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Spiny brown dinoflagellate cysts are commonly used as sea‐ice indicators in the Arctic, but their biological affinities are not well known. We present the first indication of hitherto temperate Protoperidinium tricingulatum in the Arctic based on single‐cell LSU rDNA sequencing from sediments of the Disko Bay‐Vaigat Sound, West Greenland. The morphological similarity of the sequenced cyst morphotype to the sea‐ice indicator Islandinium? cezare morphotype 1 is striking. The morphology of the isolated cysts, as well as those observed in the total cyst assemblage following standard palynological preparation, both resemble either I.? cezare morphotype 1 or P. tricingulatum, suggesting that the specimens may in fact be close morphological variants of the same species. In addition, nine LSU rDNA sequences were obtained from morphological variants assigned to Islandinium minutum s.l.: including both subspecies minutum and subspecies barbatum. The two subspecies could not be differentiated based on partial LSU rDNA sequencing. Overall, Arctic spiny brown dinoflagellate cyst species may be morphologically more diverse and taxonomically more complex than shown earlier and further genetic and morphological studies are needed. Importantly, the value of cysts as palaeoecological indicators depends on a sound understanding of their biological affinity and taxonomy. [ABSTRACT FROM AUTHOR]

Cells sense extracellular matrix‐induced biophysical signals, which are transduced into intracellular signaling cascades, and trigger a series of cell responses, including adhesion, migration, and lineage commitment. Traditionally, in in vitro context, monofactorial approaches are employed to control cell fate, despite the fact that in vivo cells are exposed simultaneously to a diverse range of signals. Herein, an overview of key mechanotransduction pathways is first provided. Conventional single‐factor and contemporary multifactorial methodologies, based on substrate rigidity and surface topography, are then reviewed to recapitulate in vitro the in vivo niche, in an attempt to elucidate the underlying mechanisms involved in human mesenchymal stromal cell‐material interactions. [ABSTRACT FROM AUTHOR]

The Pikialasorsuaq (North Water polynya) is an area of local and global cultural and ecological significance. However, over the last decades, the region has been subject to rapid warming, and in some recent years, the seasonal ice arch that has historically defined the polynya's northern boundary has failed to form. Both factors are deemed to alter the polynya's ecosystem functioning. To understand how climate‐induced changes to the Pikialasorsuaq impact the basis of the marine food web, we explored diatom community‐level responses to changing conditions, from a sediment core spanning the last 3800 years. Four metrics were used: total diatom concentrations, taxonomic composition, mean size, and diversity. Generalized additive model statistics highlight significant changes at ca. 2400, 2050, 1550, 1200, and 130 cal years BP, all coeval with known transitions between colder and warmer intervals of the Late Holocene, and regime shifts in the Pikialasorsuaq. Notably, a weaker/contracted polynya during the Roman Warm Period and Medieval Climate Anomaly caused the diatom community to reorganize via shifts in species composition, with the presence of larger taxa but lower diversity, and significantly reduced export production. This study underlines the high sensitivity of primary producers to changes in the polynya dynamics and illustrates that the strong pulse of early spring cryopelagic diatoms that makes the Pikialasorsuaq exceptionally productive may be jeopardized by rapid warming and associated Nares Strait ice arch destabilization. Future alterations to the phenology of primary producers may disproportionately impact higher trophic levels and keystone species in this region, with implications for Indigenous Peoples and global diversity. [ABSTRACT FROM AUTHOR]

