Negative feedback regulation of calcineurin-dependent Prz1 transcription factor by the CaMKK-CaMK1 axis in fission yeast

Calcium signals trigger the translocation of the Prz1 transcription factor from the cytoplasm to the nucleus. The process is regulated by the calciumactivated phosphatase calcineurin, which activates Prz1 thereby maintaining active transcription during calcium signalling. When calcium signalling ceases, Prz1 is inactivated by phosphorylation and exported to the cytoplasm. In budding yeast and mammalian cells, different kinases have been reported to counter calcineurin activity and regulate nuclear export. Here, we show that the Ca2+/calmodulin-dependent kinase Cmk1 is first phosphorylated and activated by the newly identified kinase CaMKK2 homologue, Ckk2, in response to Ca2+. Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca2+. Furthermore, Cdc25 phosphatase is also phosphorylated by Cmk1, inducing cell cycle arrest in response to an increase in Ca2+. Moreover, cmk1 deletion shows a high tolerance to chronic exposure to Ca2+, due to the lack of cell cycle inhibition and elevated Prz1 activity. This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription. These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe.

Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells.

Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington's disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150Glued complex interactions.

Pulmonary 64-MDCT angiography with 50 mL of iodinated contrast material in an unselected patient population: a feasible protocol

Abstract Objective: To propose a protocol for pulmonary angiography using 64-slice multidetector computed tomography (64-MDCT) with 50 mL of iodinated contrast material, in an unselected patient population, as well as to evaluate vascular enhancement and image quality. Materials and Methods: We evaluated 29 patients (22-86 years of age). The body mass index ranged from 19.0 kg/m2 to 41.8 kg/m2. Patients underwent pulmonary CT angiography in a 64-MDCT scanner, receiving 50 mL of iodinated contrast material via venous access at a rate of 4.5 mL/s. Bolus tracking was applied in the superior vena cava. Two experienced radiologists assessed image quality and vascular enhancement. Results: The mean density was 382 Hounsfield units (HU) for the pulmonary trunk; 379 and 377 HU for the right and left main pulmonary arteries, respectively; and 346 and 364 HU for the right and left inferior pulmonary arteries, respectively. In all patients, subsegmental arteries were analyzed. There were streak artifacts from contrast material in the superior vena cava in all patients. However, those artifacts did not impair the image analysis. Conclusion: Our findings suggest that pulmonary angiography using 64-MDCT with 50 mL of iodinated contrast can produce high quality images in unselected patient populations.

Presence of Chlamydiales DNA in samples negative by broad-range bacterial 16S rRNA PCRs: new insights into chlamydial pathogenic role.

Since routine eubacterial 16S rRNA PCR does not amplify members of the Chlamydiales order, we tested all samples received in our laboratory during a 10 months period using a pan-Chlamydiales real-time PCR. 3 of 107 samples (2.8%) revealed to be positive, suggesting a role of some Chlamydiales in the pathogenesis of chronic bronchial stenosis or bronchial stenosis superinfection and as agents of orthopaedic prosthesis infections.

Gram-negative flagella glycosylation

Protein glycosylation had been considered as an eccentricity of a few bacteria. However, through advances in analytical methods and genome sequencing, it is now established that bacteria possess both N-linked and O-linked glycosylation pathways. Both glycosylation pathways can modify multiple proteins, flagellins from Archaea and Eubacteria being one of these. Flagella O-glycosylation has been demonstrated in many polar flagellins from Gram-negative bacteria and in only the Gram-positive genera Clostridium and Listeria. Furthermore, O-glycosylation has also been demonstrated in a limited number of lateral flagellins. In this work, we revised the current advances in flagellar glycosylation from Gram-negative bacteria, focusing on the structural diversity of glycans, the O-linked pathway and the biological function of flagella glycosylation.

