Glycoprotein production in moss bioreactors

Complex multimeric recombinant proteins such as therapeutic antibodies require a eukaryotic expression system. Transgenic plants may serve as promising alternatives to the currently favored mammalian cell lines or hybridomas. In contrast to prokaryotic systems, posttranslational modifications of plant and human proteins resemble each other largely, among those, protein N-glycosylation of the complex type. However, a few plant-specific sugar residues may cause immune reactions in humans, representing an obstacle for the broad use of plant-based systems as biopharmaceutical production hosts. The moss Physcomitrella patens represents a flexible tissue-culture system for the contained production and secretion of recombinant biopharmaceuticals in photobioreactors. The recent synthesis of therapeutic proteins as a scFv antibody fragment or the large and heavily modified complement regulator factor H demonstrate the versatility of this expression system. A uniquely efficient gene targeting mechanism can be employed to precisely engineer the glycosylation machinery for recombinant products. In this way, P. patens lines with non-immunogenic optimized glycan structures were created. Therapeutic antibodies produced in these strains exhibited antibody-dependent cellular cytotoxicity superior to the same molecules synthesized in mammalian cell lines.     

Presence and distribution of serotonin in the stomach of the Antarctic silverfish <Emphasis Type="Italic">Pleuragramma antarcticum</Emphasis>

Serotonin is a signal molecule with a wide range of functions in vertebrates. In Antarctic fishes, the serotonergic system has been studied in the brain, revealing differences from temperate fishes related to the long-term cold adaptation. To date, little is known regarding the peripheral nervous system, and no information is available for the stomach. In the present work, we contribute to fill the gaps by investigating the presence and the immunohistochemical distribution of serotonin in the stomach of the Antarctic silverfish Pleuragramma antarcticum, a cold-adapted key species of the Southern Ocean shelf food web. The main aim was to investigate the serotonergic system at the gastric level, in order to reveal possible peculiarities related to long-term cold adaptation, similar to the ones seen in the central nervous system of Antarctic fishes. Serotonin immunoreactivity was detected in the pyloric and cardiac mucosa of P. antarcticum stomach with immunopositive cells in the pyloric and cardiac surface epithelia and in the tubular glands. No immunopositive fibers and neuronal cell bodies were found. Our results highlight that the serotonin distribution pattern at the gastric level is similar to that described in temperate teleosts. This finding suggests that in P. antarcticum, long-term adaptations to the Antarctic condition do not affect the serotonergic system at the gastric level. In addition, our data constitute the baseline information for further investigations aimed at clarifying the effects of short-term temperature variations on the gastric serotonergic system of Antarctic species in the frame of the global climate change.     

Diversification of genes for carotenoid biosynthesis in aphids following an ancient transfer from a fungus

The pea aphid genome was recently found to harbor genes for carotenoid biosynthesis, reflecting an ancestral transfer from a fungus. To explore the evolution of the carotene desaturase gene family within aphids, sequences were retrieved from a set of 34 aphid species representing numerous deeply diverging lineages of aphids and analyzed together with fungal sequences retrieved from databases. All aphids have at least one copy of this gene and some aphid species have up to seven, whereas fungal genomes consistently have a single copy. The closest relatives of aphids, adelgids, also have carotene desaturase; these sequences are most closely related to those from aphids, supporting a shared origin from a fungal to insect transfer predating the divergence of adelgids and aphids. Likewise, all aphids, and adelgids, have carotenoid profiles that are consistent with their biosynthesis using the acquired genes of fungal origin rather than derivation from food plants. The carotene desaturase was acquired from a fungal species outside of Ascomycota or Basidiomycota and closest to Mucoromycotina among sequences available in databases. In aphids, an ongoing pattern of gene duplication is indicated by the presence of both anciently and recently diverged paralogs within genomes and by the presence of a high frequency of pseudogenes that appear to be recently inactivated. Recombination among paralogs is evident, making analyses of patterns of selection difficult, but tests of selection for a nonrecombining region indicates that duplications tend to be followed by bouts of positive selection. Species of Macrosiphini, which often show color polymorphisms, typically have a larger number of desaturase copies relative to other species sampled in the study. These results indicate that aphid evolution has been accompanied by ongoing evolution of carotenogenic genes, which have undergone duplication, recombination, and occasional positive selection to yield a wide variety of carotenoid profiles in different aphid species.     