The organic matter content of marine sediments is often used to infer past changes in ocean conditions. However, the organic carbon pool preserved in coastal sediments is a complex mixture derived from different sources and may not reflect in situ processes. In this study, we combine taxonomic identification of reworked palynomorphs with pyrolysis organic geochemistry and reflected‐light organic petrographic microscopy to investigate the provenance, composition and preservation of organic matter in a marine sediment core retrieved from the NE Greenland shelf. Our study reveals continuous yet variable input of land‐derived organic carbon to the marine environment throughout the late Younger Dryas–Holocene, with the highest input of inert carbon in the late Younger Dryas. Although the sediments contain some recent marine palynomorphs, there is no other evidence of fresh marine organic carbon. In contrast, our results indicate that these shelf sediments represent a significant sink of recycled organic carbon. The results of pyrolysis geochemistry revealed that ~90% of the total organic carbon in the sediments is inert. The organic petrography analyses revealed that >70–84% of the organic carbon in the sediment core is terrigenous. Reworked dinoflagellate cysts showed a continuous provenance of Cretaceous land‐derived material, most likely from the nearby Clavering Island. Our study points to the importance of constraining the organic matter origin, composition and preservation in marine sediments to achieve more accurate palaeoenvironmental reconstructions based on organic proxies. [ABSTRACT FROM AUTHOR]

At the Pingorsuit Glacier in North‐West Greenland, an organic‐rich deposit that had recently emerged from the retreating ice cap was discovered at an elevation of 480 m above sea level. This paper reports on macrofossil analyses of a coarse detritus gyttja and peaty soil, which occurred beneath a thin cover of till and glacifluvial deposits. The sediments contained remains of vascular plants, mosses, beetles, caddisflies, midges, bryozoans, sponges and other invertebrates. The flora includes black spruce, tree birch, boreal shrubs and wetland and aquatic taxa, which shows that mires, lakes and ponds were present in the area. We describe a new extinct waterwort species Elatine odgaardii. The fossils were deposited in a boreal environment with a mean July air temperature that was at least 9 °C higher than at present. The fossil assemblages show strong similarities with others from Greenland that have been assigned an Early Pleistocene age, and we suggest a similar age for the sediments found at the margin of the Pingorsuit Glacier. At the Pingorsuit Glacier in North‐West Greenland, an organic‐rich deposit was discovered at an elevation of 480 m above sea level. The sediments contained remains of vascular plants, mosses, beetles, caddisflies, midges, bryozoans, sponges and other invertebrates. The fossils were deposited in a boreal environment with a mean July air temperature that was at least 9 °C higher than at present. [ABSTRACT FROM AUTHOR]

The combined effect of surface topography and substrate rigidity in stem cell cultures is still under‐investigated, especially when biodegradable polymers are used. Herein, we assessed human bone marrow stem cell response on aliphatic polyester substrates as a function of anisotropic grooved topography and rigidity (7 and 12 kPa). Planar tissue culture plastic (TCP, 3 GPa) and aliphatic polyester substrates were used as controls. Cell morphology analysis revealed that grooved substrates caused nuclei orientation/alignment in the direction of the grooves. After 21 days in osteogenic and chondrogenic media, the 3 GPa TCP and the grooved 12 kPa substrate induced significantly higher calcium deposition and alkaline phosphatase (ALP) activity and glycosaminoglycan (GAG) deposition, respectively, than the other groups. After 14 days in tenogenic media, the 3 GPa TCP upregulated four and downregulated four genes; the planar 7 kPa substrate upregulated seven genes and downregulated one gene; and the grooved 12 kPa substrate upregulated seven genes and downregulated one gene. After 21 days in adipogenic media, the softest (7 kPa) substrates induced significantly higher oil droplet deposition than the other substrates and the grooved substrate induced significantly higher droplet deposition than the planar. Our data pave the way for more rational design of bioinspired constructs. [ABSTRACT FROM AUTHOR]

