Extent of cell differentiation and capacity for cartilage synthesis in human adult adipose‐derived stem cells: Comparison with fetal chondrocytes

This study evaluated the extent of differentiation and cartilage biosynthetic capacity of human adult adipose‐derived stem cells relative to human fetal chondrocytes. Both types of cell were seeded into nonwoven‐mesh polyglycolic acid (PGA) scaffolds and cultured under dynamic conditions with and without addition of TGF‐β1 and insulin. Gene expression for aggrecan and collagen type II was upregulated in the stem cells in the presence of growth factors, and key components of articular cartilage such as glycosaminoglycan (GAG) and collagen type II were synthesized in cultured tissue constructs. However, on a per cell basis and in the presence of growth factors, accumulation of GAG and collagen type II were, respectively, 3.4‐ and 6.1‐fold lower in the stem cell cultures than in the chondrocyte cultures. Although the stem cells synthesized significantly higher levels of total collagen than the chondrocytes, only about 2.4% of this collagen was collagen type II. Relative to cultures without added growth factors, treatment of the stem cells with TGF‐β1 and insulin resulted in a 59% increase in GAG synthesis, but there was no significant change in collagen production even though collagen type II gene expression was upregulated 530‐fold. In contrast, in the chondrocyte cultures, synthesis of collagen type II and levels of collagen type II as a percentage of total collagen more than doubled after growth factors were applied. Although considerable progress has been achieved to develop differentiation strategies and scaffold‐based culture techniques for adult mesenchymal stem cells, the extent of differentiation of human adipose‐derived stem cells in this study and their capacity for cartilage synthesis fell considerably short of those of fetal chondrocytes. Biotechnol. Bioeng. 2010;107: 393–401. © 2010 Wiley Periodicals, Inc.     

Investigating the role of native propionyl‐CoA and methylmalonyl‐CoA metabolism on heterologous polyketide production in Escherichia coli

6‐Deoxyerythronolide B (6dEB) is the macrocyclic aglycone precursor of the antibiotic natural product erythromycin. Heterologous production of 6dEB in Escherichia coli was accomplished, in part, by designed over‐expression of a native prpE gene (encoding a propionyl‐CoA synthetase) and heterologous pcc genes (encoding a propionyl‐CoA carboxylase) to supply the needed propionyl‐CoA and (2S)‐methylmalonyl‐CoA biosynthetic substrates. Separate E. coli metabolism includes three enzymes, Sbm (a methylmalonyl‐CoA mutase), YgfG (a methylmalonyl‐CoA decarboxylase), and YgfH (a propionyl‐CoA:succinate CoA transferase), also involved in propionyl‐CoA and methylmalonyl‐CoA metabolism. In this study, the sbm, ygfG, and ygfH genes were individually deleted and over‐expressed to investigate their effect on heterologous 6dEB production. Our results indicate that the deletion and over‐expression of sbm did not influence 6dEB production; ygfG over‐expression reduced 6dEB production by fourfold while ygfH deletion increased 6dEB titers from 65 to 129 mg/L in shake flask experiments. It was also found that native E. coli metabolism could support 6dEB biosynthesis in the absence of exogenous propionate and the substrate provision pcc genes. Lastly, the effect of the ygfH deletion was tested in batch bioreactor cultures in which 6dEB titers improved from 206 to 527 mg/L. Biotechnol. Bioeng. 2010; 105: 567–573. © 2009 Wiley Periodicals, Inc.     

Generation of a triple‐gene knockout mammalian cell line using engineered zinc‐finger nucleases

Mammalian cells with multi‐gene knockouts could be of considerable utility in research, drug discovery, and cell‐based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events—a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc‐finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi‐locus genome engineering. Biotechnol. Bioeng. 2010; 106: 97–105. © 2009 Wiley Periodicals, Inc.     

Efficient synthesis of 6′-sialyllactose, 6,6′-disialyllactose, and 6′-KDO-lactose by metabolically engineered E. coli expressing a multifunctional sialyltransferase from the Photobacterium sp. JT-ISH-224

We have previously reported the efficient conversion of lactose into 3′-sialyllactose by high cell density cultures of a genetically engineered Escherichia coli strain expressing the Neisseria meningitidis gene for α-(2→3)-sialyltransferase [Fierfort, N.; Samain, E. J. Biotechnol. 2008, 134, 261-265.]. First attempts to use a similar strategy to produce 6′-sialyllactose with a strain expressing α-(2→6)-sialyltransferase from the Photobacterium sp. JT-ISH-224 led to the production of a trisaccharide that was identified as KDO-lactose (2-keto-3-deoxy-manno-octonyllactose). This result showed that α-(2→6)-sialyltransferase was able to use CMP-KDO as sugar donor and preferentially used CMP-KDO over CMP-Neu5Ac. By reducing the expression level of the sialyltransferase gene and increasing that of the neuABC genes, we have been able to favour the formation of 6′-sialyllactose and to prevent the formation of KDO-lactose. However, in this case, a third lactose derivative, which was identified as 6,6′-disialyllactose, was also produced. Formation of 6,6′-disialyllactose was mainly observed under conditions of lactose shortage. On the other hand, when the culture was continuously fed with an excess of lactose, 6′-sialyllactose was almost the only product detected and its final concentration was higher than 30 g/L of culture medium.     

