Qualichem In Vivo: A Tool for Assessing the Quality of In Vivo Studies and Its Application for Bisphenol A

In regulatory toxicology, quality assessment of in vivo studies is a critical step for assessing chemical risks. It is crucial for preserving public health studies that are considered suitable for regulating chemicals are robust. Current procedures for conducting quality assessments in safety agencies are not structured, clear or consistent. This leaves room for criticism about lack of transparency, subjective influence and the potential for insufficient protection provided by resulting safety standards. We propose a tool called “Qualichem in vivo” that is designed to systematically and transparently assess the quality of in vivo studies used in chemical health risk assessment. We demonstrate its use here with 12 experts, using two controversial studies on Bisphenol A (BPA) that played an important role in BPA regulation in Europe. The results obtained with Qualichem contradict the quality assessments conducted by expert committees in safety agencies for both of these studies. Furthermore, they show that reliance on standardized guidelines to ensure scientific quality is only partially justified. Qualichem allows experts with different disciplinary backgrounds and professional experiences to express their individual and sometimes divergent views—an improvement over the current way of dealing with minority opinions. It provides a transparent framework for expressing an aggregated, multi-expert level of confidence in a study, and allows a simple graphical representation of how well the study integrates the best available scientific knowledge. Qualichem can be used to compare assessments of the same study by different health agencies, increasing transparency and trust in the work of expert committees. In addition, it may be used in systematic evaluation of in vivo studies submitted by industry in the dossiers that are required for compliance with the REACH Regulation. Qualichem provides a balanced, common framework for assessing the quality of studies that may or may not be following standardized guidelines.     

A Statistical Model for In Vivo Neuronal Dynamics

Single neuron models have a long tradition in computational neuroscience. Detailed biophysical models such as the Hodgkin-Huxley model as well as simplified neuron models such as the class of integrate-and-fire models relate the input current to the membrane potential of the neuron. Those types of models have been extensively fitted to in vitro data where the input current is controlled. Those models are however of little use when it comes to characterize intracellular in vivo recordings since the input to the neuron is not known. Here we propose a novel single neuron model that characterizes the statistical properties of in vivo recordings. More specifically, we propose a stochastic process where the subthreshold membrane potential follows a Gaussian process and the spike emission intensity depends nonlinearly on the membrane potential as well as the spiking history. We first show that the model has a rich dynamical repertoire since it can capture arbitrary subthreshold autocovariance functions, firing-rate adaptations as well as arbitrary shapes of the action potential. We then show that this model can be efficiently fitted to data without overfitting. We finally show that this model can be used to characterize and therefore precisely compare various intracellular in vivo recordings from different animals and experimental conditions.     

一种体内标记转录RNA的新技术

目的:验证一种体内标记转录RNA的新技术,并用该技术观察一种长寿药物雷帕霉素对真核细胞HEK293中RNA合成的影响。方法:将尿嘧啶类似物5-乙炔尿苷(EU)和雷帕霉素加到HEK293细胞培养基中,共同孵育2h,然后在激光共聚焦显微镜下对EU标记的新合成RNA进行观察;用Image-pro plus软件对图像的荧光强度进行分析,获得反应荧光强度的平均光密度数值;用SPSS软件对数值进行统计分析。结果:激光共聚焦显微镜下,可见EU标记的新合成RNA主要分布在胞质和核仁中,以核仁中荧光最强;Image-pro plus软件和SPSS软件分析表明,加入雷帕霉素前后,细胞新合成的RNA平均光密度无明显差别。结论:EU是一种安全、简单、快速而灵敏的检测新合成RNA的新技术,用该技术检测表明长寿药物雷帕霉素不影响真核细胞HEK293中总RNA的合成。     

A Cremophor-Free Self-Microemulsified Delivery System for Intravenous Injection of Teniposide: Evaluation In Vitro and In Vivo