The expansion of mcr‐carrying bacteria is a well‐recognized public health problem. Measures to contain mcr spread have mainly been focused on the food–animal production sector. Nevertheless, the spread of MCR producers at the environmental interface particularly driven by the increasing population of gulls in coastal cities has been less explored. Occurrence of mcr‐carrying Escherichia coli in gull's colonies faeces on a Portuguese beach was screened over 7 months. Cultural, molecular and genomic approaches were used to characterize their diversity, mcr plasmids and adaptive features. Multidrug‐resistant mcr‐1‐carrying E. coli were detected for 3 consecutive months. Over time, multiple strains were recovered, including zoonotic‐related pathogenic E. coli clones (e.g. B2‐ST131‐H22, A‐ST10 and B1‐ST162). Diverse mcr‐1 genetic environments were mainly associated with ST2/ST4‐HI2 (ST10, ST131, ST162, ST354 and ST4204) but also IncI2 (ST12990) plasmids or in the chromosome (ST656). Whole‐genome sequencing revealed enrichment of these strains on antibiotic resistance, virulence and metal tolerance genes. Our results underscore gulls as important spreaders of high‐priority bacteria and genes that may affect the environment, food–animals and/or humans, potentially undermining One‐Health strategies to reduce colistin resistance. [ABSTRACT FROM AUTHOR]

Summary: Expansion of mcr‐carrying Enterobacteriaceae (MCR‐E) is a well‐recognized problem affecting animals, humans and the environment. Ongoing global control actions involve colistin restrictions among food‐animal production, but their impact on poultry‐derived products is largely unknown, justifying comprehensive farm‐to‐fork studies. Occurrence of MCR‐E among 53 chicken‐meat batches supplied from 29 Portuguese farms shortly after colistin withdrawal was evaluated. Strains (FT‐IR/MLST/WGS), mcr plasmids and their adaptive features were characterized by cultural, molecular and genomic approaches. We found high rates of chicken‐meat batches (80%–100% – 4 months; 12% – the last month) with multiple MDR + mcr‐1‐carrying Escherichia coli (Ec‐including ST117 and ST648‐Cplx) and Klebsiella pneumoniae (Kp‐ST147‐O5:K35) clones, some of them persisting over time. The mcr‐1 was located in the chromosome (Ec‐ST297/16‐farms) or dispersed IncX4 (Ec‐ST602/ST6469/5‐farms), IncHI2‐ST2/ST4 (Ec‐ST533/ST6469/5 farms and Kp‐ST147/6‐farms) or IncI2 (Ec‐ST117/1‐farm) plasmids. WGS revealed high load and diversity in virulence, antibiotic resistance and metal tolerance genes. This study supports colistin withdrawal potential efficacy in poultry production and highlights both poultry‐production chain as a source of mcr‐1 and the risk of foodborne transmission to poultry‐meat consumers. Finally, in the antibiotic reduction/replacement context, other potential co‐selective pressures (e.g., metals‐Cu as feed additives) need to be further understood to guide concerted, effective and durable actions under 'One Health' perspective. [ABSTRACT FROM AUTHOR]

Climate warming is rapidly reshaping the Arctic cryosphere and ocean conditions, with consequences for sea ice and pelagic productivity patterns affecting the entire marine food web. To predict how ongoing changes will impact Arctic marine ecosystems, concerted effort from various disciplines is required. Here, we contribute multi‐decadal reconstructions of changes in diatom production and sea‐ice conditions in relation to Holocene climate and ocean conditions off northwest Greenland. Our multiproxy study includes diatoms, sea‐ice biomarkers (IP25 and HBI III) and geochemical tracers (TOC [total organic carbon], TOC:TN [total nitrogen], δ13C, δ15N) from a sediment core record spanning the last c. 9,000 years. Our results suggest that the balance between the outflow of polar water from the Arctic, and input of Atlantic water from the Irminger Current into the West Greenland Current is a key factor in controlling sea‐ice conditions, and both diatom phenology and production in northeastern Baffin Bay. Our proxy record notably shows that changes in sea‐surface conditions initially forced by Neoglacial cooling were dynamically amplified by the shift in the dominant phase of the Arctic Oscillation (AO) mode that occurred at c. 3,000 yr BP, and caused drastic changes in community composition and a decline in diatom production at the study site. In the future, with projected dominant‐positive AO conditions favored by Arctic warming, increased water column stratification may counteract the positive effect of a longer open‐water growth season and negatively impact diatom production. [ABSTRACT FROM AUTHOR]

