The SSV-Seq 2.0 PCR-Free Method Improves the Sequencing of Adeno-Associated Viral Vector Genomes Containing GC-Rich Regions and Homopolymers

Adeno-associated viral vectors (AAV) are efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs must be accompanied by the development of appropriate quality control (QC) assays. Given the potential risk of co-transfer of oncogenic or immunogenic sequences with therapeutic vectors, accurate methods to assess the level of residual DNA in AAV vector stocks are particularly important. An assay based on high-throughput sequencing (HTS) to identify and quantify DNA species in recombinant AAV batches is developed. Here, it is shown that PCR amplification of regions that have a local GC content >90% and include successive mononucleotide stretches, such as the CAG promoter, can introduce bias during DNA library preparation, leading to drops in sequencing coverage. To circumvent this problem, SSV-Seq 2.0, a PCR-free protocol for sequencing AAV vector genomes containing such sequences, is developed. The PCR-free protocol improves the evenness of the rAAV genome coverage and consequently leads to a more accurate relative quantification of residual DNA. HTS-based assays provide a more comprehensive assessment of DNA impurities and AAV vector genome integrity than conventional QC tests based on real-time PCR and are useful methods to improve the safety and efficacy of these viral vectors.     

Current standards and pitfalls associated with the transfection of primary fibroblast cells

Cultured fibroblast cells, especially dermal cells, are used for various types of scientific research, particularly within the medical field. Desirable features of the cells include their ease of isolation, rapid cellular growth, and high degree of robustness. Currently, fibroblasts are mainly used to obtain pluripotent cells via a reprogramming process. Dermal fibroblasts, are particularly useful for gene therapies used for promoting wound healing or minimizing skin aging. In recent years, fibroblast transfection efficiencies have significantly improved. In order to introduce molecules (most often DNA or RNA) into cells, viral-based systems (transduction) or non-viral methods (transfection) that include physical/mechanical processes or lipid reagents may be used. In this article, we describe critical points that should be considered when selecting a method for transfecting fibroblasts. The most effective methods used for the transfection of fibroblasts include both viral-based and non-viral nucleofection systems. These methods result in a high level of transgene expression and are superior in terms of transfection efficacy and viability.     

Profile of micronucleus frequencies and nuclear abnormalities in different species of electric fishes (Gymnotiformes) from the Eastern Amazon

The frequency of spontaneous micronucleus (MN) formation in fish species needs to be determined to evaluate their usefulness for genotoxic biomonitoring. The definition of a good bioindicator takes into account the current knowledge of its metabolic traits as well as other factors including its feeding behavior and relationship to the environment. In this study, we compared the basal frequencies of micronucleated erythrocytes and nuclear abnormalities (NA) among different species of the fish Order Gymnotiformes (Rhamphichthys marmoratus, Steatogenys elegans, Sternopygus macrurus, Parapteronotus hasemani, Gymnotus mamiraua, Gymnotus arapaima, Brachyhypopomus beebei, Brachyhypopomus n. sp. BENN) sampled in several localities of the Eastern Amazon. A baseline of MN and NA frequency in these fish was determined, enabling the identification of potentially useful species as models for genotoxicity studies. Only one impacted sample collected at a site in the River Caripetuba showed a significant number of NAs, which may be due to the release of wastewater by neighbouring mining industries and by the burnt fuel released by the small boats used by a local community. Our results may provide support for further studies in areas of the Eastern Amazon affected by mining, deforestation and other anthropogenic activities.     

基于2A肽策略构建多基因表达载体的研究进展

多基因表达载体的构建在生物技术领域尤其是基因治疗方面显得日趋重要。目前常用的多基因表达载体多存在载体容量小、上下游基因表达失衡、蛋白活性低或蛋白空间位置不理想等缺陷。2A肽是近年使用较多的一种构建多基因载体的工具,与其它多基因构建策略相比,2A肽策略具有更明显优势。就(类)2A肽的来源、剪切机制、生物学特征、与蛋白定位的关系以及应用方面做一综述。     

Modelling paralytic shellfish toxins (PSTs) transfer and accumulation in populations of two planktonic grazers

A model was developed in this paper in order to study and compare the paralytic shellfish toxin (PST) transfer and accumulation of two different potential PST vectors in the planktonic community, the heterotrophic dinoflagellate in its red form Noctiluca scintillans and the copepod Acartia clausi. Different factors that influence the toxin transfer such as toxin synthesis, grazing on toxic and non-toxic food and population size of PST producers and vectors were considered in the model. Moreover, a laboratory experiment was conducted in order to calculate the detoxification rates of Noctiluca fed on Alexandrium catenella. According to the model results, the two grazers showed a significant difference mainly in the timing of the PST accumulation. Noctiluca exhibited a rapid response to the grazing of Alexandrium with high initial toxin accumulation, followed by a reduction to zero concentration of toxins in a period of almost two days. In contrast, Acartia showed a considerable delay in comparison to Noctiluca to accumulate the same amount of toxin in the population. This delay is linked to the slower reproduction rates that characterize the copepod. The range of the initial values used for the sensitivity analysis of the model is representative of the coastal environment of a Galician ria (embayment located at the NW of Iberian Peninsula), where the two grazers and Alexandrium may co-exist. The model for Acartia showed less sensitivity to these key parameters probably due to the time delay in accumulation of significant amount of toxins. Both grazers showed a rapid (50 h) reduction of ingested toxin, suggesting inefficiency to transfer toxins through predation in the food web.     