Harvesting and cryopreservation of lymphatic endothelial cells for lymphatic tissue engineering

In order to provide a suitable source of cells for lymphatic tissue engineering, the present study was designed to investigate techniques for harvesting and cryopreservation of human dermal lymphatic endothelial cells (LECs) in vitro. The LECs were isolated from children’s foreskins and then cultured in endothelial growth medium-2 MV (EGM-2-MV) with 5% FBS. The second passage LECs were suspended in cryopreservation solution containing 40% FBS and 10% Me₂SO in EGM-2-MV, cooled to −80 °C at about 1 °C/min and stored in liquid nitrogen. Samples were thawed quickly in a 37 °C water bath, and the cryoprotectant was removed by serial elution. The membrane integrity of thawed LECs was determined by trypan blue staining exclusion, and their proliferation was evaluated using the MTT method. The expanded cells of two groups were identified using immunofluorescence staining and RT-PCR with lymphatic-specific markers such as Podoplanin and VEGFR-3. Uptake of fluorescent DiI-Ac-LDL and microtubular formation in three-dimensional cultures were used to detect the function of LECs. Flow cytometry was applied to identify cells and to measure the apoptosis rate as well. Cryopreservation resulted in a retrieval of 67 ± 4% and an intact cell rate of 80 ± 3%. The early apoptosis rate of thawed LECs (9.15 ± 0.34%) was higher than that of fresh control LECs (5.31 ± 0.23%). The growth curves of thawed LECs were similar to those of fresh LECs. The thawed LECs were propagated for at least 6-7 passages without alterations in phenotype and function. Highly purified LECs can be isolated by immunomagnetic beads from human dermis. The cryopreserved/thawed and recultivated LECs are proven to have high vitality and growth potential in vitro and may be considered suitable seed cells for lymphatic tissue engineering.     

An energy budget approach for evaluating the biocontrol potential of cotton aphid (Aphis gossypii) by the ladybeetle Propylaea japonica

Biological control of economically important crop pests is an important component of integrated pest management (IPM) strategies. Predator–prey energy relationships are critical to the success of biocontrol strategies; however, these relationships are often ignored in many IPM programs. In this study, the biocontrol potential of cotton aphid, Aphis gossypii (Glover) (Hemiptera: Aphididae), by the ladybeetle Propylaea japonica (Thunberg) (Coleoptera: Coccinellidae) was estimated in terms of energy budgets calculated at 27 ± 1 °C. The energy equivalent of prey subjects (aphids) consumed was estimated from bomb calorimetry and partitioned into the energy associated with ingestion, assimilation, respiration, reproduction, and waste for each developmental stage of the lady beetle. The average assimilation efficiencies for larval and adult ladybeetles were 88.2 and 91.1%, respectively, whereas net ecological efficiencies were 17.6% for larvae and 2.6% for adults. Similarly, assimilation efficiencies of cotton aphids were 71.5 and 74.4% for nymphs and adults, respectively. Based on energy budget calculations, approximately 520, 3‐day‐old aphids and 5 356, 3‐day‐old aphids were estimated to be consumed by the ladybeetle larval stage and the female adult stage, respectively. These estimates were similar to the actual number of aphids consumed by the ladybeetles, based on actual counts. The current data demonstrate that P. japonica is an important natural enemy of the cotton aphid, and that predator–prey energy relationships can play a critical role in biocontrol strategies and IPM programs.     

ADAPTATION TO DESICCATION FAILS TO GENERATE PRE‐ AND POSTMATING ISOLATION IN REPLICATE DROSOPHILA MELANOGASTER LABORATORY POPULATIONS

Many laboratory speciation experiments have raised allopatric populations in different environments to determine whether reproductive isolation evolves as a byproduct of adaptation (a form of ecological speciation). Few, however, have addressed the evolution of both pre‐ and postmating isolation or investigated the conditions affecting the process. We present results of an evolution experiment in which 12 lines of Drosophila melanogaster were derived from a common population and then independently evolved for more than 57 generations under alternative selection treatments. Six “desiccation” populations had access to food and water removed during a period of their adult lives generating strong desiccation selection, and six “starvation” populations had access to food but not water removed for the same period, generating a mild starvation stress. Substantial divergence of cuticular hydrocarbons occurred between the desiccation and starvation populations, key traits that have been implicated in sexual isolation in Drosophila. Despite this divergence, there was no detectable premating isolation between desiccation and starvation populations and postmating isolation was asymmetrical: the fitness of F1 hybrids was reduced in the desiccation but not the starvation environment. This asymmetry was likely caused by the absence of divergent selection: adaptation to desiccation appears to have come at no cost to performance in the starvation environment. Novel environments are thought to promote the evolution of reproductive isolation. Understanding the conditions that favor or hamper this remains a key challenge for speciation research.     