Summary: Emergence and expansion of frequent multidrug‐resistant (MDR) major Salmonella clones/serotypes has been a significant threat in the last years. Metal compounds, such as copper, commonly used in animal‐production settings, have been pointed out as possible contributors for the selection of such strains/clones. However, the scarcity of studies limits our understanding of the impact of other metal environmental contaminants as arsenic (used in insecticides/herbicides/coccidiostats). We analysed arsenic tolerance (AsT) dispersion by phenotypic and genotypic (PCR/sequencing/I‐CeuI/S1/XbaI‐PFGE/hybridization) assays among Salmonella with diverse epidemiological and genetic backgrounds. Then, to better understand ars operon genetic contexts, the whole genome of five representative strains was sequenced. We found a high dispersion of ars operons conferring AsT, especially among copper‐tolerant and relevant serotypes/clones related to pig‐production setting. The acr3‐type was found dispersed in the chromosome of diverse serotypes, including the emergent S. Rissen. Conversely, arsBII was almost confined to the MDR ST34 European clone of S. Typhimurium/S. 4,[5],12:i:‐, always along with copper/silver tolerance sil + pco clusters in an integrative conjugative element. These data suggest that AsT is an essential adaptive feature for the ecological success of these epidemic clones/serotypes and alerts for global strategies to reduce arsenic‐based compounds' impact thus preventing environmental/food contamination with frequent MDR foodborne pathogens. [ABSTRACT FROM AUTHOR]

Abstract: The eastern north coast of Greenland is considered to be highly sensitive to the ongoing Arctic warming, but there is a general lack of data on modern conditions and in particular on the modern distribution of climate and environmental proxies to provide a baseline and context for studies on past variability. Here we present a detailed investigation of 11 biogenic proxies preserved in surface sediments from the remote High Arctic Wandel Sea shelf, the entrance to the Independence, Hagen, and Danmark fjords. The composition of organic matter (organic carbon, C:N ratios, δ13C, δ15N, biogenic silica, and IP25) and microfossil assemblages revealed an overall low primary production dominated by benthic diatoms, especially at the shallow sites. While the benthic and planktic foraminiferal assemblages underline the intrusion of chilled Atlantic waters into the deeper parts of the study area, the distribution of organic‐walled dinoflagellate cysts is controlled by the local bathymetry and sea ice conditions. The distribution of the dinoflagellate cyst Polarella glacialis matches that of seasonal sea ice and the specific biomarker IP25, highlighting the potential of this species for paleo sea ice studies. The information inferred from our multiproxy study has important implications for the interpretation of the biogenic‐proxy signal preserved in sediments from circum‐Arctic fjords and shelf regions and can serve as a baseline for future studies. This is the first study of its kind in this area. [ABSTRACT FROM AUTHOR]

ABSTRACT: In the past decade, research on long‐term persistence of phytoplankton resting stages has intensified. Simultaneously, insight into life‐cycle variability in the diverse groups of phytoplankton has also increased. Aquatic ‘seed banks’ have tremendous significance and show many interesting parallels to terrestrial seed beds of vascular plants, but are much less studied. It is therefore timely to review the phenomenon of long‐term persistence of aquatic resting stages in sediment seed banks. Herein we compare function, morphology and physiology of phytoplankton resting stages to factors central for persistence of terrestrial seeds. We review the types of resting stages found in different groups of phytoplankton and focus on the groups for which long‐term (multi‐decadal) persistence has been shown: dinoflagellates, diatoms, green algae and cyanobacteria. We discuss the metabolism of long‐term dormancy in phytoplankton resting stages and the ecological, evolutionary and management implications of this important trait. Phytoplankton resting stages exhibiting long‐term viability are characterized by thick, often multi‐layered walls and accumulation vesicles containing starch, lipids or other materials such as pigments, cyanophycin or unidentified granular materials. They are reported to play central roles in evolutionary resilience and survival of catastrophic events. Promising areas for future research include the role of hormones in mediating dormancy, elucidating the mechanisms behind metabolic shut‐down and testing bet‐hedging hypotheses. [ABSTRACT FROM AUTHOR]