Estudo comparativo da seletividade de um método cinético-catalítico: a determinação de Cr(VI) pela oxidação da o-dianisidina pelo peróxido de hidrogênio

Selectivity studies for the determination of Cr(VI) using the catalytic oxidation of the o-dianisidine by hydrogen peroxide showed two distincts situations. In the first, when interferents were studied by a univariate procedure, Cr(III) and Cu(II) cause serious interferences even at the 2:1 proportion, relative to Cr(VI), while Fe(III) interfered at the 15:1 ratio and EDTA at the 10:1 ratio. On the other hand, when a multivariate investigation was performed, Cr(III) did not present any significant principal effects and its significant interaction effects were negative, in contrast to EDTA, that presented positive interaction effects although, like Cr(III), did not show significant interaction effects. In view of the interferent's action it become necessary to separate Cr(VI) by extraction with methylisobutylketone in a chloridric acid medium before its determination in vegetals and in wastewater from a cellulose industry samples. Using this procedure, the method precision is ±0,5% at the 10 ng/mL Cr(VI) concentration level. The detection and quantification limits, calculated by means of absorbance measurements of ten replicates of blank reagents were 1,1 and 3,2 ng/mL, respectively. The results obtained with real samples showed a relative standard deviation between 1,2% and 3,0% relative to their reference values.

Negative Regulation of Receptor Tyrosine Kinases by T-cell Protein Tyrosine Phosphatase

Protein tyrosine phosphorylation controls a wide array of cellular responses such as growth, migration, proliferation, differentiation, metabolism and cytoskeletal organisation. Tyrosine phosphorylation is a dynamic process involving the competing activities of protein tyrosine kinases and protein tyrosine phosphatases. The protein tyrosine kinases are further divided into non-receptor- and receptor tyrosine kinases. The latter are transmembrane glycoproteins activated by the binding of specific ligands, mostly growth factors, to their extracellular domain, transmitting different signals to the cell. Growth factor receptors such as the epidermal growth factor receptor, vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor β, belong to the receptor tyrosine kinases, the signalling of which is often disturbed in various diseases, including cancer. This has led to the development of receptor tyrosine kinase antagonists for use as anti-cancer drugs. As the receptor tyrosine kinases, also the protein tyrosine phosphatases can be divided into receptor- and non-receptor types. The protein tyrosine phosphatases have attained much less attention than the receptor tyrosine kinases partly because they were identified later. However, accumulating evidence shows that the protein tyrosine phosphatases have important roles as specific and active regulators of tyrosine phosphorylation in cells and of physiological processes. Consequently, the protein tyrosine phosphatases are receiving arising interest as novel drug targets. The aim of this work was to elucidate the negative regulation of receptor tyrosine kinases by one non-receptor protein tyrosine phosphatase, T-cell protein tyrosine phosphatase TCPTP. The results show that TCPTP activated by cell adhesion receptor integrin α1 functions as a negative regulator of the epidermal growth factor receptor. It was also found that TCPTP affects vascular endothelial growth factor receptor 2 signalling and angiogenesis. Lastly, a High-throughput screen with 64,280 compounds was performed to identify novel TCPTP activators, resulting in identification of one small molecule compound capable of exerting similar effects on TCPTP signalling as integrin α1. This compound is shown to downregulate signalling of epidermal growth factor receptor and platelet-derived growth factor receptor β, as well as to inhibit cell proliferation and angiogenesis. Our results suggest that a suitable small-molecule TCPTP activator could be utilized in the development of novel anti-cancer drugs.

