Oocyte selection: a new model for the maternal-age dependence of Down syndrome

Summary Previously proposed mechanisms for Down syndrome (trisomy 21) have generally invoked a progressive increase in meiotic nondisjunction to explain maternal-age dependence, but models of this sort have failed to predict the observed patterns of marker segregation. Here we propose instead that age-dependent trisomy 21 results primarily from a mechanism that favors maturation and utilization of euploid oocytes in preference to the pre-existing aneuploid products of mitotic (premeiotic) nondisjunction. The increased utilization of aneuploid oocytes at later stages of maternal life would result from their increased proportion following many progressive cycles of selection against their maturation in earlier stages. Derivation of a quantitative model and evaluation of existing data indicate that the pattern of marker segregation associated with agedependent trisomy 21 supports the proposed mechanism.     

Changing the landscape: a new strategy for estimating large phylogenies

In this paper we describe a new heuristic strategy designed to find optimal (parsimonious) trees for data sets with large numbers of taxa and characters. This new strategy uses an iterative searching process of branch swapping with equally weighted characters, followed by swapping with reweighted characters. This process increases the efficiency of the search because, after each round of swapping with reweighted characters, the subsequent swapping with equal weights will start from a different group (island) of trees that are only slightly, if at all, less optimal. In contrast, conventional heuristic searching with constant equal weighting can become trapped on islands of suboptimal trees. We test the new strategy against a conventional strategy and a modified conventional strategy and show that, within a given time, the new strategy finds trees that are markedly more parsimonious. We also compare our new strategy with a recent, independently developed strategy known as the Parsimony Ratchet.     

Microalgae in the postgenomic era: a blooming reservoir for new natural products

Bacteria, fungi, algae and higher plants are the most prolific producers of natural products (secondary metabolites). Compared to macroalgae, considerably fewer natural products have been isolated from microalgae, which offer the possibility of obtaining sufficient and well-defined biological material from laboratory cultures. Interest in microalgae is reinforced by large-scale data sets from genome sequencing projects and the development of genetic tools such as transformation protocols. This review highlights what is currently known about the biosynthesis and biological role of natural products in microalgae, with examples from isoprenoids, complex polyketides, nonribosomal peptides, polyunsaturated fatty acids and oxylipins, alkaloids, and aromatic secondary metabolites. In addition, we introduce a bioinformatic analysis of available genome sequences from totally 16 microalgae, belonging to the green and red algae, heterokonts and haptophytes. The results suggest that the biosynthetic potential of microalgae is underestimated and many microalgal natural products remain to be discovered.     

Microbiome of fungus-growing termites: a new reservoir for lignocellulase genes

Fungus-growing termites play an important role in lignocellulose degradation and carbon mineralization in tropical and subtropical regions, but the degradation potentiality of their gut microbiota has long been neglected. The high quality and quantity of intestinal microbial DNA are indispensable for exploring new cellulose genes from termites by function-based screening. Here, using a refined intestinal microbial DNA extraction method followed by multiple-displacement amplification (MDA), a fosmid library was constructed from the total microbial DNA isolated from the gut of a termite growing in fungi. Functional screening for endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase resulted in 12 β-glucosidase-positive clones and one xylanase-positive clone. The sequencing result of the xylanase-positive clone revealed an 1,818-bp open reading frame (ORF) encoding a 64.5-kDa multidomain endo-1,4-β-xylanase, designated Xyl6E7, which consisted of an N-terminal GH11 family catalytic domain, a CBM_4_9 domain, and a Listeria-Bacteroides repeat domain. Xyl6E7 was a highly active, substrate-specific, and endo-acting alkaline xylanase with considerably wide pH tolerance and stability but extremely low thermostability.     

