A strategy for the production of soluble human senescence marker protein-30 in Escherichia coli

Senescence marker protein-30 (SMP30) has been reported to hydrolyze diisopropyl fluorophosphate (DFP), a surrogate compound of chemical warfare nerve agents. Thus, SMP30 has the potential to be useful as a prophylactic against chemical warfare nerve agent toxicity. Our efforts to generate human SMP30 in bacteria using a variety of expression vectors invariably resulted in insoluble and inactive preparations. In this study, properly folded and active recombinant human SMP30 (rHuSMP30) was produced in Escherichia coli by coexpressing it with molecular chaperones in a combined strategy. The coexpression of rHuSMP30 with GroES/GroEL/Tf at 15 °C, combined with the addition of a membrane fluidizer, increased osmolytes, and a two-step expression resulted in the highest enhancement of solubility and DFPase activity. Our results pave the way for exploring the use of rHuSMP30 against organophosphate and nerve agent toxicity.     

Marker assisted selection in crop plants

Genetic mapping of major genes and quantitative traits loci (QTLs) for many important agricultural traits is increasing the integration of biotechnology with the conventional breeding process. Exploitation of the information derived from the map position of traits with agronomical importance and of the linked molecular markers, can be achieved through marker assisted selection (MAS) of the traits during the breeding process. However, empirical applications of this procedure have shown that the success of MAS depends upon several factors, including the genetic base of the trait, the degree of the association between the molecular marker and the target gene, the number of individuals that can be analyzed and the genetic background in which the target gene has to be transferred. MAS for simply inherited traits is gaining increasing importance in breeding programs, allowing an acceleration of the breeding process. Traits related to disease resistance to pathogens and to the quality of some crop products are offering some important examples of a possible routinary application of MAS. For more complex traits, like yield and abiotic stress tolerance, a number of constraints have determined severe limitations on an efficient utilization of MAS in plant breeding, even if there are a few successful applications in improving quantitative traits. Recent advances in genotyping technologies together with comparative and functional genomic approaches are providing useful tools for the selection of genotypes with superior agronomical performancies.     

Assessment of the potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery failure of 99m-technetium marker to represent intact liposomes in lymph nodes

The in vivo fate of subcutaneously injected neutral SUV liposomes in rats was examined using a membrane marker, ^99mTc, and an aqueous marker, ¹²⁵I-labelled poly(vinyl pyrrolidone). Liposomes with entrapped ¹²⁵I-labelled poly(vinyl pyrrolidone) were labelled with ^99mTc by the SnCl₂ method [2]. ^99mTc-radioactivity was localized several-fold more in the primary and secondary regional lymph nodes than ¹²⁵I-labelled poly(vinyl pyrrolidone)-radioactivity. Similarly, ^99mTc-radioactivity appeared and was subsequently cleared from the circulation much more rapidly than ¹²⁵I-labelled poly(vinyl pyrrolidone). The gel chromatography of the lymph node homogenate revealed that 60–70% of ¹²⁵I-labelled poly(vinyl pyrrolidone)-radioactivity was in the liposome fractions, whereas only 3% of ^99mTc-radioactivity was co-eluted with liposomes. Thus, the two markers have different fates in the lymphatics, and the presence of all ^99mTc-radioactivity does not represent the 60–70% of intact liposomes present in lymph nodes. Using the aqueous marker ¹²⁵I-labelled poly(vinyl pyrrolidone), the lymphnode localization of positive, negative and neutral small unilamellar vesicles was studied, and it was found that ¹²⁵I-radioactivity was more localized from negative liposomes than from positive liposomes, which in turn was more localized than that from neutral liposomes. Thus, these findings differ from those reported earlier [2], where the authors used ^99mTc as a liposomal marker. In vitro studies showed that liposomes of preparations containing 20 mol% cholesterol became ‘leaky’ to low-molecular-weight drugs, for example, methotrexate (M_r 454) to a much greater extent than with a large-molecular-weight substance, ¹²⁵I-labelled poly(vinyl pyrrolidone) (M_r 30 000–40 000), when incubated with rat lymph at 37°C. Using the two markers ^99mTc and ¹²⁵I-labelled poly(vinyl pyrrolidone) it was found that the localization of both radioactivities was reduced in lymph nodes draining λ-carrageenan-treated footpads. In conclusion, it is suggested that liposomes can be used for the delivery of drugs to diseased lymph nodes, and it would be worthwhile examining the possibilities of using alternative methods of labelling liposomes with ^99mTc rather than using the SnCl₂ technique [2], or using other radionuclides as markers for γ-scan imaging.     

Genotypic diversity and structure of Satureja mutica revealed by inter-simple sequence repeat markers

Satureja mutica (Lamiaceae) is an herbaceous medicinal plant which grows in Iran. The objective of the study was to obtain an overview of the genetic relatedness among and within seven populations of this species using inter-simple sequence repeat (ISSR). Fourteen ISSR primers amplified a total of 197 DNA fragments of which 176 (88.91%) were polymorphic. All ISSR primers were highly effective in discriminating among the populations. Genetic similarity coefficients ranged from 0.45 to 0.94, indicating considerable distance and diversity in the germplasm and were confirmed by clustering analysis. The dendrogram showed a clear clustering pattern of plants indicating a significant association between genetic similarity and geographical distance. Analysis of molecular variance revealed that a greater proportion of total genetic variation existed within populations (75%) rather than among populations (25%). The study indicated that ISSR markers were effective and reliable for assessing the degree of genetic variation of S. mutica. These findings can support future research on the selection of S. mutica for breeding and medicinal plant development.     