A journey through the Amazon Middle Earth reveals Aspidoras azaghal (Siluriformes: Callichthyidae), a new species of armoured catfish from the rio Xingu basin,Brazil

Aspidoras azaghal n. sp. was discovered during a multitaxonomic scientific expedition to the remote Amazon Terra do Meio region in tributaries to the rio Xingu basin, Pará, Brazil. The new species can be promptly distinguished from its congeners by the following combination of features: (a) absence of the first dorsal-fin element; (b) parieto-supraoccipital fontanel located medially on bone; (c) absence of a longitudinal dark-brown or black stripe along flank midline; (d) ventral surface of trunk covered by clearly smaller, irregular and/or roundish platelets; (e) inner laminar expansion of infraorbital 1 well developed; (f) relatively wide frontal bone, with width equal to half of entire length; (g) absence of a thick, longitudinal conspicuous dark-brown stripe along dorsal portion of flank; and (h) poorly developed serrations on posterior margin of the pectoral-fin spine. Besides morphological evidence, the molecular analyses indicated significant differences between the new species and its congeners, with A. albater and A. raimundi as its closest species, showing 6.53% of genetic differentiation in both cases. The intraspecific molecular data revealed gene flow (peer fixation index, FST = 0.05249, P > 0.05, for the cytochrome oxidase I (COI) marker and FST = -0.01466, P > 0.05, for the control region) between specimens upstream and downstream from a 30-m height waterfall at the type-locality, which therefore represent a single population. Furthermore, it was possible to observe a unidirectional gene flow pattern, with genetic diversity increasing in the downstream direction.     

A new Hemigrammus from the upper Río Vaupés,Colombia (Characiformes: Characidae), with a discussion on the presence of an enlarged urogenital papilla in the family

Hemigrammus xaveriellus sp. nov. is described from the upper Río Vaupés basin (Amazon basin), Departamento Guaviare, Colombia. It is distinguished from all congeners by the combination of the following characters: presence of a conspicuous, dark, longitudinal midlateral stripe extending along the body; presence of a conspicuous rounded, horizontally elongated humeral blotch anterior to the beginning of the dark midlateral stripe; seven scale rows between the dorsal fin and lateral line (vs. five to six); and five scale rows between the lateral line and pelvic-fin insertion (vs. three to four). The single mature male of He. xaveriellus possessed a well-developed urogenital papilla, an unusual feature among characids. The presence of an enlarged urogenital papilla in the family is discussed, and comments regarding the putative relationships of the new species are presented.     

A new rheophilic South American darter (Crenuchidae: Characidium) from the rio Juruena basin,Brazil, with comments on morphological adaptations to life in fast-flowing waters

Characidium iaquira, a new species from the upper rio Juruena, rio Tapajós basin, Brazil, is described. The new species can be promptly distinguished from all congeners by having a unique v-shaped dark mark lying along the caudal-fin extension, in medium- and large-sized specimens, and a remarkable iridescent green colouration in life. Characidium iaquira is closely related to Characidium crandellii and Characidium declivirostre by sharing unambiguous synapomorphies such as branchiostegal membranes united to each other across the isthmus, a scaleless area extending from the isthmus to the pectoral girdle, and dermal flaps surrounding anterior and posterior naris independent, but touching each other distally. Morphological specializations of the paired fins in the three riffle-dwellers species are discussed, including the wing-like shape, robustness, and inclination of the pectoral fin.     

A molecular assessment of species diversity in Tympanopleura and Ageneiosus catfishes (Auchenipteridae: Siluriformes)

In order to test the congruence of genetic data to the morphologically defined Neotropical catfish genera Tympanopleura and Ageneiosus and explore species diversity, we generated 17 DNA barcodes from five of six species of Tympanopleura and 12 of 13 species of Ageneiosus. To discriminate limits between species, an automatic barcode gap discovery (ABGD), a generalised mixed yule-coalescent model (GYMC) and fixed distance thresholds Kimura two-parameter (K2P; 3%) were used to discriminate putative species limits from the DNA barcodes. The ABGD, GMYC and K2P methods agreed by each generating 13 clusters: six in Tympanopleura (five nominal plus one undescribed species) and seven in Ageneiosus. These clusters corresponded broadly to the described species, except in the case of the Ageneiosus ucayalensis group (A. akamai, A. dentatus, A. intrusus, A. ucayalensis, A. uranophthalmus and A. vittatus). Haplotype sharing and low divergences may have prevented molecular methods from distinguishing these species. We hypothesise that this is the result of a recent radiation of a sympatric species group distributed throughout the Amazon Basin. One putative new species of Tympanopleura was also supported by the molecular data. These results taken together highlight the utility of molecular methods such as DNA barcoding in understanding patterns of diversification across large geographic areas and in recognising overlooked diversity.