Telomere length is associated with types of chromosome 21 nondisjunction: a new insight into the maternal age effect on Down syndrome birth

Advanced maternal age is a well-documented risk factor of chromosome 21 nondisjunction in humans, but understanding of this association at the genetic level is still limited. In particular, the state of maternal genetic age is unclear. In the present study, we estimated maternal genetic age by measuring telomere length of peripheral blood lymphocytes among age-matched mothers of children with Down syndrome (cases: N = 75) and mothers of euploid children (controls: N = 75) in an age range of 18–42 years. All blood samples were taken within 1 week of the birth of the child in both cases and controls. The telomere length estimation was performed by restriction digestion—Southern blot hybridization method. We stratified the cases on the basis of centromeric STR genotyping into maternal meiosis I (N = 48) and maternal meiosis II (N = 27) nondisjunction groups and used linear regression to compare telomere length as a function of age in the euploid, meiosis I and meiosis II groups. Our results show that all three groups have similar telomere length on average for younger mothers. As age increases, all groups show telomere loss, but that loss is largest in the meiosis II mother group and smallest in the euploid mother group with the meiosis I mother group in the middle. The regression lines for all three were statistically significantly different from each other (p < 0.001). Our results do not support the theory that younger women who have babies with Down syndrome do so because are ‘genetically older’ than their chronological age, but we provide the first evidence that older mothers who have babies with Down syndrome are “genetically older” than controls, who have euploid babies at the same age. We also show for the first time that telomere length attrition may be associated in some way with meiosis I and meiosis II nondisjunction of chromosome 21 and subsequent Down syndrome births at advanced maternal age.     

Marine biofilms on submerged surfaces are a reservoir for Escherichia coli and Vibrio cholerae

The enteric bacterium and potential human pathogen, Escherichia coli, is known to persist in tropical soils and coastal waters. Vibrio cholerae causes the disease cholera and inhabits marine environments including microbial films on submerged surfaces. The abundances of E. coli and V. cholerae were quantified in biofilm and water-column samples from three harbors in Honolulu, Hawai‘i, which differ in their local and international ship traffic. E. coli and, in some cases V. cholerae, occurred in relatively high abundances in marine biofilms formed on abiotic surfaces, including the exterior hulls of ships. The community fingerprints of the biofilms and the water harboring these pathogens were further analyzed. The community compositions of biofilms from different locations were more similar to each other than to water-column communities from the same locations. These results suggest that biofilms are an overlooked reservoir and a source of dissemination for E. coli and V. cholerae.     

Potential for ammonia recapture by farm woodlands: design and application of a new experimental facility

There has been increasing pressure on farmers in Europe to reduce the emissions of ammonia from their land. Due to the current financial climate in which farmers have to operate, it is important to identify ammonia control measures that can be adopted with minimum cost. The planting of trees around farmland and buildings has been identified as a potentially effective and low-cost measure to enhance ammonia recapture at a farm level and reduce long-range atmospheric transport. This work assesses experimentally what fraction of ammonia farm woodlands could potentially remove from the atmosphere. We constructed an experimental facility in southern Scotland to simulate a woodland shelterbelt planted in proximity to a small poultry unit. By measuring horizontal and vertical ammonia concentration profiles within the woodland, and comparing this to the concentration of an inert tracer (SF6) we estimate the depletion of ammonia due to dry deposition to the woodland canopy. Together with measurements of mean ammonia concentrations and throughfall fluxes of nitrogen, this information is used to provide a first estimate of the fraction of emitted ammonia that is recaptured by the woodland canopy. Analysis of these data give a lower limit of recapture of emitted ammonia, at the experimental facility, of 3%. By careful design of shelterbelt woodlands this figure could be significantly higher.     

Music notation: a new method for visualizing social interaction in animals and humans

Background

Researchers have developed a variety of techniques for the visual presentation of quantitative data. These techniques can help to reveal trends and regularities that would be difficult to see if the data were left in raw form. Such techniques can be of great help in exploratory data analysis, making apparent the organization of data sets, developing new hypotheses, and in selecting effects to be tested by statistical analysis. Researchers studying social interaction in groups of animals and humans, however, have few tools to present their raw data visually, and it can be especially difficult to perceive patterns in these data. In this paper I introduce a new graphical method for the visual display of interaction records in human and animal groups, and I illustrate this method using data taken on chickens forming dominance hierarchies.