Identification and characterization of 21 polymorphic microsatellite loci from the mycophagous fly Drosophila neotestacea

Drosophila neotestacea is a mushroom‐feeding fly that is common in the temperate and boreal forests of North America. Here I describe the isolation and characterization of 21 polymorphic microsatellite loci from the D. neotestacea genome. In a sample of flies from Rochester, New York, the expected and observed heterozygosities ranged from 0.344 to 1.000 and from 0.384 to 0.923, respectively. Of the 21 markers, six were likely X‐linked, seven showed a departure from Hardy–Weinberg equilibrium, and none were found to be in linkage disequilibrium. These loci will facilitate future ecological and population genetic studies of D. neotestacea.     

Recent insertion/deletion (reINDEL) mutations: increasing awareness to boost molecular‐based research in ecology and evolution

Today, the comparative analysis of DNA molecules mainly uses information inferred from nucleotide substitutions. Insertion/deletion (INDEL) mutations, in contrast, are largely considered uninformative and discarded, due to our lacking knowledge on their evolution. However, including rather than discarding INDELs would be relevant to any research area in ecology and evolution that uses molecular data. As a practical approach to better understanding INDEL evolution in general, we propose the study of recent INDEL (reINDEL) mutations – mutations where both ancestral and derived state are seen in the sample. The precondition for reINDEL identification is knowledge about the pedigree of the individuals sampled. Sound reINDEL knowledge will allow the improved modeling needed for including INDELs in the downstream analysis of molecular data. Both microsatellites, currently still the predominant marker system in the analysis of populations, and sequences generated by next‐generation sequencing, a promising and rapidly developing range of technologies, offer the opportunity for reINDEL identification. However, a 2013 sample of animal microsatellite studies contained unexpectedly few reINDELs identified. As most likely explanation, we hypothesize that reINDELs are underreported rather than absent and that this underreporting stems from common reINDEL unawareness. If our hypothesis applies, increased reINDEL awareness should allow gathering data rapidly. We recommend the routine reporting of either the absence or presence of reINDELs together with standardized key information on the nature of mutations when they are detected and the use of the keyword “reINDEL” to increase visibility in both instances of successful and unsuccessful search.     

昆虫遗传转化品系的常用标记

遗传转化标记是将遗传修饰昆虫从野生型种群中分辨出来的根据,遗传转化昆虫的鉴定、转化品系的维持及其遗传稳定性的监测都依赖于可靠的标记系统,发展易于应用和监测的转化标记能够极大地促进害虫遗传防治的相关研究。用于遗传修饰昆虫的转化标记主要有昆虫眼睛颜色标记基因、抗药性标记基因和荧光蛋白标记基因等。非果蝇类昆虫首个遗传转化品系的鉴定是通过眼睛颜色突变而实现,但大多数昆虫物种没有可用的突变体或缺少相应基因的信息,从而限制了眼睛颜色标记的应用。抗药性基因标记虽然能够通过对转化昆虫进行集体选择而大幅度提高筛选转化体的效率,但由于其鉴定的准确性不高且存在安全性问题,未得到广泛应用。荧光蛋白标记基因的发展则显著拓宽了能够转化的昆虫种类。从水母分离的绿色荧光蛋白(GFP)经突变方法获得了多种不同荧光性质的突变体,经人为修饰后与适宜的强启动子构成转化标记载体,能够有效鉴定更多昆虫物种的遗传转化个体,其中应用较多的是增强型绿色荧光蛋白(EGFP)。此外,从珊瑚属海葵中分离得到的红色DsRed标记基因提供了多样化的红色荧光蛋白选择,在某些生物中DsRed与GFP联合应用的表现明显优于GFP突变体,所以其应用前景也非常广泛。本文着重从眼睛颜色、抗药性和荧光蛋白等3个方面阐述了标记基因的发展历史与现状,并对其今后的发展方向进行了展望。     

Measurement of Dynamic Scapular Kinematics Using an Acromion Marker Cluster to Minimize Skin Movement Artifact

The measurement of dynamic scapular kinematics is complex due to the sliding nature of the scapula beneath the skin surface. The aim of the study was to clearly describe the acromion marker cluster (AMC) method of determining scapular kinematics when using a passive marker motion capture system, with consideration for the sources of error which could affect the validity and reliability of measurements. The AMC method involves placing a cluster of markers over the posterior acromion, and through calibration of anatomical landmarks with respect to the marker cluster it is possible to obtain valid measurements of scapular kinematics. The reliability of the method was examined between two days in a group of 15 healthy individuals (aged 19-38 years, eight males) as they performed arm elevation, to 120°, and lowering in the frontal, scapular and sagittal planes. Results showed that between-day reliability was good for upward scapular rotation (Coefficient of Multiple Correlation; CMC = 0.92) and posterior tilt (CMC = 0.70) but fair for internal rotation (CMC = 0.53) during the arm elevation phase. The waveform error was lower for upward rotation (2.7° to 4.4°) and posterior tilt (1.3° to 2.8°), compared to internal rotation (5.4° to 7.3°). The reliability during the lowering phase was comparable to results observed during the elevation phase. If the protocol outlined in this study is adhered to, the AMC provides a reliable measurement of upward rotation and posterior tilt during the elevation and lowering phases of arm movement.