Acute Infection in Total Knee Arthroplasty: Diagnosis and Treatment

Infection is one of the most serious complications after total knee arthroplasty (TKA). The current incidence of prosthetic knee infection is 1-3%, depending on the series(.) For treatment and control to be more cost effective, multidisciplinary groups made up of professionals from different specialities who can work together to eradicate these kinds of infections need to be assembled. About the microbiology, Staphylococcus aureus and coagulase-negative staphylococcus were among the most frequent microorganisms involved (74%). Anamnesis and clinical examination are of primary importance in order to determine whether the problem may point to a possible acute septic complication. The first diagnosis may then be supported by increased CRP and ESR levels. The surgical treatment for a chronic prosthetic knee infection has been perfectly defined and standardized, and consists in a two-stage implant revision process. In contrast, the treatment for acute prosthetic knee infection is currently under debate. Considering the different surgical techniques that already exist, surgical debridement with conservation of the prosthesis and polythene revision appears to be an attractive option for both surgeon and patient, as it is less aggressive than the two-stage revision process and has lower initial costs. The different results obtained from this technique, along with prognosis factors and conclusions to keep in mind when it is indicated for an acute prosthetic infection, whether post-operative or haematogenous, will be analysed by the authors.

Interactions of bacteria and fungi on decomposing litter: differential extracellular enzyme activities

Fungi and bacteria are key agents in plant litter decomposition in freshwater ecosystems. However, the specific roles of these two groups and their interactions during the decomposition process are unclear. We compared the growth and patterns of degradativeenzymes expressed by communities of bacteria and fungi grown separately and in coexistence on Phragmites leaves. The two groups displayed both synergistic and antagonistic interactions. Bacteria grew better together with fungi than alone. In addition, there was a negative effect of bacteria on fungi, which appeared to be caused by suppression of fungal growth and biomass accrual rather than specifically affecting enzyme activity. Fungi growing alone had a high capacity for the decomposition of plant polymers such as lignin, cellulose, and hemicellulose. In contrast, enzyme activities were in general low when bacteria grew alone, and the activity of key enzymes in the degradation of lignin and cellulose (phenol oxidase and cellobiohydrolase) was undetectable in the bacteria-only treatment. Still, biomass-specific activities of most enzymes were higher in bacteria than in fungi. The low total activity and growth of bacteria in the absence of fungi in spite of apparent high enzymatic efficiency during the degradation of many substrates suggest that fungi provide the bacteria with resources that the bacteria were not able to acquire on their own, most probably intermediate decomposition products released by fungi that could be used by bacteria

Avaliação sensorial de cachaça

The hedonic level of commercial cachaças, was evaluated by consumers and by a tasters. The results of sensorial methods analyzed trough Principal Components Analysis, Hierarchical Cluster Analysis and the Pearson linear correlation indicated that the best classified cachaças were produced in copper stills and aged in oak casks. By contrast the worst classified exhibited as the main features be not aged and high alcohol percentage. The index of preference is positively correlated with the intensity of yellow color, wood flavor, sweetness and fruit aroma. There is a negative preference correlation with the acidity, the taste of alcohol and bitterness.

Päämääriä, ajelehtimista, tietämättömyyttä, etsintää: Koulutusvalinnat, opinnot ja koulutuksen vaihtaminen yliopisto-opiskelijoiden kokemana 2000 -luvun taitteessa