Results

This new method presents data in a way that can help researchers immediately to see patterns and connections in long, detailed records of interaction. I show a variety of ways in which this new technique can be used: (1) to explore trends in the formation of both group social structures and individual relationships; (2) to compare interaction records across groups of real animals and between real animals and computer-simulated animal interactions; (3) to search for and discover new types of small-scale interaction sequences; and (4) to examine how interaction patterns in larger groups might emerge from those in component subgroups. In addition, I discuss how this method can be modified and extended for visualizing a variety of different kinds of social interaction in both humans and animals.

Conclusion

This method can help researchers develop new insights into the structure and organization of social interaction. Such insights can make it easier for researchers to explain behavioural processes, to select aspects of data for statistical analysis, to design further studies, and to formulate appropriate mathematical models and computer simulations.     

The effects of large and small-scale random events on the synchrony of metapopulation dynamics: a theoretical analysis

We present an analysis of the conditions under which migration and global random factors may determine large scale synchrony in the dynamics of spatially structured populations. We derive an analytic approximation which describes how the desynchronizing influence of local environmental stochasticity combines with the synchronizing influences of larger scale environmental stochastic variation and migration to determine population cross correlation coefficients. Despite the simplifications made by this analysis, computer simulations show that the behaviour of more complicated models is well described by our approximation over considerable regions of parameter space. We conclude that population synchrony is largely determined by the coefficients of variation (CVs) of the local and larger scale stochastic processes, and that migration alone is only likely to maintain population synchrony when the CV of the local stochastic process is very small.     