Superhelical DNA as a preferential binding target of 14-3-3γ protein

The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.     

Alcohol consumption and the risk of incident atrial fibrillation among people with cardiovascular disease

Background:

Moderate alcohol consumption may reduce cardiovascular events, but little is known about its effect on atrial fibrillation in people at high risk of such events. We examined the association between moderate alcohol consumption and the risk of incident atrial fibrillation among older adults with existing cardiovascular disease or diabetes.

Methods:

We analyzed data for 30 433 adults who participated in 2 large antihypertensive drug treatment trials and who had no atrial fibrillation at baseline. The patients were 55 years or older and had a history of cardiovascular disease or diabetes with end-organ damage. We classified levels of alcohol consumption according to median cut-off values for low, moderate and high intake based on guidelines used in various countries, and we defined binge drinking as more than 5 drinks a day. The primary outcome measure was incident atrial fibrillation.

Results:

A total of 2093 patients had incident atrial fibrillation. The age- and sex-standardized incidence rate per 1000 person-years was 14.5 among those with a low level of alcohol consumption, 17.3 among those with a moderate level and 20.8 among those with a high level. Compared with participants who had a low level of consumption, those with higher levels had an increased risk of incident atrial fibrillation (adjusted hazard ratio [HR] 1.14, 95% confidence interval [CI] 1.04–1.26, for moderate consumption; 1.32, 95% CI 0.97–1.80, for high consumption). Results were similar after we excluded binge drinkers. Among those with moderate alcohol consumption, binge drinkers had an increased risk of atrial fibrillation compared with non–binge drinkers (adjusted HR 1.29, 95% CI 1.02–1.62).

Interpretation:

Moderate to high alcohol intake was associated with an increased incidence of atrial fibrillation among people aged 55 or older with cardiovascular disease or diabetes. Among moderate drinkers, the effect of binge drinking on the risk of atrial fibrillation was similar to that of habitual heavy drinking.A trial fibrillation is associated with an increased risk of stroke and a related high burden of mortality and morbidity, both in the general public and among patients with existing cardiovascular disease.^1,2 The prevalence of atrial fibrillation increases steadily with age, as do the associated risks, and atrial fibrillation accounts for up to 23.5% of all strokes among elderly people.³Moderate alcohol consumption has been reported to be associated with a reduced risk of cardiovascular disease and all-cause death,^1,2 whereas heavy alcohol intake and binge drinking have been associated with an increased risk of stroke,⁴ cardiovascular disease and all-cause death.^5,6 Similarly, heavy drinking and binge drinking are associated with an increased risk of incident atrial fibrillation in the general population.⁷ However, the association between moderate drinking and incident atrial fibrillation is less consistent and not well understood among older people with existing cardiovascular disease.In this analysis, we examined whether drinking moderate quantities of alcohol, and binge drinking, would be associated with an increased risk of incident atrial fibrillation in a large cohort of people with existing cardiovascular disease or diabetes with end-organ damage who had been followed prospectively in 2 long-term antihypertensive drug treatment trials.     