Tutkimuksessa tarkastellaan yliopisto-opiskelua, opintojen kokemista ja niiden kulkua alkaen koulutusvalinnoista. Lisäksi tutkimuksessa pyritään ymmärtämään ja kuvaamaan yliopisto-opintojen aikaista koulutuksen vaihtamista prosessina. Kyseessä on seurantatutkimus Turun yliopistossa vuonna 1998 aloittaneesta opiskelijakohortista. Tutkittavat opiskelijat valittiin sillä perusteella, että he vastasivat kolmeen opintoja koskevaan kyselyyn ja olivat vähintään kolmen vuoden ajan läsnä olevina opiskelijoina. Opiskelijat (n=440) jaettiin neljään vertailuryhmään: 1) opintoihinsa tyytyväiset, 2) ensimmäisen vuoden jälkeen koulutusta vaihtaneet, 3) toisen vuoden jälkeen koulutusta vaihtaneet ja 4) koulutusta vaihtaa halunneet. Tutkimusaineisto koostuu kolmesta kyselystä, opintorekisteriaineistosta ja koulutusta opintojen aikana vaihtaneiden opiskelijoiden haastatteluista (n=22). Kyselyt tehtiin ensimmäisen, toisen ja kolmannen opintovuoden kevätlukukausilla ja haastattelut vuonna 2002, jolloin opiskelijat opiskelivat Turun yliopistossa neljättä tai viidettä vuotta. Kyselyiden avulla tutkittiin eroavatko koulutusta vaihtaneet opiskelijat muista opiskelijoista rakenteellisilta tekijöiltään tai opintokokemuksissaan. Opintorekisteriaineiston perusteella tutkittiin eroavatko koulutusta vaihtaneet opiskelijat koulutuksessa pysyneistä opiskelijoista opintojen kulussa. Kyselyiden ja haastatteluiden avulla tutkittiin minkälaisia tietoja ja odotuksia opiskelijoilla oli yliopisto-opiskelusta ollut ennen opintojen aloittamista. Haastatteluiden perusteella tutkittiin mitä syitä koulutuksen vaihtamisen taustalla oli ja minkälaisina kertomuksina koulutuksen vaihtamisprosessi näyttäytyi. Koulutusta vaihtaneet opiskelijat erosivat koulutuksessa pysyneistä opiskelijoista aiemman koulutuksen määrän, iän ja perheellisyyden suhteen. Verrattaessa ensimmäisen ja toisen vuoden jälkeen koulutusta vaihtaneita opiskelijoita keskenään havaittiin näillä ryhmillä olevan erilainen perhetausta. Koulutusta valittaessa tärkein tekijä oli kiinnostus alaa kohtaan. Toiseksi voimakkain hakutekijä oli muilla paitsi ensimmäisen vuoden jälkeen koulutusta vaihtaneilla valmiudet haluttuun ammattiin. Vuorostaan ensimmäisen vuoden jälkeen koulutusta vaihtaneilla toiseksi tärkein tekijä hakeutumisessa oli helppo sisäänpääsy opintoihin. Suurin osa opiskelijoista oli päässyt ensisijaisesti haluamaansa koulutukseen. Koulutusta myöhemmin vaihtaneissa ryhmissä ja koulutuksen vaihtoa halunneiden ryhmässä ensisijaisesti haluttuun koulutukseen päässeitä oli alle puolet. Odotukset opiskelusta eivät täysin täyttyneet ensimmäisen puolen vuoden opiskelun aikana. Tutkittujen ryhmien välillä havaittiin eroja kokemuksissa koulutuksen sopivuudesta itselle. Koulutuksen vaihtaminen oli myönteinen kokemus, ja uusi koulutus koettiin aiempaa koulutusta sopivammaksi itselle. Kolmannen vuoden kevätlukukaudella omaa koulutusta kohtaan koettu kiinnostus oli voimakasta muissa ryhmissä paitsi koulutuksen vaihtoa halunneissa. Harvimmin positiivisia tunteita opiskelua kohtaan kokivat vaihtoa halunneet opiskelijat. Koulutuksen vaihtaminen vähensi opintoihin kiinnittymättömyyttä. Koulutuksen vaihtoa halunneiden kokemukset nykyisen koulutuksensa ilmapiiristä olivat negatiivisimpia. Koulutuksen vaihtaminen vaikutti myönteisesti myös opetuksen kokemiseen. Opinnot etenivät kaikissa tutkituissa ryhmissä tasaisesti. Koulutuksen vaihtamisen jälkeisenä vuonna opinnot etenivät vaihtaneissa ryhmissä keskimääräistä enemmän. Haastatteluiden mukaan pääasiallisia syitä koulutuksen vaihtamiselle olivat päätyminen toissijaiseen koulutukseen ja pettyminen aloittamaansa koulutukseen. Narratiivien analyysin avulla tutkimuksessa löydettiin neljä erilaista tarinaa vaihtamisprosessista: 1) toissijaisesta koulutuksesta ensisijaiseen koulutukseen vaihtaminen, 2) ensisijaiseen koulutukseen pettyminen, 3) kiinnostavampaan koulutukseen ajautuminen ja 4) oman alan löytyminen etsinnän kautta. Vaikka koulutuspolitiikassa koulutuksen vaihtaminen nähdään ongelmana, oli se tämän tutkimuksen mukaan opiskelijalle myönteinen kokemus. Tämän tutkimuksen perusteella tulisikin kiinnittää huomiota opiskelijoihin, jotka haluaisivat vaihtaa koulutuksesta toiseen. Heillä opintojen eteneminen osoittautui hitaimmaksi ja opintokokemukset negatiivisimmiksi. Koulutusta vaihtaneiden opiskelijoiden opintojen eteneminen näytti hitaalta verrattuna opintoihinsa tyytyväisiin opiskelijoihin. Koulutuksen vaihtamisen jälkeen opintojen eteneminen kuitenkin nopeutui ja opintotyytyväisyys lisääntyi.