discs large in the Drosophila testis: An old player on a new task

Gamete development requires a coordinated soma-germ line interaction that ensures renewal and differentiation of germline and somatic stem cells. The physical contact between the germline and somatic cell populations is crucial because it allows the exchange of diffusible signals among them. The tumor suppressor gene discs large (dlg) encodes a septate junction protein with functions in epithelial cell polarity, asymmetric neuroblast division and formation of neuromuscular junctions. Our recent work reveals a new role of dlg in the Drosophila testis, as mutations in dlg lead to testis defects and cell death. Dlg is required throughout spermatogenesis in the somatic lineage and its localization changes from a uniform distribution along the plasma membrane of somatic cells in the testis apex, to a restricted localization on the distally located somatic cell in growing cysts. The extensive defects in dlg testis underline the importance of the somatic cells in the establishment and maintenance of the male stem cell niche and somatic cell differentiation. Here, we discuss our latest findings on the role of dlg in the Drosophila testis, supporting the view that junction proteins are dynamic structures, which can provide guiding cues to recruit scaffold proteins or other signaling molecules.Key words: dlg, Drosophila melanogaster, germ cell differentiation, septate junctions, somatic stem cells, somatic cyst cellsThe discovery of mutations causing neoplasia during Drosophila development¹ followed by the molecular characterization of these genes has shown that cell polarity is critically affected in the tumor cells. Three of these genes, lethal (2) giant larvae (lgl), discs large-1 (dlg) and scribble (scrib), encode scaffolding proteins, associated with either the cytoskeleton matrix or septate junctions.^2–9 Analysis over the last decades revealed that these proteins act as more than just static barriers limiting the diffusion of other components along the cortical cell domains. In particular, they function as dynamic organizing centers, targeting site-specific proteins in discrete domains and provide guiding cues for signaling molecules and insertion of membrane components.¹⁰ Nowadays, several studies place Dlg as a key player in numerous tissues at different time points throughout development, contributing to epithelial polarity establishment, polarized membrane insertion, asymmetric neuroblast division, formation of neuromuscular junctions (NMJ) and planar cell polarity in vertebrates.^{4,7,9,11–15} Interestingly, the four mammalian homologs of the Drosophila dlg are also involved in cell polarity and become downregulated in a series of human cancers. Moreover, a mammalian dlg-1 transgene can substitute a defective dlg gene in Drosophila and rescue the development of dlg mutant animals.^8,9,16 Therefore, it is perfectly plausible to envisage that Drosophila functions uncovered in other tissues may be similarly conserved in other species.Similar to vertebrates, the Drosophila testis consists of germ cells and somatic cells. The somatic cells of the hub form the organizing center at the apex of the testis and recruit germline stem cells (GSCs) (Fig. 1A), giving rise to the male stem cell niche. Each GSC attached to the hub is surrounded by two somatic stem cells (SSCs). Upon asymmetric stem cell division, each GSC produces a new GSC attached to the hub and a distally located gonialblast, whereas each SSC pair divides to generate two SSCs and two somatic cyst cells (SCCs).^17–19 The gonialblast divides mitotically four more times to give rise to 16 interconnected spermatogonial cells, forming a cyst surrounded by the two SCCs.²¹ Then, the spermatogonial cyst grows markedly in size and differentiates to primary spermatocytes that enter the pre-meiotic phase (Fig. 1A, B and D–F).¹⁹ We have recently investigated a new role of dlg in the Drosophila testis.²⁰ In contrast to the overgrowth phenotypes observed in imaginal discs and brain hemispheres,^4,6,21 dlg inactivation leads to testis degeneration during early larval development. The dlg testes are extremely small and contain a reduced number of GSCs loosely attached to the hub (Fig. 1C).²⁰ In addition, the few spermatogonial cysts, which become formed, fully degenerate during the second and third larval instars.Open in a separate windowFigure 1(A) Diagram depicting early spermatogenesis in Drosophila. The red line indicates the Dlg distribution in the hub, SSCs, early and late SCCs. GSCs, germline stem cells; SCCs, somatic cyst cells; SSCs, somatic stem cells. (B) Apex of wild-type 3^rd instar larval testis and (C) dlg^m52 3^rd instar larval testis displaying a reduced number of GSCs, spermatogonial and spermatocyte cysts. In mutant dlg^m52 testes, SSCs and early SCCS positively stained for Traffic-jam are still present. However, late SCCs identified by staining for Eye absent remain undetectable.²⁰ Vasa (red), Traffic-jam (green) and Arm + α-Spectrin (blue). (D–F) Pattern of Dlg distribution in 3^rd instar larval testis. (E and F) are enlargements at different optical sections of the testis shown in (D), displaying Dlg staining in the hub region and growing spermatocyte cysts, respectively. Testis hub is oriented towards the left.Recent advances in Drosophila spermatogenesis and the male stem cel niche have clearly shown that the intrinsic signals of the germ cells are important but not sufficient to support stem cell homeostasis. Signals emanating from SSCs and SCCs are also required for testis development. Physical contacts among the cell populations in the Drosophila testis allow the exchange of signals, which promote tissue survival and set the balance between stem cell identity and differentiation.