In order to tackle the problems on low water solubility of teniposide, involvement of toxic surfactant in its injection, and the poor stability during infusion, a Cremophor-free teniposide self-microemulsified drug delivery system (TEN-SMEDDS) was prepared for the first time, characterized, and evaluated in comparison with teniposide injection (VUMON) in vitro and in vivo. The optimized formulation contained N, N-dimethylacetamide, medium-chain triglyceride, lecithin, and dehydrated alcohol besides teniposide. The TEN-SMEDDS could form fine droplets with mean diameter of 282 ± 21 nm and zeta potential of −7.5 ± 1.7 mV after dilution with 5% glucose, which were stable within 4 h. The release of teniposide from TEN-SMEDDS and VUMON was similar. However, the pharmacokinetic behavior of TEN-SMEDDS in rats was different from that of VUMON, evidenced by the lower area under the concentration–time curve and larger volume of distribution in emulsion group. Finally, TEN-SMEDDS was found to distribute more teniposide in most tissues, especially in reticuloendothelial system, after intravenous administration to rats. Importantly, brain drug level in TEN-SMEDDS group was higher than or similar to that in control group, although the emulsion system had a lower plasma drug concentration. In conclusion, the novel SMEDDS prepared here, without toxic surfactant and as an oil solution before use, may be potential for clinical use due to its low toxicity and high store stability. It may be favorable for the treatment of some tumors like cerebroma, since it may achieve the relatively higher drug level in brain but lower blood concentration.KEY WORDS: characterization, pharmacokinetics, self-microemulsified drug delivery system, teniposide, tissue distribution     

Formation of Formaldehyde from Adrenaline In Vivo; a Potential Risk Factor for Stress-Related Angiopathy

Cardiovascular and cerebrovascular disorders are well known to be associated with stress related behaviors. Stress enhances excretion of adrenaline, which is deaminated by monoamine oxidase and methylamine is formed. This product can be further deaminated by semicarbazide-sensitive amine oxidase (SSAO) and converted to toxic formaldehyde, hydrogen peroxide and ammonia. SSAO is located in the cardiovascular smooth muscles and circulated in the blood. We investigated whether formaldehyde can be derived from adrenaline in vivo. Methylamine was confirmed to be a product of adrenaline catalyzed by type A monoamine oxidase (MAO-A). Irreversible and long-lasting radioactive residual activity was detected in different tissues following administration of 1-[N-methyl-³H]-adrenaline. Such irreversible linkage could be blocked by selective MAO-A or SSAO inhibitors. Endothelial cells are quite sensitive to formaldehyde and relatively resistant to hydrogen peroxide. It is possible that stimulation of adrenaline excretion by chronic stress could increase the levels of circulatory formaldehyde. Such chronic formaldehyde stress may be involved in the initiation of endothelial injury and subsequently angiopathy.     

生物活性玻璃提高骨质疏松绵羊椎弓根螺钉稳定性的体内实验研究

目的：研究生物活性玻璃（Bioactive Glass,BG）在骨质疏松绵羊体内强化椎弓根螺钉固定的力学效果,并观察钉道界面及材料吸收等情况。方法：4只成年雌性小尾寒羊,采用去势联合激素注射方法建立骨质疏松绵羊模型。选择绵羊L2至L5双侧椎弓根,随机化选取一侧直接拧入椎弓根螺钉（空白组）,对侧采用BG强化钉道后拧入椎弓根螺钉（实验组）。术后3月随机选取6个椎体（12枚椎弓根螺钉）,对螺钉骨质界面行显微CT分析和组织学观察。对剩余10个椎体（20枚椎弓根螺钉）行轴向拔出实验,分析螺钉固定强度。结果：术前绵羊腰椎BMD为0.818±0.0310 g/cm2,建模完成后为1.000±0.0316 g/cm2,BMD平均值下降22.38%,差异有统计学意义（P〈0.05）。实验组螺钉骨质界面的Tb.Th、Tb.N组较对照组分别增加143.60%和33.56%,差异有统计学意义（P〈0.05）。实验组钉道周围材料绝大部分已经降解吸收,大量新生骨组织紧密包裹螺钉;对照组钉道周围骨量较少,钉骨结合不紧密,实验组螺钉骨质界面结合优于对照组。实验组的最大轴向拔出力为1083.04±86.37N,空白组为871.76±79.03N,前者较后者提高25.26%,差异有统计学意义（P〈0.05）。结论：生物活性玻璃能显著改善骨质疏松情况下螺钉骨质界面的骨微观结构,进而提高椎弓根螺钉的把持力。     

In Vivo Muscle Electroporation Threshold Determination: Realistic Numerical Models and In Vivo Experiments

In vivo electroporation is used as an effective technique for delivery of therapeutic agents such as chemotherapeutic drugs or DNA into target tissue cells for different biomedical purposes. In order to successfully electroporate a target tissue, it is essential to know the local electric field distribution produced by an application of electroporation voltage pulses. In this study three-dimensional finite element models were built in order to analyze local electric field distribution and corresponding tissue conductivity changes in rat muscle electroporated either transcutaneously or directly (i.e., two-plate electrodes were placed either on the skin or directly on the skeletal muscle after removing the skin). Numerical calculations of electroporation thresholds and conductivity changes in skin and muscle were validated with in vivo measurements. Our model of muscle with skin also confirms the in vivo findings of previous studies that electroporation “breaks” the skin barrier when the applied voltage is above 50?V.     