Passive acoustic density estimation of sperm whales in the Tongue of the Ocean,Bahamas

Long‐term passive acoustic monitoring of marine mammals on navy ranges provides the opportunity to better understand the potential impact of sonar on populations. The navy range in Tongue of the Ocean (TOTO), Bahamas contains extensive hydrophone arrays, potentially allowing estimation of the density of deep diving, vocally active species such as the sperm whale (Physeter macrocephalus). Previous visual surveys in TOTO have been of limited spatio–temporal coverage and resulted in only sporadic sightings of sperm whales, whereas passive acoustic observations suggest the species is present year round. However, until now the means of acoustically determining the specific number of individuals in each cluster has been limited. We used recently developed algorithms to identify the number of echolocating whales present during a 42 d study period. We screened a 297 h acoustic data set to determine the proportion of time animals were present; fifty 10 min samples during presence were analyzed to estimate the number of individuals vocalizing during each sample. These counts were combined with an independent estimate of the proportion of 10 min periods when tagged animals vocalize. The estimated average density was 0.16 whales/1,000 km² (CV 27%; 95% CI 0.095–0.264). The method is potentially applicable to other areas containing dense hydrophone arrays.     

Thermographic mapping of the skin surface of the head in bald-headed male subjects

Thermal patterns on the skin are related to skin blood perfusion. While knowledge concerning thermal distribution patterns of the face are well established, the thermal distribution patterns of the skin surface of other aspects of the head has only been investigated once previously in a study employing low resolution thermographic equipment [van Dulken, H., van Voss, S.F.C.H, 1971. The normal thermographic patterns of the human head. Acta Neurochir. 23, 247–58.] The purpose of the study was to re-examine the thermal distribution patterns of the surface of the entire head in 12 healthy bald headed male subjects (mean age 39±7.8 (SD) years) using a high definition infrared (IR) thermographic camera. The thermal distribution patterns were monitored using dynamic infrared thermography (DIRT) before, during and after a 2 min local skin cooling procedure with a pair of fans. Infrared thermal images (thermograms) were taken of the superior, anterior, lateral and posterior aspects of the head. The thermal patterns in general followed the anatomical location of the main superficial arteries and veins of the head as described in anatomy books although there were some variations, especially with regard to the forehead and top of the head (superior aspect), ranging from an asymmetrical distribution pattern to a lack of a clear thermal pattern. The thermal patterns in the left and right lateral thermograms in each individual were relatively symmetrical. During the rewarming period the helix, auricular lobule of the auricles and the nose were relatively cold. The frontal thermograms were characterized by cool nasal and cheek areas and a clear warm area surrounding the eyes, especially around the inner canthus.     

BDNF and DYRK1A Are Variable and Inversely Correlated in Lymphoblastoid Cell Lines from Down Syndrome Patients

Down syndrome or trisomy 21 is the most common genetic disorder leading to mental retardation. One feature is impaired short- and long-term spatial memory, which has been linked to altered brain-derived neurotrophic factor (BDNF) levels. Mouse models of Down syndrome have been used to assess neurotrophin levels, and reduced BDNF has been demonstrated in brains of adult transgenic mice overexpressing Dyrk1a, a candidate gene for Down syndrome phenotypes. Given the link between DYRK1A overexpression and BDNF reduction in mice, we sought to assess a similar association in humans with Down syndrome. To determine the effect of DYRK1A overexpression on BDNF in the genomic context of both complete trisomy 21 and partial trisomy 21, we used lymphoblastoid cell lines from patients with complete aneuploidy of human chromosome 21 (three copies of DYRK1A) and from patients with partial aneuploidy having either two or three copies of DYRK1A. Decreased BDNF levels were found in lymphoblastoid cell lines from individuals with complete aneuploidy as well as those with partial aneuploidies conferring three DYRK1A alleles. In contrast, lymphoblastoid cell lines from individuals with partial trisomy 21 having only two DYRK1A copies displayed increased BDNF levels. A negative correlation was also detected between BDNF and DYRK1A levels in lymphoblastoid cell lines with complete aneuploidy of human chromosome 21. This finding indicates an upward regulatory role of DYRK1A expression on BDNF levels in lymphoblastoid cell lines and emphasizes the role of genetic variants associated with psychiatric disorders.