¹⁸ Interestingly, the Dlg protein is present in all somatic cells including the hub, SSCs and SCCs (Fig. 1D–F) and a specific requirement of dlg in these cells is further supported by the finding that the mutant phenotype could be reverted by expressing dlg in somatic cells but not in germ cells.²⁰ Further analysis points out that the mutant GSCs are significantly larger than in wild-type, lower in number and loosely attached to the hub.²⁰ Preliminary results indicate a defective orientation of the daughter centrosome and absence of mitotic spindle in dividing GSCs, which together with the increased GSC size, allows us to speculate that GSCs may grow but fail to undergo mitosis. Similar phenotypes are observed in mutations affecting the insulin pathway,²² further stressing the importance of cell communication between germ cells and somatic cells. However, a functional connection between dlg and the insulin pathway remains yet to be experimentally determined. The defects detected in the dlg mutant testis place dlg as a key regulator in the early development of spermatogonial cysts.During testis differentiation, the Dlg protein displays a dynamic change in its intracellular localization. First, Dlg is uniformly associated with the plasma membrane on all somatic cells in the male stem cell niche and early spermatogonial cysts, and then becomes restricted to the most proximal SCC in late spermatogonial cysts and growing spermatocyte cysts (Fig. 1D–F). The transition from a uniform to a restricted distribution is achieved between the 8- to 16-cell cyst stages, when one of the two SCCs caps the distal side of the growing cyst. Interestingly, the capping corresponds to the axis of cyst growth and points out the direction of cyst expansion. A restoration of nearly normal testis morphology can be obtained by expressing a dlg transgene in SSCs and early SCCs. In contrast, expression of a dlg transgene in later SCCs can still restore the development of already formed cysts, some of which may reach an advanced post-meiotic stage, but the testis is generally depleted in early cysts.²⁰ These data indicate that dlg is required for the differentiation of the somatic cell lineage and, therefore, the early differentiation of the germline into spermatogonial cells. Results of RNAi experiments provide also evidence that dlg silencing in late SSCs results in a fragmentation of the cysts in advanced stages.²⁰ The specific recruitment of Dlg on the membrane of distal SCCs remains an open question, although it is possible to envisage that phosphorylation of Dlg by the PAR-1 kinase may play a role, as it has been shown in the case of postsynaptic targeting of Dlg in NMJs.²³Therefore, Dlg may exert different functions in the somatic cells that are required for (1) GSC attachment to the hub and proper asymmetric GSC division, (2) the architecture and early differentiation of the spermatogonial cysts and (3) the expansion and growth of the spermatocyte cysts. Presumably, dlg is required for establishing and maintaining a tight connection between GSCs and SSCs around the hub. The connection between gonialblast and SCC is also maintained during the mitotic divisions. In SSCs and early SCCs, dlg acts critically to establish a normal cyst structure, whereas in further spermatogonial and spermatocyte stages dlg is critical for the survival, growth and expansion of the cyst. Our rescue experiments further suggest that if proper cyst architecture is not established when the two stem cell populations move away from the hub, it cannot be re-established at later stages. Moreover, the restricted Dlg localization in the distal SCC suggests that dlg may be necessary for the polarized growth of spermatocyte cysts and thus act as a critical factor for planar cell polarity. In the second phase, dlg is involved in spermatogonial and spermatocyte cyst growth, viability and differentiation. Further RNA silencing experiments using GAL4-drivers that target dlg in SCCs during late spermatocyte growth, meiosis and post-meiotic stages may further provide insights into dlg requirement during the whole spermatogenesis. Preliminary results indicate that Dlg is similarly produced and localized on the distal SCC in spermatocyte and spermatid cysts of adult testes, suggesting that dlg may be required from the early stages, from the establishment of male stem cell niche and SCC survival, up to the later stages of sperm formation.An unexpected finding of our analysis deals with the formation of wavy and ruffled plasma membrane in dlg overexpressing cells capping the spermatocyte cysts. One way to interpret this result would be to consider that Dlg regulates the intensity of germ cell encapsulation through the Egfr pathway, which is the major signaling pathway active at the microenvironment of the spermatogonial cysts.^24,25 Membrane ruffling, detected in somatic cells upon dlg overexpression, is highly reminiscent of the formation of lammellipodia-like structures formed upon upregulation of Rac1 in SCCs.²⁶ Rac1 is a downstream component of the Egfr pathway and acts antagonistically to Rho to regulate germ cell encapsulation. As the Dlg protein plays a central role in the organization of epithelial junctions and in signal transduction at sites of cell-cell contact, it is possible to envisage that the C-terminal tail of Egfr interacts with one of the PDZ domains of Dlg.⁹ In this way, dlg inactivation would result in a disruption of the Egfr protein complexes, block the Egfr pathway and impair Rac1 function. Based on these data, we hypothesize that Dlg may act on the cytoskeleton of the somatic cells to mediate cell-shape changes leading to either cellular extensions over the spermatogonial and spermatocyte cysts or reinforcing cell-to-cell contact with the growing germ cells.A second possibility would imply a general role for Dlg in membrane proliferation and expansion of the SCCs. It has already been shown that Dlg regulates membrane proliferation in a subset of NMJ in a dose-dependent fashion.