Quality of life and menopausal symptoms in women with liver transplants

PURPOSE: To assess quality of life and climacteric symptoms in women with and without liver transplants. METHODS: This was a cross-sectional study of 52 women undergoing follow-up at a university hospital in southeastern Brazil from February 4th, 2009 to January 5th, 2011. Twenty-four of these women were 35 years old or older and had undergone liver transplantation at least one year before study entry. The remaining 28 women had no liver disease and were matched by age and menstrual patterns to the patients with transplants. The abbreviated version of the World Health Organization (WHOQOL-BREF) questionnaire was used to assess quality of life. Menopausal symptoms were assessed using the Menopause Rating Scale (MRS). Statistical analysis was carried out by Student's t-test, Mann-Whitney test and analysis of variance. Correlations between MRS and the WHOQOL-BREF were established by correlation coefficients. RESULTS: The mean age of the women included in the study was 52.2 (±10.4) years and the mean time since transplantation was 6.1 (±3.3) years. Women with liver transplants had better quality of life scores in the environment domain (p=0.01). No difference was noted between the two groups in any domain of the MRS. For women in the comparison group, there was a strongly negative correlation between somatic symptoms in the MRS and the physical domain of the WHOQOL-BREF (p<0.01; r=-0.8). In contrast, there was only a moderate association for women with liver transplants (p<0.01; r=-0.5). CONCLUSIONS: Women with liver transplants had better quality of life scores in the domain related to environment and did not exhibit more intense climacteric symptoms than did those with no liver disease. Climacteric symptoms negatively influenced quality of life in liver transplant recipients, although less intensely than in women without a history of liver disease.

A Brazilian glycoprotein E-negative bovine herpesvirus type 1.2a (BHV-1.2a) mutant is attenuated for cattle and induces protection against wild-type virus challenge

The authors previously reported the construction of a glycoprotein E-deleted (gE-) mutant of bovine herpesvirus type 1.2a (BHV-1.2a). This mutant, 265gE-, was designed as a vaccinal strain for differential vaccines, allowing the distinction between vaccinated and naturally infected cattle. In order to determine the safety and efficacy of this candidate vaccine virus, a group of calves was inoculated with 265gE-. The virus was detected in secretions of inoculated calves to lower titres and for a shorter period than the parental virus inoculated in control calves. Twenty one days after inoculation, the calves were challenged with the wild type parental virus. Only mild signs of infection were detected on vaccinated calves, whereas non-vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Six months after vaccination, both vaccinated and control groups were subjected to reactivation of potentially latent virus. The mutant 265gE- could not be reactivated from vaccinated calves. The clinical signs observed, following the reactivation of the parental virus, were again much milder on vaccinated than on non-vaccinated calves. Moreover, parental virus shedding was considerably reduced on vaccinated calves at reactivation. In view of its attenuation, immunogenicity and protective effect upon challenge and reactivation with a virulent BHV-1, the mutant 265gE- was shown to be suitable for use as a BHV-1 differential vaccine virus.