THE BIOLOGY OF SPECIATION

Since Darwin published the “Origin,” great progress has been made in our understanding of speciation mechanisms. The early investigations by Mayr and Dobzhansky linked Darwin's view of speciation by adaptive divergence to the evolution of reproductive isolation, and thus provided a framework for studying the origin of species. However, major controversies and questions remain, including: When is speciation nonecological? Under what conditions does geographic isolation constitute a reproductive isolating barrier? and How do we estimate the “importance” of different isolating barriers? Here, we address these questions, providing historical background and offering some new perspectives. A topic of great recent interest is the role of ecology in speciation. “Ecological speciation” is defined as the case in which divergent selection leads to reproductive isolation, with speciation under uniform selection, polyploid speciation, and speciation by genetic drift defined as “nonecological.” We review these proposed cases of nonecological speciation and conclude that speciation by uniform selection and polyploidy normally involve ecological processes. Furthermore, because selection can impart reproductive isolation both directly through traits under selection and indirectly through pleiotropy and linkage, it is much more effective in producing isolation than genetic drift. We thus argue that natural selection is a ubiquitous part of speciation, and given the many ways in which stochastic and deterministic factors may interact during divergence, we question whether the ecological speciation concept is useful. We also suggest that geographic isolation caused by adaptation to different habitats plays a major, and largely neglected, role in speciation. We thus provide a framework for incorporating geographic isolation into the biological species concept (BSC) by separating ecological from historical processes that govern species distributions, allowing for an estimate of geographic isolation based upon genetic differences between taxa. Finally, we suggest that the individual and relative contributions of all potential barriers be estimated for species pairs that have recently achieved species status under the criteria of the BSC. Only in this way will it be possible to distinguish those barriers that have actually contributed to speciation from those that have accumulated after speciation is complete. We conclude that ecological adaptation is the major driver of reproductive isolation, and that the term “biology of speciation,” as proposed by Mayr, remains an accurate and useful characterization of the diversity of speciation mechanisms.     

GEOGRAPHIC VARIATION IN THE SONGS OF NEOTROPICAL SINGING MICE: TESTING THE RELATIVE IMPORTANCE OF DRIFT AND LOCAL ADAPTATION

Patterns of geographic variation in communication systems can provide insight into the processes that drive phenotypic evolution. Although work in birds, anurans, and insects demonstrates that acoustic signals are sensitive to diverse selective and stochastic forces, processes that shape variation in mammalian vocalizations are poorly understood. We quantified geographic variation in the advertisement songs of sister species of singing mice, montane rodents with a unique mode of vocal communication. We tested three hypotheses to explain spatial variation in the song of the lower altitude species, Scotinomys teguina: selection for species recognition in sympatry with congener, S. xerampelinus, acoustic adaptation to different environments, and stochastic divergence. Mice were sampled at seven sites in Costa Rica and Panamá; genetic distances were estimated from mitochondrial control region sequences, between‐site differences in acoustic environment were estimated from climatic data. Acoustic, genetic and geographic distances were all highly correlated in S. teguina, suggesting that population differentiation in song is largely shaped by genetic drift. Contrasts between interspecific genetic‐acoustic distances were significantly greater than expectations derived from intraspecific contrasts, indicating accelerated evolution of species‐specific song. We propose that, although much intraspecific acoustic variation is effectively neutral, selection has been important in shaping species differences in song.     

沿海榆毡蚧的生物学特性及防治

园林重要害虫沿海榆毡蚧Eriococcus costatus(Danzig)在山西太谷1年发生1代,以2龄若虫及预蛹越冬。雌虫经卵、1龄若虫、2龄若虫、成虫完成生活史。雄虫则经卵、1龄若虫、2龄若虫、预蛹、冬前蛹(无翅型)、无翅雄成虫和部分若虫到冬后化蛹(有翅型)、有翅雄成虫,完成其生活史。影响该虫种群动态的主要生态因子包括温度、湿度、食料、天敌等,其中湿度在初孵若虫期是最主要的影响因子。4月上中旬为该虫的最佳防治时期,杀虫剂宜采用内吸性、触杀型的,且脂溶性效果较好。