Undisturbed records of resting stages produced in the past and stored in coastal sediments are very valuable to science, because they may provide unique insights into past evolutionary and ecological trajectories. Within marine phytoplankton, multidecadal time series of monoclonal strains germinated from resting stages have been established for diatoms ( Skeletonema marinoi) and dinoflagellates ( Pentapharsodinium dalei), spanning ca. a century. Phenotypic and genotypic analyses of these time series have revealed effects of past environmental changes on population genetic structure. Future perspectives include direct comparisons of phenotypes and genotypic data of populations, for example, by genomewide assays that can correlate phenotypic trends with genotypes and allele frequencies in temporally separated strains. Besides their usefulness as historical records, 'seed' banks of phytoplankton resting stages also have the potential to provide an inoculum that influences present populations through 'dispersal from the past' (the storage effect) and are important for adaptation to future environments through their standing genetic diversity. [ABSTRACT FROM AUTHOR]

The emergence of antimicrobial resistant infectious diseases remains a major threat to worldwide public health, in developed and in developing countries. Therefore, new antimicrobial agents acting by new mechanisms of action are urgently needed. As plants used in traditional medicine may help to overcome these problems, Justicia subsessilis, Platostoma rotundifolium, Pavetta ternifolia, Stomatanthes africanus, and Virectaria major (plants highly cited to be used against microbial infections in traditional Burundian medicine) were studied to assess their traditional use efficacy. We conducted a preliminary phytochemical screening of the extracts, as well as their direct and indirect (effect on antibiotic resistance) antibacterial activity on four bacterial strains (Staphylococcus sp. and Escherichia coli) by broth microdilution methods. All five medicinal plants investigated in this work were found to have direct antibacterial activity against all tested bacterial strains (minimum inhibitory concentration = 62.5-1000 μg/mL) that may support the use of these species in traditional Burundian medicine. Extracts (with no direct antibacterial activity), tested at 200 μg/mL, decreased the MIC values of β-lactams and aminoglycoside antibiotics by a factor of 2 to 64-fold. These interactions between plant extracts and antibiotics could open an avenue of research against antibiotic resistance. Copyright © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Many marine protists form resting stages that can remain viable in coastal sediments for several decades. Their long-term survival offers the possibility to explore the impact of changes in environmental conditions on population dynamics over multidecadal time scales. Resting stages of the phototrophic dinoflagellate Pentapharsodinium dalei were isolated and germinated from five layers in dated sediment cores from Koljö fjord, Sweden, spanning ca. 1910-2006. This fjord has, during the last century, experienced environmental fluctuations linked to hydrographic variability mainly driven by the North Atlantic Oscillation. Population genetic analyses based on six microsatellite markers revealed high genetic diversity and suggested that samples belonged to two clusters of subpopulations that have persisted for nearly a century. We observed subpopulation shifts coinciding with changes in hydrographic conditions. The large degree of genetic diversity and the potential for both fluctuation and recovery over longer time scales documented here, may help to explain the long-term success of aquatic protists that form resting stages. [ABSTRACT FROM AUTHOR]

