A fluorescence method to detect cyanophycin in the symbiotic cyanobacterium, Anabaena azollae.

The cyanophycin content of the heterocystous nitrogen-fixing symbiotic cyanobacterium, Anabaena azollae, which inhabits an ovoid cavity in the dorsal leaf lobes of the fern, Azolla filiculoides, is seldom analyzed. To study the cyanophycin content in vegetative cells and heterocysts of A. azollae, we used three fluorochromes: aluminum trichloride, lead citrate and Wilson citroboric solution and Coomassie brilliant blue. Blue and yellow fluorescence were emitted from the polar nodes and cytoplasm cyanophycin granules of the heterocysts when stained with the three fluorochromes. The cyanophycin observed without staining or with Coomassie brilliant blue staining did not alter the results obtained using the fluorochromes. We found that aluminum trichloride, lead acetate and Wilson citroboric solution could be used to detect cyanophycin.

Graphene woven fabric-polydimethylsiloxane piezoresistive films for smart multi-stimuli responses.

The outstanding properties of graphene, including its electromechanical property, could be engineered for wearable electronic sensor platforms. The tubular graphene weaved into a mesh or graphene woven fabrics (GWF) has been reported as one of the most sensitive materials for deformation detection, as well as a promising temperature sensor. Herein, we present the performance of our developed flexible, stretchable, and multiple sensitive sensors fabricated from GWF embedded in polydimethylsiloxane (PDMS) film substrate. The GWF/PDMS sensor shows a pressure sensitivity of 0.0142 kPa-1 in a wide linearity range of 0-20 kPa, an outstanding Gauge factor (GF) of 582 at a strain of 6.2 %, and a very high positive sensitivity of 0.0238 °C-1 in the temperature range of 25-80 °C. A practical application as a high sensitivity air pressure sensor able to measure low pressures (in the range of Pa to kPa) was also demonstrated. This sensor platform having desirable performance characteristics is an excellent candidate for wearable devices in the healthcare sector.

Genomic and Phenotypic Characterization of Campylobacter fetus subsp. venerealis Strains.

The pathogenesis mechanisms of Campylobacter fetus subsp. venerealis (Cfv), the etiologic agent of Bovine Genital Campylobacteriosis remain elusive. This study evaluated the virulence potential and biovar characteristics of Cfv isolates (n = 13) by PCR screening of putative virulence-factor (VF) genes, Multilocus Sequence Typing (MLST) analysis, antimicrobial susceptibility to tetracycline, penicillin, enrofloxacin and streptomycin testing and whole-genome sequencing (WGS; n = 5), also comparing the latter with 26 other whole-genome sequences of Cfv strains. The putative VF genes encoding type IV secretion system of Cfv (virB2-virB11/virD4) were absent in 92% of isolates, including isolates from aborted foetuses, evidencing that these VF genes are not essential for Cfv pathogenicity. The parA gene, used as a Cfv diagnostic molecular target, was detected in only 3 of 13 isolates, invalidating its use for diagnosis purposes. Three novel sequence types were identified by MLST. Although no in vitro antimicrobial resistance was detected, WGS identified antimicrobial resistance-related genes, including those encoding the multidrug efflux pumps CmeABC and YkkCD, indicating that their presence is not enough to provide antimicrobial resistance. The SNP and accessory protein families analysis segregated the Cfv and Cfv biovar intermedius (Cfvi) strains into different clusters. In conclusion, this study evidenced virulence potential and biovar characteristics of Cfv and Cfvi, which are of relevance for the control of Bovine Genital Campylobacteriosis.

Prevalence of underweight, overweight, and obesity in children and adolescents with type 1 diabetes: Data from the international SWEET registry.