²⁷ Recent focus on membrane growth during cellularization indicates again that Dlg is an important player in the process of polarized membrane insertion.^11,28–30 Up to now, there is no mechanism describing how SCCs in Drosophila testis expand, elongate and envelop germ cell cysts, and how the SCCs direct sperm differentiation and individualization. Membrane proliferation during tissue spreading and cell surface extensions is frequently associated with the formation of membrane ruffles.^31,32 The finding that dlg overexpression in the distal SCC leads to membrane ruffling indicates that Dlg may mediate membrane growth and membrane extension over the cysts but not necessarily at the expense of the proximal SCC devoid of Dlg. Therefore, there should be a physical limitation in the expansion of the dlg-expressing cell, independent of the amount of synthesized Dlg. Further analyses of components at the junctions between the distal and proximal SCCs or components exhibiting a complementary distribution to Dlg may provide ways to identify further regulators of testis morphogenesis.If Dlg defines sites of membrane addition it may provide a link between membrane trafficking and insertion of polarized membrane components. In NMJs, the postsynaptic distribution of the t-SNARE protein Gtaxin depends on its direct interaction to the Dlg GUK domain,¹² whereas in early embryogenesis Dlg genetically interacts with Exo84.³³ Moreover, the Dlg-Strabismus complex recruits membrane associated proteins and lipids from internal membranes to sites of new plasma membrane formation.¹¹ The occurrence of similar proteins in testis was reported in humans where the SNARE-associated component Snapin binds Pumilio2 and Nanos1 proteins in the male germ cells.³⁴ It would be interesting to know whether Dlg plays a similar role in Drosophila testis, in guiding t-SNARE proteins and components of the exocyst complex into intracellular membranes, either directly or indirectly by regulating the distribution of their direct binding partners. Although Dlg may bind to different proteins in epithelial cells, neuroblasts and NMJ according to the protein availability in these tissues, the function of the Dlg protein may be still conserved in a broader sense. Through its PDZ domains Dlg may bind to numerous transmembrane proteins and receptors, and may link them to the cytoskeleton or signaling pathways. The knowledge gained on the role of Dlg in these systems will allow us to study how Dlg mediates membrane proliferation in the early germ cells in male gonads.Recent work has showed that Zero population growth (Zpg), the Drosophila gap junction Innexin 4, is localized to the spermatogonia surface, primarily on the sides adjacent to SCCs³⁵ and is required for the survival and differentiation of early germ cells in both sexes.^35–37 In zpg testes, the spermatogonia are unable to differentiate and are progressively lost, leading to the formation of tiny testes containing a small number of GSCs and germline clusters devoid of branching fusome,³⁵ resembling the dlg phenotype. In contrast, the SCCs that die through apoptosis in dlg testes are present in zpg, indicating that Dlg acts primarily on SCCs and Zpg on the germ cells.^20,35 Moreover, zpg testes display often a considerably enlarged hub. However, a direct comparison of the effect of the two proteins on the hub cannot be made because the null dlg^m52 allele produces a truncated non-functional Dlg protein that could still be detected in the hub.²⁰ Apparently this protein, which contains the PDZ1 and PDZ2 domains, could be recognized by a monoclonal antibody against the PDZ2 domain (data not shown).²⁰ This observation raises the possibility that the truncated Dlg protein may maintain some of its binding properties, which prevents the hub structure from falling apart. Further studies will be performed to determine the requirement of dlg in hub formation and structure.Our results, complementary to current researches conducted in this field, point out the importance of the somatic cell contribution in the organization of the Drosophila testis and the differentiation of the male germline. In mammals, spermatogenesis depends also on interactions between somatic Sertoli cells and germ cells. Sertoli cells act as supportive somatic cells and contain junction proteins with a high degree of similarity to Dlg. These proteins play a critical role in mammalian spermatogenesis.^38,39 Furthermore, the identification of mammalian genes with known function in Drosophila spermatogenesis and the evolutionary conservation among the Dlg proteins suggests that the pathways regulating the balance between stem cell renewal and differentiation might be similarly conserved. Interestingly, recent observations in mammals indicate that Dlg homologs play a role in the formation of mouse gonads and interact with gap junction proteins.^13,40 In addition, Dlg is required for smooth muscle orientation in the mouse ureter¹³ and interacts with the gap protein Connexin 32,⁴¹ whereas ZO-1, a MAGUK protein bearing similarity to Dlg and associated with tight junctions in mammalian Sertoli cells,³⁹ binds also to gap junction proteins, among them connexin 43, which is the predominant gap junction protein in the testis.^38,39,42 All these observations point out to functional similarities between Drosophila and vertebrate Dlg and provide strong indications that our findings in Drosophila may be extended to higher organisms.     