A Fluorescent Brightener used for Pollen Tube Identification In Vivo

Squash preparations of styles stained in watersoluble aniline blue and viewed under ultra-violet illumination are regularly used for examining pollen tubes because the callose plugs fluoresce brightly under these conditions. Tubes are therefore clearly distinguish from the astylar tissue and may be readily counted and measured. This method has proved to be quite unsatisfactory for plum pollen tubes, since they contain very few cause plugs and better results have been obtained with a mixed stain of 0.1% aniline blue and 0.07% of the fluorescent brightener 'Calcofluor White M2R New'. Styles are softened by autoclaving in 50 g/1 sodium sulphite, rinsed and stained for ten minutes, then squashed and examined with a fluorescence microscope in the usual way. Callose deposits, when present, fluoresce bright yellow, but lengths of tube with no deposits can also be clearly identified and followed, permitting easier, faster and more accurate assessments of pollen tube length and numbers in plum and pear styles.     

A Comparison of In Vitro- and In Vivo Phosphorylated Neurofilament Polypeptides

Abstract: Neurofilament polypeptides phosphorylated in vitro by incubation of neurofilament-enriched preparations from rat CNS with [γ-³²P]ATP were compared with the corresponding polypeptides labeled in vivo by injection of ³²P_i into the lateral ventricles of rats. Autoradiography of sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed that the major phosphorylated species in both preparations were the three neurofilament subunits, which have molecular weights of 200K, 145K, and 68K. However, the relative levels of ³²P detected in the three in vitro -labeled subunits differed from the relative in vivo levels. The two larger neurofilament polypeptides displayed similar ³²P isoprotein distribution patterns on two-dimensional gels, whereas additional isoproteins were seen in the in vitro -labeled 68K species. Limited proteolysis in SDS-polyacrylamide gels revealed the presence of common phosphopeptides in the corresponding pairs of in vitro- and in vivo-labeled subunits, but the in vivo -labeled 145K and in vitro -labeled 200K polypeptides contained additional digestion products. Two-dimensional peptide mapping of the 68K polypeptide digested with a mixture of trypsin and chymotrypsin indicated that this component was phosphorylated at a single, identical site, both in vivo and in vitro. These results indicate that the protein kinase that copurifies with neurofilament preparations may be involved in their in vivo phosphorylation.     

A High Throughput In Vivo Assay for Taste Quality and Palatability

Taste quality and palatability are two of the most important properties measured in the evaluation of taste stimuli. Human panels can report both aspects, but are of limited experimental flexibility and throughput capacity. Relatively efficient animal models for taste evaluation have been developed, but each of them is designed to measure either taste quality or palatability as independent experimental endpoints. We present here a new apparatus and method for high throughput quantification of both taste quality and palatability using rats in an operant taste discrimination paradigm. Cohorts of four rats were trained in a modified operant chamber to sample taste stimuli by licking solutions from a 96-well plate that moved in a randomized pattern beneath the chamber floor. As a rat’s tongue entered the well it disrupted a laser beam projecting across the top of the 96-well plate, consequently producing two retractable levers that operated a pellet dispenser. The taste of sucrose was associated with food reinforcement by presses on a sucrose-designated lever, whereas the taste of water and other basic tastes were associated with the alternative lever. Each disruption of the laser was counted as a lick. Using this procedure, rats were trained to discriminate 100 mM sucrose from water, quinine, citric acid, and NaCl with 90-100% accuracy. Palatability was determined by the number of licks per trial and, due to intermediate rates of licking for water, was quantifiable along the entire spectrum of appetitiveness to aversiveness. All 96 samples were evaluated within 90 minute test sessions with no evidence of desensitization or fatigue. The technology is capable of generating multiple concentration–response functions within a single session, is suitable for in vivo primary screening of tastant libraries, and potentially can be used to evaluate stimuli for any taste system.     

