Microphytoplankton assemblages in shallow waters at Admiralty Bay (King George Island,Antarctica) during the summer 2002–2003

Microphytoplankton populations were studied in shallow coastal water (<60 m) near the Brazilian Antarctic Station Comandante Ferraz (EACF) and three reference areas in Admiralty Bay in early and late summer (2002–2003). Phytoplankton was diverse (113 taxa), but not abundant (10³ cells l⁻¹). The highest abundances (>10⁴ cells l⁻¹) were caused by pennate benthic diatoms (Fragilaria striatula Lyngbye) that occurred mainly in early summer, associated with the presence of ice. In late summer, when the water temperature (−0.4 to 1.5°C), salinity (34 to 35), and phosphate (2.6 to 4.5 μmol l⁻¹) were highest and the dissolved oxygen was lowest (6.4 to 2.9 ml l⁻¹), centric diatoms (Thalassiosira spp.) were more abundant, suggesting an influence of oceanic waters. Phytoplankton abundance (≤10² cells l⁻¹) and chlorophyll a concentrations (0.22 μg l⁻¹) were lowest close to EACF. Pennate diatoms were dominant close to shore and in surface waters elsewhere, probably because of ice melting or sediment resuspension caused by water mixing.     

Time-course determination of plasmid content in eukaryotic and prokaryotic cells using Real-Time PCR

A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1 derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires neither specific cell lysis nor DNA purification and can be performed in <30 min with dynamic ranges covering 0.9 pg–55 ng, and 5.0 pg–2.5 ng of plasmid DNA (pDNA) for E. coli and CHO cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h post-transfection was around 3 × 10⁴ copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and around 100 copies/cell were still detected 6 days after transfection.     

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

Spheroids have emerged as in vitro models that reproduce in a great extent the architectural microenvironment found in human tissues. However, the imaging of 3D cell cultures is highly challenging due to its high thickness, which results in a light-scattering phenomenon that limits light penetration. Therefore, several optical clearing methods, widely used in the imaging of animal tissues, have been recently explored to render spheroids with enhanced transparency. These methods are aimed to homogenize the microtissue refractive index (RI) and can be grouped into four different categories, namely (a) simple immersion in an aqueous solution with high RI; (b) delipidation and dehydration followed by RI matching; (c) delipidation and hyperhydration followed by RI matching; and (d) hydrogel embedding followed by delipidation and RI matching. In this review, the main optical clearing methods, their mechanism of action, advantages, and disadvantages are described. Furthermore, the practical examples of the optical clearing methods application for the imaging of 3D spheroids are highlighted.     

Monochromatic light increases anthocyanin content during fruit development in bilberry

Background

Light is one of the most significant environmental factors affecting to the accumulation of flavonoids in fruits. The composition of the light spectrum has been shown to affect the production of phenolic compounds during fruit ripening. However, specific information on the biosynthesis of flavonoids in fruits in response to different wavelengths of light is still scarce. In the present study bilberry (Vaccinium myrtillus L.) fruits, which are known to be rich with anthocyanin compounds, were illuminated with blue, red, far-red or white light during the berry ripening process. Following the illumination, the composition of anthocyanins and other phenolic compounds was analysed at the mature ripening stage of fruits.

Results

All the three monochromatic light treatments had significant positive effect on the accumulation of total anthocyanins in ripe fruits compared to treatment with white light or plants kept in darkness. The elevated levels of anthocyanins were mainly due to a significant increase in the accumulation of delphinidin glycosides. A total of 33 anthocyanin compounds were detected in ripe bilberry fruits, of which six are novel in bilberry (cyanidin acetyl-3-O-galactose, malvidin acetyl-3-O-galactose, malvidin coumaroyl-3-O-galactose, malvidin coumaroyl-3-O-glucose, delphinidin coumaroyl-3-O-galactose, delphinidin coumaroyl-3-O-glucose).

Conclusions

Our results indicate that the spectral composition of light during berry development has significant effect on the flavonoid composition of ripe bilberry fruits.

     

Early Initiation of Smoking and Alcohol Drinking as a Predictor of Lower Forearm Bone Mineral Density in Late Adolescence: A Cohort Study in Girls

Background

Adolescence is a critical stage for bone accrual. It is also decisive for the establishment of behaviors such as smoking and alcohol drinking.

Objective

To quantify the short- and long-term associations between smoking and drinking initiation and bone mineral density in adolescent girls.

