the very low micromolar level against tachyzoites of T. gondii. Both of these drugs are interesting examples of effective antiparasitic agents that have outstanding potential not only as lead drugs but also to be used for further in vivo studies.     

昆虫病原微生物及其在蝗灾治理中的应用

本文重点介绍了蝗虫痘病毒、蝗虫微孢子虫、蝗噬虫霉、绿僵菌、白僵菌、米曲霉等的生物学特性、侵染机制、流行病学和应用潜力。蝗虫痘病毒和蝗噬虫霉分别由于生产成本过高和分生孢子在离体条件下存活时间过短而难以开发应用;蝗虫微孢子虫和绿僵菌已广泛应用到蝗灾治理中,有关绿僵菌致病机理的研究最深入全面;白僵菌也已成功应用到蝗灾治理中;介绍了一种新病原——米曲霉对飞蝗的毒力以及产孢量和耐热性等生物学特性。最后,提出蝗虫新病原微生物资源的开发、提高生产工艺水平、研发新的制剂和延长储存时间等是今后蝗虫微生物农药的发展方向。     

普鲁士蓝纳米粒子在生物医学诊断和治疗中的应用及进展

随着生物医学诊断和治疗的持续深入研究,出现了多种医学诊断和治疗新方法,为人类的健康提供了更大的保证,其中纳米生物技术在生物医学诊断和治疗中的应用日益增多,基于纳米技术,开发传统材料的生物医学新应用成为了人们的研究热点。普鲁士蓝是一种历史悠久的蓝色染料,其制备过程简单、绿色、成本低,化学结构稳定,具有优良的物理、化学、光学以及磁性等性能,已经在许多领域得到了广泛的应用。近年来,普鲁士蓝开始在生物医学诊断和治疗领域中崭露头角,它已经成功的被开发为新型的核磁共振造影剂和光声成像造影剂,并且在药物输送系统和光热治疗等领域也开始占有一席之地,开发基于纳米技术的普鲁士蓝的生物医学应用已经成为极具吸引力的研究方向。本文对普鲁士蓝在生物医学诊断和治疗中的应用及进展进行综述。