Direct Fluorination of Phenolsulfonphthalein: A Method for Synthesis of Positron-Emitting Indicators for In Vivo pH Measurement

We report a reaction of direct electrophilic fluorination of phenolsulfonphthalein at mild conditions. This reaction affords the synthesis of novel positron-emitting ¹⁸F-labeled pH indicators. These compounds are useful for non-invasive in vivo pH measurement in biological objects.     

A mariner-Based Transposon System for In Vivo Random Mutagenesis of Clostridium difficile

Understanding the molecular basis of Clostridium difficile infection is a prerequisite to the development of effective countermeasures. Although there are methods for constructing gene-specific mutants of C. difficile, currently there is no effective method for generating libraries of random mutants. In this study, we developed a novel mariner-based transposon system for in vivo random mutagenesis of C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, the BI/NAP1/027 epidemic strain at the center of the C. difficile outbreaks in Stoke Mandeville, United Kingdom, in 2003 to 2004 and 2004 to 2005. Transposition occurred at a frequency of 4.5 (±0.4) × 10⁻⁴ per cell to give stable insertions at random genomic loci, which were defined only by the nucleotide sequence TA. Furthermore, mutants with just a single transposon insertion were generated in an overwhelming majority (98.3% in this study). Phenotypic screening of a C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 random mutant library yielded a sporulation/germination-defective clone with an insertion in the germination-specific protease gene cspBA and an auxotroph with an insertion in the pyrimidine biosynthesis gene pyrB. These results validate our mariner-based transposon system for use in forward genetic studies of C. difficile.Clostridium difficile infection is widely recognized as the leading cause of health care-associated diarrhea in North America and Europe. Infection usually follows antibiotic treatment, which disrupts the native gastrointestinal microflora and thus allows C. difficile to proliferate. The emergence of so-called “epidemic” or “hypervirulent” strains of C. difficile over the last 5 to 10 years has compounded an already serious problem. Classed as BI/NAP1/027, these epidemic strains are believed to cause a more severe disease and lead to increased mortality and relapse rates (11, 20, 24).Understanding the genetic and molecular basis of C. difficile infection will be a crucial step in the development of effective countermeasures. Methods for directed gene inactivation in C. difficile have recently been described (7, 21). This has opened the way for reverse genetic studies, in which the exact role of a specific gene, hypothesized to be important in a given phenotype, can be elucidated experimentally. By way of contrast, forward genetic studies aim to identify the genetic basis of a particular phenotype without making any assumptions about the genes involved. In forward genetic studies, transposons are often used to generate libraries of random insertion mutants. Libraries are then screened to identify mutants that are defective in a particular phenotype. Identification of the gene or genes which have been inactivated by transposon insertion then implicates them as having a role in that particular phenotype. Recently, just such an approach was used to identify a novel toxin-regulatory locus in Clostridium perfringens (29). This study elegantly demonstrated the power of forward genetic studies in bacterial pathogens.A number of transposon mutagenesis systems have been described for Gram-positive bacteria (2, 3, 15, 16, 29, 32). Two different systems have recently been developed for use in C. perfringens (15, 29). Both are in vitro mutagenesis systems which rely on being able to transform the recipient organism. As such, they are not suitable for use in C. difficile because in the laboratory at present, recombinant DNA can be transferred into C. difficile only via conjugation. The conjugative transposons Tn916 and Tn5397 have been studied in C. difficile, but both have been found either to have a strong target site preference or to yield multiple insertions in individual clones (9, 30). Therefore, neither is well suited to generating libraries of random C. difficile mutants.We reasoned that a mariner-based transposon mutagenesis system would be an effective tool for generating libraries of random C. difficile mutants. The mariner-transposable element Himar1 has been shown to insert randomly into the genomes of many bacterial species (3, 6, 16, 17, 32). The cognate Himar1 transposase is the only factor required for transposition, which occurs via a cut-and-paste mechanism (13, 14). The transposon itself is defined by inverted terminal repeats (ITRs) at either end and inserts into a TA target site. This is highly appropriate for an organism with a low-GC content such as C. difficile. In this study, we have developed a novel mariner-based transposon system for in vivo random mutagenesis of C. difficile. Moreover, we have demonstrated the system in C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, the BI/NAP1/027 epidemic strain at the center of the C. difficile outbreaks in Stoke Mandeville, United Kingdom, in 2003 to 2004 and 2004 to 2005. This new genetic tool opens the way for forward genetic studies of C. difficile.