OBJECTIVE: To assess the prevalence of underweight (UW), overweight (OW), and obesity in children and adolescents with type 1 diabetes (T1D). METHODS: An international cross-sectional study including 23 026 T1D children (2-18 years, duration of diabetes ≥1 year) participating in the SWEET prospective, multicenter diabetes registry. Body mass index SD score (BMI-SDS) was calculated using the World Health Organization BMI charts. Children were categorized as UW (BMI-SDS < -2SD), OW (+1SD < BMI-SDS ≤ +2SD), and obese (OB) (BMI-SDS > +2SD). Hierarchic regression models were applied with adjustment for sex, age, and duration of diabetes. RESULTS: The prevalence of UW, OW, and obesity was: 1.4%, 22.3%, and 7.3% in males and 0.6%, 27.2%, and 6.8% in females. Adjusted BMI-SDS was significantly higher in females than in males (mean ± SEM: 0.54 ± 0.05 vs 0.40 ± 0.05, P < 0.0001). In males, BMI-SDS significantly decreased by age (P < 0.0001) in the first three age categories 0.61 ± 0.06 (2 to <10 years), 0.47 ± 0.06 (10 to <13 years), 0.34 ± 0.05 (13 to <16 years). In females, BMI-SDS showed a U-shaped distribution by age (P < 0.0001): 0.54 ± 0.04 (2 to <10 years), 0.39 ± 0.04 (10 to <13 years), 0.55 ± 0.04 (13 to <16 years). BMI-SDS increased by diabetes duration (<2 years: 0.38 ± 0.05, 2 to <5 years: 0.44 ± 0.05, and ≥5 years: 0.50 ± 0.05, P < 0.0001). Treatment modality did not affect BMI-SDS. Adjusted HbA1c was significantly higher in females than in males (8.20% ± 0.10% vs 8.06% ± 0.10%, P < 0.0001). In both genders, the association between HbA1c and BMI-SDS was U-shaped with the highest HbA1c in the UW and obesity groups. CONCLUSIONS: The high rate of OW and obesity (31.8%) emphasize the need for developing further strategies to prevent and treat excess fat accumulation in T1D.

Cyanobacterial diversity held in microbial biological resource centers as a biotechnological asset: the case study of the newly established LEGE culture collection.

Cyanobacteria are a well-known source of bioproducts which renders culturable strains a valuable resource for biotechnology purposes. We describe here the establishment of a cyanobacterial culture collection (CC) and present the first version of the strain catalog and its online database (http://lege.ciimar.up.pt/). The LEGE CC holds 386 strains, mainly collected in coastal (48%), estuarine (11%), and fresh (34%) water bodies, for the most part from Portugal (84%). By following the most recent taxonomic classification, LEGE CC strains were classified into at least 46 genera from six orders (41% belong to the Synechococcales), several of them are unique among the phylogenetic diversity of the cyanobacteria. For all strains, primary data were obtained and secondary data were surveyed and reviewed, which can be reached through the strain sheets either in the catalog or in the online database. An overview on the notable biodiversity of LEGE CC strains is showcased, including a searchable phylogenetic tree and images for all strains. With this work, 80% of the LEGE CC strains have now their 16S rRNA gene sequences deposited in GenBank. Also, based in primary data, it is demonstrated that several LEGE CC strains are a promising source of extracellular polymeric substances (EPS). Through a review of previously published data, it is exposed that LEGE CC strains have the potential or actual capacity to produce a variety of biotechnologically interesting compounds, including common cyanotoxins or unprecedented bioactive molecules. Phylogenetic diversity of LEGE CC strains does not entirely reflect chemodiversity. Further bioprospecting should, therefore, account for strain specificity of the valuable cyanobacterial holdings of LEGE CC.

Assessment of uptake and phytotoxicity of cyanobacterial extracts containing microcystins or cylindrospermopsin on parsley (Petroselinum crispum L.) and coriander (Coriandrum sativum L).

Blooms of harmful cyanobacteria that synthesize cyanotoxins are increasing worldwide. Agronomic plants can uptake these cyanotoxins and given that plants are ultimately ingested by humans, this represents a public health problem. In this research, parsley and coriander grown in soil and watered through 7 days with crude extracts containing microcystins (MCs) or cylindrospermopsin (CYN) in 0.1-1 µg mL-1 concentration range were evaluated concerning their biomass, biochemical parameters and uptake of cyanotoxins. Although biomass, chlorophylls (a and b), carotenoids and glutathione-S-transferase of parsley and coriander exposed to the crude extracts containing MC or CYN had shown variations, these values were not statistically significantly different. Protein synthesis is not inhibited in coriander exposed to MC or CYN and in parsley exposed to MC. Also, glutathione reductase (GR) and glutathione peroxidase (GPx) in parsley and coriander was not affected by exposure to MC, and in coriander, the CYN did not induce statistically significant differences in these two antioxidative enzymes. Only parsley showed statistically significant increase in protein content exposed to 0.5 µg CYN mL-1 (3.981 ± 0.099 mg g-1 FW) compared to control (2.484 ± 0.145 mg g-1 FW), statistically significant decrease in GR exposed to 0.1 µg CYN mL-1 (0.684 ± 0.117 nmol min-1 mg-1 protein) compared to control (1.30 ± 0.06 nmol min-1 mg-1 protein) and statistically significant increase in GPx exposed to 1 µg CYN mL-1 (0.054 ± 0.026 nmol min-1 mg-1 protein) compared to 0.5 µg CYN mL-1 (0.003 ± 0.001 nmol min-1 mg-1 protein). These changes may be due to the induction of defensive mechanisms by plants by the presence of toxic compounds in the soil or probably to a low generation of reactive oxygen species. Furthermore, the parsley and coriander leaves and stems after 10 days of exposure did not accumulate microcystins or cylindrospermopsin.