Mean body size decreases with increasing temperature in a variety of organisms. This size-temperature relationship has generally been tested through space but rarely through time. We analyzed the sedimentary archive of dinoflagellate cysts in a sediment record taken from the West Greenland shelf and show that mean cell size decreased at both intra- and interspecific scales in a period of relatively warm temperatures, compared with a period of relatively cold temperatures. We further show that intraspecific changes accounted for more than 70% of the change in community mean size, whereas shifts in species composition only accounted for about 30% of the observed change. Literature values on size ranges and midpoints for individual taxa were in several cases not representative for the measured sizes, although changes in community mean size, calculated from literature values, did capture the direction of change. While the results show that intraspecific variation is necessary to accurately estimate the magnitude of change in protist community mean size, it may be possible to investigate general patterns, that is relative size differences, using interspecific-level estimates. [ABSTRACT FROM AUTHOR]

The purpose of this study is to examine the motivation to transfer training in a multidimensional way. It investigates autonomous and controlled motivation and explores the difference in motivation to transfer according to whether the employee is enrolled in training on a voluntary or mandatory basis. This is a cross-sectional hypotheses-testing study. Data were collected at a large insurance company involving employees who had participated in training programs in the previous 6 months. Findings show that when considering autonomous motivation to transfer, results support the argument that being voluntarily enrolled in the training program shows a higher impact on motivation to transfer than being mandatorily enrolled. When considering controlled motivation to transfer, results do not support such an argument, probably because of the nature of controlled motivation to transfer that in practice reduces the difference in results between the two types of enrollment. [ABSTRACT FROM AUTHOR]

Background Chylothorax, an uncommon cause of pleural effusion, results from the accumulation of lymph in the pleural space due to damage or obstruction of the thoracic duct. The high content of triglycerides and the presence of chylomicrons in the pleural fluid sets the diagnosis. Objective To present a case report of a chylothorax due to superior vena cava compression or a thoracic aortic aneurysm, discuss the particularities, the investigation of chylothorax as well as its treatment options. Methods A review of the literature on chylothorax was performed using PubMed to assess the different aetiologies, investigation and treatments usually performed. Conclusion Chylothorax is usually secondary to malignancy, trauma, congenital diseases and infections. However, less common causes are also described, as the ones described in our case report. The gold standard for diagnosis is the identification of chylomicrons in the pleural fluid. Conservative management is recommended in most cases of chylothorax. [ABSTRACT FROM AUTHOR]

Protoperidinium species with two anterior intercalary plates were originally classified by Jörgensen in the subgenus Archaeperidinium and assigned to the sections Excentrica, Avellana and Archaeperidinium by Taylor, on the basis of the relative size of anterior intercalary plates and the extent of cingulum displacement. Phylogenetic relationships among these three sections have not been fully explored. Recently, Archaeperidinium was reinstated as a genus, but several species fitting the emended description have not been transferred formally as molecular data were not available. In the present study we examined the cyst-theca relationship of seven species with two anterior intercalary plates: four species assigned to Protoperidinium and three to Archaeperidinium, from the China Sea. Partial large subunit ribosomal DNA sequences were obtained from these seven species by single-cell polymerase chain reaction, and for the cyst of Protoperidinium stellatum from France. Two new species, Archaeperidinium bailongense and Protoperidinium fuzhouense, were described based on both theca and cyst morphology, and the cyst-theca relationships of A. constrictum and P. abei var. rotunda were established for the first time. Maximum-likelihood and Bayesian inference analyses revealed that P. fuzhouense was nested within the Protoperidinium sensu stricto clade despite having only six postcingular plates, and sections Excentrica, Avellana and the genus Archaeperidinium were all monophyletic. Our results suggest that the presence/absence of a sulcal fin and antapical horns and the displacement of the cingulum are the most stable characteristics of the motile stages within the respective clades. [ABSTRACT FROM AUTHOR]