Methods

We used prospective data from 731 girls identified in public and private schools in Porto, Portugal. Evaluations were conducted when participants were 13 and 17 years old. Bone mineral density (BMD) was measured at the forearm by dual-energy X-ray absorptiometry and weight, height and fat-free mass were measured. Pubertal development status was estimated using menarche age. Self-administered questionnaires were used to collect data on smoking and alcohol drinking, physical exercise and calcium and vitamin D intakes. BMD in early and late adolescence was analyzed as a continuous or dichotomous (Z-score cutoff: −1.0) variable. Associations were calculated using linear or logistic regression.

Results

Over one quarter of these girls had tried smoking by 13, while 59% had drunk alcoholic beverages and 20% had experienced both behaviors by that age. Lower mean BMD at 17 years of age was observed in girls who had ever smoked by 13, as well as in those who reported drinking at that age. There were no significant cross-sectional associations between experience and frequency of smoking or drinking and BMD at 13 years of age. However, we observed significant associations between BMD z-score<−1 in late adolescence and having ever smoked by 13, after adjustment for menarche age and sports practice, (OR = 1.92; 95% CI: 1.21, 3.05) and with ever smoking and drinking in the same period (OR = 2.33; 95% CI: 1.36, 4.00).

Conclusion

Our study adds prospective evidence to the role of early initiation of smoking and alcohol drinking as relevant markers of lower bone mineral density in late adolescence.     

Involvement of mitochondria in endoplasmic reticulum stress-induced apoptotic cell death pathway triggered by the prion peptide PrP(106-126)

Prion disorders are progressive neurodegenerative diseases characterized by extensive neuronal loss and by the accumulation of the pathogenic form of prion protein, designated PrP^Sc. Recently, we have shown that PrP_106–126 induces endoplasmic reticulum (ER) stress, leading to mitochondrial cytochrome c release, caspase 3 activation and apoptotic death. In order to further clarify the role of mitochondria in ER stress-mediated apoptotic pathway triggered by the PrP peptide, we investigated the effects of PrP_106–126 on the Ntera2 human teratocarcinoma cell line that had been depleted of their mitochondrial DNA, termed NT2 ρ0 cells, characterized by the absence of functional mitochondria, as well as on the parental NT2 ρ+ cells. In this study, we show that PrP_106–126 induces ER stress in both cell lines, given that ER Ca²⁺ content is low, glucose-regulated protein 78 levels are increased and caspase 4 is activated. Furthermore, in parental NT2 ρ+ cells, PrP_106–126-activated caspase 9 and 3, induced poly (ADP-ribose) polymerase cleavage and increased the number of apoptotic cells. Dantrolene was shown to protect NT2 ρ+ from PrP_106–126-induced cell death, demonstrating the involvement of Ca²⁺ release through ER ryanodine receptors. However, in PrP_106–126-treated NT2 ρ0 cells, apoptosis was not able to proceed. These results demonstrate that functional mitochondria are required for cell death as a result of ER stress triggered by the PrP peptide, and further elucidate the molecular mechanisms involved in the neuronal loss that occurs in prion disorders.     

Artificial laminin polymers assembled in acidic pH mimic basement membrane organization

Natural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, laminin can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 +/- 5 and 86 +/- 3 nm of height, which approximately correspond to the sizes of the short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment E1', which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, involve only interactions between the short arms. The electrostatic surface map of laminin alpha1 LG4-5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin in the future.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected. An erratum to this article can be found at     

First occurrences of non-neosauropod eusauropod procoelous caudal vertebrae in the Portuguese Upper Jurassic record

The Upper Jurassic of the Lusitanian Basin (Portugal) is particularly rich in sauropod fossil remains, with four established taxa: Dinheirosaurus, Lusotitan, Lourinhasaurus and Zby. The presence of sauropod caudal procoelous vertebrae is reported for the first time in the Upper Jurassic of Portugal, with specimens described from the localities of Baleal, Paimogo, Praia da Areia Branca, Porto das Barcas, and Praia da Corva. The presence of slightly procoelous centra and fan-shaped caudal ribs with smooth prezygapophyseal centrodiapophyseal fossa in the more anterior caudal vertebrae allows for the assignment of these specimens to an indeterminate eusauropod, probably belonging to a non-neosauropod eusauropod form. The absence of several features in the Portuguese specimens that are common in diplodocids, mamenchisaurids and titanosaurs, prevents the establishment of sound relationships with these clades. The described specimens are almost identical to the anterior caudal vertebrae of the Iberian turiasaur Losillasaurus. During the Iberian Late Jurassic, Turiasauria is the only Iberian group of sauropods, which shares this type of morphology with the Baleal, Paimogo, Praia da Areia Branca, Porto das Barcas and Praia da Corva specimens. These specimens represent one of the four anterior caudal vertebral morphotypes recorded in the Upper Jurassic of the Lusitanian Basin and briefly described herein.