Zigzag phosphorene nanoribbons: one-dimensional resonant channels in two-dimensional atomic crystals.

We theoretically investigate phosphorene zigzag nanoribbons as a platform for constriction engineering. In the presence of a constriction at one of the edges, quantum confinement of edge-protected states reveals conductance peaks, if the edge is uncoupled from the other edge. If the constriction is narrow enough to promote coupling between edges, it gives rise to Fano-like resonances as well as antiresonances in the transmission spectrum. These effects are shown to mimic an atomic chain like behavior in a two dimensional atomic crystal.

Biochemical and growth performance of the aquatic macrophyte Azolla filiculoides to sub-chronic exposure to cylindrospermopsin.

Physiological and biochemical effects of cylindrospermopsin (CYN), a cyanobacterial toxin that inhibits protein synthesis and released during a harmful cyanobacterial bloom, has been overlooked in plants. Therefore, at the present research, the toxic effects (physiological and biochemical) of a crude extract containing CYN were assessed in the aquatic fern Azolla filiculoides exposed to three concentrations (0.05, 0.5 and 5 µg CYN mL(-1)). At 5 µg CYN mL(-1), fern growth rate has showed a drastic decrease (0.001 g g(-1) day(-1)) corresponding to a 99.8% inhibition, but at the concentrations of 0.05 and 0.5 µg CYN mL(-1) the growth rate was similar to the control plants. Growth rate also indicated a IC50 of 2.9 µg CYN mL(-1). Those data point to the presence of other compounds in the crude extract may stimulate the fern growth and/or the fern is tolerant to CYN. Chlorophyll (a and b), carotenoids and protein content as well as the activities of glutathione reductase (GR) and glutathione-S-transferase (GST) has increased at 5 µg CYN mL(-1) which may indicate that photosynthesis and protein synthesis are not affected by CYN and the probable activation of defense and detoxifying mechanisms to overcome the effects induced by the presence of CYN. Low uptake of cylindrospermopsin (1.314 µg CYN g(-1) FW) and low bioconcentration factor (0.401) point towards to a safe use of A. filiculoides as biofertilizer and as food source, but also indicate that the fern is not suitable for CYN phytoremediation.

Mitosis. Microtubule detyrosination guides chromosomes during mitosis.

Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination.

Classification and phylogeny of the cyanobiont Anabaena azollae Strasburger: an answered question?

The symbiosis Azolla-Anabaena azollae, with a worldwide distribution in pantropical and temperate regions, is one of the most studied, because of its potential application as a biofertilizer, especially in rice fields, but also as an animal food and in phytoremediation. The cyanobiont is a filamentous, heterocystic cyanobacterium that inhabits the foliar cavities of the pteridophyte and the indusium on the megasporocarp (female reproductive structure). The classification and phylogeny of the cyanobiont is very controversial: from its morphology, it has been named Nostoc azollae, Anabaena azollae, Anabaena variabilis status azollae and recently Trichormus azollae, but, from its 16S rRNA gene sequence, it has been assigned to Nostoc and/or Anabaena, and from its phycocyanin gene sequence, it has been assigned as non-Nostoc and non-Anabaena. The literature also points to a possible co-evolution between the cyanobiont and the Azolla host, since dendrograms and phylogenetic trees of fatty acids, short tandemly repeated repetitive (STRR) analysis and restriction fragment length polymorphism (RFLP) analysis of nif genes and the 16S rRNA gene give a two-cluster association that matches the two-section ranking of the host (Azolla). Another controversy surrounds the possible existence of more than one genus or more than one species strain. The use of freshly isolated or cultured cyanobionts is an additional problem, since their morphology and protein profiles are different. This review gives an overview of how morphological, chemical and genetic analyses influence the classification and phylogeny of the cyanobiont and future research.

Microtubule plus-end tracking proteins and their roles in cell division.

Microtubules are cellular components that are required for a variety of essential processes such as cell motility, mitosis, and intracellular transport. This is possible because of the inherent dynamic properties of microtubules. Many of these properties are tightly regulated by a number of microtubule plus-end-binding proteins or +TIPs. These proteins recognize the distal end of microtubules and are thus in the right context to control microtubule dynamics. In this review, we address how microtubule dynamics are regulated by different +TIP families, focusing on how functionally diverse +TIPs spatially and temporally regulate microtubule dynamics during animal cell division.

The microtubule plus-end tracking protein CLASP2 is required for hematopoiesis and hematopoietic stem cell maintenance.

Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators of microtubule behavior has been studied mainly in cultured cells. We show here that absence of murine CLASP2 in vivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization of microtubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.

Dynamic culturing of cartilage tissue: the significance of hydrostatic pressure.

Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×10(6) cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10×10(6) cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and amplitude-dependant manner.

The body in persons with an amputation.

This study reports on a comparison of how two different groups of people with an amputation view their bodies and perceive how others view them. One group has a history of sport participation, while the other has not. The analysis is based on 14 semistructured interviews with people with amputations: 7 were engaged in sport and 7 were not. The following themes emerged: Body, Prosthesis, Independence, Huma Person, and Social Barriers. One could conclude that participation in sport influences how people with an amputation perceive their body as they live with their body in a more positive way and they better accept their new body condition and their being-in-the-world. The social barriers that people with an amputation have to face daily were evident, and one of the most significant ideas was the importance of being recognized and treated as a person and not as a person with a disability.

Expression of cytokeratins 10, 13, 14 and 19 in oral lichen planus.

OLP is a chronic inflammatory disease of unknown etiology that may develop into squamous-cell carcinoma. Cytokeratins, which are important components of the cytoskeleton, are excellent epithelial differentiation markers used to study neoplastic and inflammatory diseases. To study the profile of cytokeratins in OLP and their possible association with dysplastic alterations, monoclonal antibodies were used for cytokeratins 10, 13, 14 and 19, in 26 samples of OLP. The streptavidin-biotin technique was employed in paraffin-embedded tissue sections. Sample analysis revealed suprabasal expression of cytokeratin 10 in 16/17 samples, 14 of them with reduced expression; suprabasal expression of cytokeratin 13 in 18/23, 16 of them with delay; basal and suprabasal expression of cytokeratin 14 in all samples; and focal basal expression of cytokeratin 19 in 4/21. Expression of cytokeratins 10, 13 and 14 was altered in OLP lesions. The inflammatory process and hyperkeratosis or parakeratosis seem to have interfered with the expression of these CKs. Cytokeratin 19 was expressed in the lesions, in a pattern similar to that mentioned in the literature for the non-keratinized oral mucosa. The presence of mild dysplasia did not change the expression of the cytokeratins studied. No differences in pattern of expression were observed between keratinized and non-keratinized areas in the lesions caused by OLP.

Motor-independent targeting of CLASPs to kinetochores by CENP-E promotes microtubule turnover and poleward flux.

Efficient chromosome segregation during mitosis relies on the coordinated activity of molecular motors with proteins that regulate kinetochore attachments to dynamic spindle microtubules [1]. CLASPs are conserved kinetochore- and microtubule-associated proteins encoded by two paralog genes, clasp1 and clasp2, and have been previously implicated in the regulation of kinetochore microtubule dynamics [2-4]. However, it remains unknown how CLASPs work in concert with other proteins to form a functional kinetochore microtubule interface. Here we have identified mitotic interactors of human CLASP1 via a proteomic approach. Among these, the microtubule plus-end-directed motor CENP-E [5] was found to form a complex with CLASP1 that colocalizes to multiple structures of the mitotic apparatus in human cells. We found that CENP-E recruits both CLASP1 and CLASP2 to kinetochores independently of its motor activity or the presence of microtubules. Depletion of CLASPs or CENP-E by RNA interference in human cells causes a significant and comparable reduction of kinetochore microtubule poleward flux and turnover rates and rescues spindle bipolarity in Kif2a-depleted cells. We conclude that CENP-E integrates two critical functions that are important for accurate chromosome movement and spindle architecture: one relying directly on its motor activity, and the other involving the targeting of key microtubule regulators to kinetochores.

Mammalian CLASP1 and CLASP2 cooperate to ensure mitotic fidelity by regulating spindle and kinetochore function.

CLASPs are widely conserved microtubule plus-end-tracking proteins with essential roles in the local regulation of microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles remain unknown. Here, we show that CLASP2 localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1, both showing fast microtubule-independent turnover rates. Strikingly, primary fibroblasts from Clasp2 knockout mice show numerous spindle and chromosome segregation defects that can be partially rescued by ectopic expression of Clasp1 or Clasp2. Moreover, chromosome segregation rates during anaphase A and B are slower in Clasp2 knockout cells, which is consistent with a role of CLASP2 in the regulation of kinetochore and spindle function. Noteworthy, cell viability/proliferation and spindle checkpoint function were not impaired in Clasp2 knockout cells, but the fidelity of mitosis was strongly compromised, leading to severe chromosomal instability in adult cells. Together, our data support that the partial redundancy of CLASPs during mitosis acts as a possible mechanism to prevent aneuploidy in mammals.