Ribeiro, S., Moros, M., Ellegaard, M. & Kuijpers, A. 2012 (January): Climate variability in West Greenland during the past 1500 years: evidence from a high-resolution marine palynological record from Disko Bay. Boreas, Vol. 41, pp. 68-83. 10.1111/j.1502-3885.2011.00216.x. ISSN 0300-9483. Here we document late-Holocene climate variability in West Greenland as inferred from a marine sediment record from the outer Disko Bay. Organic-walled dinoflagellate cysts and other palynomorphs were used to reconstruct environmental changes in the area through the last c. 1500 years at 30-40 years resolution. Sea ice cover and primary productivity were identified as the two main factors driving dinoflagellate cyst community changes through time. Our data provide evidence for an opposite climate trend in West Greenland relative to the NE Atlantic region from c. AD 500 to 1050. For the same period, sea-surface temperatures in Disko Bay are out-of-phase with Greenland ice-core reconstructed temperatures and marine proxy data from South and East Greenland. This is probably governed by an NAO-type pattern, which results in warmer sea-surface conditions with less extensive sea ice in the area for the later part of the Dark Ages cold period ( c. AD 500 to 750) and cooler conditions with extensive sea ice inferred for the first part of the Medieval Climate Anomaly (MCA) ( c. AD 750 to 1050). After c. AD 1050, the marine climate in Disko Bay becomes in-phase with trends described for the NE Atlantic, reflected in the warmer interval for the remainder of the MCA ( c. AD 1050-1250), followed by cooling towards the onset of the Little Ice Age at c. AD 1400. The inferred scenario of climate deterioration and extensive sea ice is concomitant with the collapse of the Norse Western Settlement in Greenland at c. AD 1350. [ABSTRACT FROM AUTHOR]

Background Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non-viral vectors. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease-rich environments. Homopurine-rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance. Methodology The effect of modifications in the poly A sequence of a model pDNA vector (pVAX1GFP) on nuclease resistance and transgene expression was investigated. Four poly A sequences were studied: bovine growth hormone (BGH), mutant BGH, SV40 and a synthetic poly A. Plasmid resistance (half-life) was assessed through in vitro incubations with mammalian nucleases. The impact in transgene expression was studied by quantifying pDNA, mRNA, and GFP expression in CHO, hybridoma and HeLa cells. Results and conclusions In vitro and cell culture studies indicate that plasmids containing the SV40 and the synthetic poly A sequences present significant improvements in nuclease resistance (up to two-fold increase in half-life). However, RT-PCR analysis demonstrated that significant reduction in mRNA steady-state levels were responsible for a decrease in transgene expression and detected transfection level of CHO and hybridoma cells when using the more resistant plasmids. Interestingly, transfection of HeLa cells demonstrated that both poly A efficiency and plasmid resistance interfere significantly in transgene expression. The results strongly suggest that the choice of the poly A is important, not only for mRNA maturation/stability, but also for pDNA resistance, and should thus be taken into consideration in the design and evaluation of pDNA vectors. Copyright © 2007 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background Nuclease degradation of plasmid DNA (pDNA) vectors after delivery and during trafficking to the nucleus is a barrier to gene expression. This barrier may be circumvented by shielding the pDNA from the nuclease-rich cell environment with adjuvants or by using nuclease inhibitors. A different alternative that is explored in this work is to make pDNA vectors more nuclease-resistant a priori. Methods and results The hypothesis that a significant part of nuclease attack is directed towards certain labile sequences in a pDNA model (pVAX1/ lacZ) was first tested. Homopurine-rich tracts in the bovine growth hormone polyadenylation signal (BGH poly A) were identified as labile sequences using S1 nuclease as a probe. Two pDNA variants were then created by replacing the BGH poly A region with the SV40 or a synthetic poly A signal. A study of plasmid degradation in eukaryotic cell lysates and mice plasma showed that the half-life of the supercoiled isoforms of the new vectors was always higher when compared with the control plasmid. An in vitro assay of the reporter β-galactosidase in transfected CHO cells further showed that gene expression with the new pDNA variants was not affected negatively by the plasmid modifications. Conclusions The replacement of labile sequences in plasmid DNA vectors improves resistance towards nuclease attack as shown by the increased half-lives of supercoiled plasmid isoforms incubated with endo/lysosomal, cytoplasmatic and blood plasma enzymes. Copyright © 2004 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The effector region of the elongation factor Tu (EF-Tu) from Thermus thermophilus was modified by limited proteolysis or via site-directed mutagenesis. The biochemical properties of the obtained EF-Tu variants were investigated with respect to partial reactions of the functional cycle of EF-Tu. EF-Tu that was cleaved at the Arg59-Gly60 peptide bond [EF-Tu-(1–59)/EF-Tu-(60–405)] bound GDP. EF-Ts and aminoacyl-tRNA, had normal intrinsic GTPase activity and was active in poly(U)-dependent poly(Phe) synthesis. However, the GTPase activity of EF-Tu-(1–59)/EF-Tu-(60–405) was not stimulated by T. thermophilus 70S ribosomes, and its GTP-dissociation rate was increased compared with that of intact EF-Tu. EF-Tu cleaved at the Lys52-Ala53 peptide bond has properties similar to EF-Tu-(1–59)/EF-Tu-(60–405). By means of site-directed mutagenesis, Glu55 was replaced by Leu, Glu56 by Ala and Arg59 by Thr in T. thermophilus EF-Tu. These amino acid substitutions did not substantially affect either the affinity of EF-Tu · GTP for aminoacyl-tRNA or the interactions with GDP, GTP or EF-Ts. Similarly the intrinsic GTPase activity is not influenced. Replacement of Glu56 by Ala led to strong reduction ill the ribosome-induced GTPase activity. This effect is specific since replacement of the neighbouring Glu55 by Leu did not affect the ribosome-induced GTPase activity. The results demonstrate that the structure of the effector region of EF-Tu in the vicinity of Arg59 is important for the control of the GTPase activity by ribosomes. [ABSTRACT FROM AUTHOR]