A large reservoir as a source of zooplankton for the river: structure of the populations and influence of fish predation

Within an extensive programme on the Seine river, including the large reservoirs in the upstream basin, zooplankton populations were investigated at the reservoir-river interface to find out their fate when released into the river. Zooplankton structure, abundance and biomass were analysed in the river upstream and downstream from the reservoir, in the release canal and in the reservoir. Whereas the river zooplankton are dominated by rotifers, microcrustaceans represent a large part of the population in the reservoir, because of the longer residence time (6 months). We have shown that the reservoir is a source of zooplankton; we calculated a biomass export of 30-60 tons C year^-1, which is, however, rapidly lost in the river. One of the main loss factors was found to be selective fish predation on larger zooplankton species developed in the reservoir.      

iCatch:a new strategy for capturing large DNA fragments using homing endonucleases

Natural genetic materials contain many biosynthetic gene clusters encoding potentially valuable natural products,many of which can be used directly without codon optimization or other manipulations.With the development of synthetic biology,several DNA assembly standards have been proposed,conveniently facilitating the reuse of natural materials.Among these standards,the iBrick assembly standard was developed by our laboratory to manipulate large DNA fragments,employing two homing endonucleases.Considering the difficulty of cloning large iBrick parts using conventional endonuclease-mediated restriction and ligation methods,we herein present a new method,known as iCatch,which readily captures biosynthetic gene clusters.As the clusters cloned by iCatch have the prefix and suffix of the iBrick standard,they serve as new iBrick parts and are therefore conducive to further editing and assembly with the iBrick standard.iCatch employs the natural homologous recombination system to flank the region of interest with I-Scel and PI-Pspl recognition sites,after which the genome is digested with I-Scel or PI-Pspl and the fragments are then self-ligated to clone the target DNA fragments.We used this method to successfully capture the actinorhodin biosynthetic cluster from Streptomyces coelicolor and then heterologously expressed this cluster in a thermophilic Streptomyces strain.We propose that iCatch can be used for the cloning of DNA sequences that are dozens of kilobases in length,facilitating the heterologous expression of microbial natural products.Moreover,this cloning methodology can be a complementary tool for the iBrick standard,especially in applications requiring the manipulation of large DNA fragments.     