Investigates the properties of isolated domains of the elongation factor Tu from Thermus Thermophilus HB8. Importance of the interdomain interactions for the stabilization of the structure of domain 1; Effect of the nucleotide exchange factor Ts on the nucleotide exchange from EF-Tu[sup 1].GDP and EF-Tu.

Analyzes the process of self-directed mutagenesis observed in the Thermus thermophilus organism. Replacement of the elongation factor by glutamine, leucine and glycine residues; Degradation due to disturbed interactions between the domains that cover protease-sensitive cleavage site.

Antithrombin (AT), the most important coagulation serine proteases inhibitor, plays an important role in maintaining the hemostatic balance. Inherited AT deficiency, mainly characterized by predisposition to recurrent venous thromboembolism, is transmitted in an autosomal dominant manner. In this study, we analyzed the underlying genetic alterations in 12 unrelated Portuguese thrombophilic families with AT deficiency. At the same time, the modulating effect of the FV Leiden mutation, PT 20210A, PAI-1 4G, and MTHFR 677T allelic variants, on the thrombotic risk of AT deficient patients was also evaluated. Three novel frameshift alterations, a 4-bp deletion in exon 4 and two 1-bp insertions in exon 6, were identified in six unrelated type I AT deficient families. A novel missense mutation in exon 3a, which changes the highly conserved F147 residue, and a novel splice site mutation in the invariant acceptor AG dinucleotide of intron 2 were also identified in unrelated type I AT deficient families. In addition to these, two previously reported missense mutations changing the AT reactive site bond (R393-S394) and leading to type II-RS deficiency, and a previously reported cryptic splice site mutation (IVS4-14G-->A), were also identified. In these families, increased thrombotic risk associated with co-inheritance of the FV Leiden mutation and of the PAI-1 4G variant was also observed. In conclusion, we present the first data regarding the underlying genetic alterations in Portuguese thrombophilic families with AT deficiency, and confirm that the FV Leiden mutation and probably the PAI-1 4G variant represent additional thrombotic risk factors in these families., (Copyright 2004 Wiley-Liss, Inc.)