Vimentin filaments in fibroblasts are a reservoir for SNAP23, a component of the membrane fusion machinery

Soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) are core machinery for membrane fusion during intracellular vesicular transport. Synaptosome-associated protein of 23 kDa (SNAP23) is a target SNARE previously identified at the plasma membrane, where it is involved in exocytotic membrane fusion. Here we show that SNAP23 associates with vimentin filaments in a Triton X-100 insoluble fraction in fibroblasts in primary culture and HeLa cells. Upon treatment of human fibroblasts with N-ethyl-maleimide, SNAP23 dissociates from vimentin filaments and forms a protein complex with syntaxin 4, a plasma membrane SNARE. The vimentin-associated pool of SNAP23 can therefore be a reservoir, which would supply the plasma membrane fusion machinery, in fibroblasts. Our observation points to a yet unexplored role of intermediate filaments.     

Building a MCHR1 homology model provides insight into the receptor-antagonist contacts that are important for the development of new anti-obesity agents

Melanin-concentrating hormone (MCH) regulates feeding and energy homeostasis through interaction with its receptor, the melanin-concentrating receptor 1 (MCHR1), making it a target in the treatment of obesity. Molecular modeling and docking studies were performed in order to find a binding model for the docking of two new series of MCHR1 antagonists to the receptor. Results suggested interactions between the ligands and two glutamines (Gln5.42 and Gln6.55) not conserved in many of the GPCRs family members. Histamine 3 receptor (HRH3) presents two apolar residues in the aforementioned positions and the available biological data against this receptor supported the role of the two glutamines in the binding of antagonists to the MCHR1. This knowledge could be useful in the development of new, more active and more selective MCHR1 antagonists.     

Use and non-use values as motivational construct dimensions for farm animal welfare: impacts on the economic outcome for the farm

This study explored how farmers’ motivation in terms of use values and/or non-use values to work with farm animal welfare are associated with the economic outcome for the farm. Use values in farm animal welfare refer to economic value derived from productivity and profitability considerations. Non-use values in farm animal welfare refer to economic value derived from good animal welfare, irrespective of the use the farmer derives from the animal, currently or in the future. The analysis was based on detailed information about the income statements of a sample of Swedish dairy farmers, obtained from the Swedish Farm Economic Survey, complemented with survey information about their perceived use and non-use values in farm animal welfare. The findings suggest that farm economic outcome is significantly associated with motivation in terms of use values, but not so much with motivation in terms of non-use values. This is interesting from a policy point of view, because it indicates that farmers with different approaches to farm animal welfare may experience different economic outcomes for their farms. Findings can, for instance, be used to strengthen farmers’ engagement in various private quality assurance standards, which generally focus on values of non-use type, by pointing to that realisation of such values will not impair the economic outcome of the farms. Moreover, findings also suggest that farmers’ economic incentives for engagement in such standards may need to be further strengthened in order to become more attractive, as findings point to that a focus on non-use values generally is not associated with more favourable economic outcomes.     

The importomer peroxins are differentially required for peroxisome assembly and meiotic development in Podospora anserina: insights into a new peroxisome import pathway

Peroxisome biogenesis relies on two known peroxisome matrix protein import pathways that are mediated by the receptors PEX5 and PEX7. These pathways converge at the importomer, a peroxisome‐membrane complex that is required for protein translocation into peroxisomes and consists of docking and RING–finger subcomplexes. In the fungus Podospora anserina, the RING–finger peroxins are crucial for meiocyte formation, while PEX5, PEX7 or the docking peroxin PEX14 are not. Here we show that PEX14 and the PEX14‐related protein PEX14/17 are differentially involved in peroxisome import during development. PEX14/17 activity does not compensate for loss of PEX14 function, and elimination of both proteins has no effect on meiocyte differentiation. In contrast, the docking peroxin PEX13, and the peroxins implicated in peroxisome membrane biogenesis PEX3 and PEX19, are required for meiocyte formation. Remarkably, the PTS2 coreceptor PEX20 is also essential for meiocyte differentiation and this function does not require PEX5 or PEX7. This finding suggests that PEX20 can mediate the import receptor activity of specific peroxisome matrix proteins. Our results suggest a new pathway for peroxisome import, which relies on PEX20 as import receptor and which seems critically required for specific developmental processes, like meiocyte differentiation in P. anserina.