Responsiveness of dark-adaptation threshold to vitamin A and beta-carotene supplementation in pregnant and lactating women in Nepal.

BACKGROUND: Impaired dark adaptation occurs commonly in vitamin A deficiency. OBJECTIVE: We sought to examine the responsiveness of dark-adaptation threshold to vitamin A and beta-carotene supplementation in Nepali women. DESIGN: The dark-adapted pupillary response was tested in 298 pregnant women aged 15-45 y in a placebo-controlled trial of vitamin A and beta-carotene; 131 of these women were also tested at 3 mo postpartum. Results were compared with those for 100 nonpregnant US women of similar age. The amount of light required for pupillary constriction was recorded after bleaching and dark adaptation. RESULTS: Pregnant women receiving vitamin A had better dark-adaptation thresholds (-1.24 log cd/m(2)) than did those receiving placebo (-1.11 log cd/m(2); P: = 0. 03) or beta-carotene (-1.13 log cd/m(2); P: = 0.05) (t tests with Bonferroni correction). Dark-adaptation threshold was associated with serum retinol concentration in pregnant women receiving placebo (P: = 0.001) and in those receiving beta-carotene (P: = 0.003) but not in those receiving vitamin A. Among women receiving placebo, mean dark-adaptation thresholds were better during the first trimester (-1.23 log cd/m(2)) than during the second and third trimesters (-1.03 log cd/m(2); P: = 0.02, t test). The mean threshold of nonpregnant US women (-1.35 log cd/m(2)) was better than that of all 3 Nepali groups (P: < 0.001, t test, for all 3 groups). CONCLUSIONS: During pregnancy, pupillary dark adaptation was strongly associated with serum retinol concentration and improved significantly in response to vitamin A supplementation. This noninvasive testing technique is a valid indicator of population vitamin A status in women of reproductive age.

Modifications of Surface Properties of Beta Ti by Laser Gas Diffusion Nitriding

Beta-type Ti-alloy is a promising biomedical implant material as it has a low Young’s modulus and is also known to have inferior surface hardness. Various surface treatments can be applied to enhance the surface hardness. Physical vapor deposition and chemical vapor deposition are two examples of this but these techniques have limitations such as poor interfacial adhesion and high distortion. Laser surface treatment is a relatively new surface modification method to enhance the surface hardness but its application is still not accepted by the industry. The major problem of this process involves surface melting which results in higher surface roughness after the laser surface treatment. This paper will report the results achieved by a 100 W continuous wave (CW) fiber laser for laser surface treatment without the surface being melted. Laser processing parameters were carefully selected so that the surface could be treated without surface melting and thus the surface finish of the component could be maintained. The surface and microstructural characteristics of the treated samples were examined using x-ray diffractometry, optical microscopy, three-dimensional surface profile and contact angle measurements, and nanoindentation test.

Carbapenemases of Serratia fonticola UTAD54

As carbapenemases, serínicas e metalo-β-lactamases (MBLs), formam um grupo cada vez mais importante de β-lactamases capazes de tornar as bactérias resistentes a antibióticos β-lactâmicos, incluindo carbapenemos utilizados como antibióticos de último recurso no tratamento de infecções causadas por bactérias multirresistentes. De modo a compreender melhor a relação estrutura-função deste grupo de enzimas, prosseguimos com a caracterização bioquímica e estrutural das carbapenemases SFC-1 e Sfh-I específicas de Serratia fonticola UTAD54, uma estirpe ambiental isolada previamente de águas de consumo não tratadas no Nordeste de Portugal. Ambas as β-lactamases foram sobre-expressas em Escherichia coli e purificadas por cromatografia líquida. A SFC-1 recombinante, uma carbapenemase serínica, hidrolisa eficientemente antibióticos β-lactâmicos de todas as classes e exibe, comparativamente a enzimas relacionadas (ex. KPC), uma maior eficiência contra a ceftazidima e uma menor susceptibilidade aos inibidores convencionais das β-lactamases. As estruturas do cristal da SFC-1 nativa e de complexos de mutantes, obtidos por mutagénese dirigida, com o meropenemo não hidrolisado e na forma de acetilenzima foram determinados por substituição molecular utilizando cristalografia de raios-X. A estrutura da SFC-1 contém todas as características conservadas do centro activo das carbapenemases de classe A. Nas estruturas dos mutantes o meropenemo aparece orientado no centro activo por Thr236 e Thr238, posicionando-o próximo da Ser130 para a transferência do protão. Nas enzimas de classe A inibidas por carbapenemos, a interacção com a Arg244 impõe uma orientação diferente do meropenemo ligado, prejudicando a transferência do protão. Estas constituem as primeiras estruturas de uma carbapenemase de classe A com um carbapenemo no centro activo e revelam que estas enzimas alteram a orientação do meropenemo ligado para promover a catálise, sem alteração significativa da estrutura geral. A Sfh-I, tal como as outras MBLs da subclasse B2, apresenta um perfil de substratos reduzido, que inclui maioritariamente os carbapenemos. A Sfh-I hidrolisa imipenemo e meropenemo com um kcat de 51 e 109 s-1 e um KM de 79 e 215 μM, respectivamente. A Sfh-I liga um equivalente de zinco, como demonstrado por espectrometria de massa. Contrariamente a enzimas da subclasse B2 previamente caracterizadas, a Sfh-I hidrolisa a cefepima, mostrando que a Sfh-I é uma MBL da subclasse B2 com propriedades únicas. Por espectroscopia de fluorescência mostrou-se que a Sfh-I é capaz de ligar até 3 equivalentes de zinco (Kd2 = 95 μM; Kd3 = 2.3 mM). A estrutura do cristal da Sfh-I, determinada por substituição molecular utilizando a CphA como modelo, é a primeira para uma MBL da subclasse B2 não ligada. Esta estrutura revela a disposição das moléculas de água no centro activo corroborando um mecanismo catalítico para as MBLs da subclasse B2 no qual a His118, em vez do Asp120 proposto anteriormente, activa a molécula de água nucleofílica.

Loss of Aggressiveness of Phytophthora cinnamomi (Beta-Cinnamomin Silenced Strain) in the Infection of Castanea sativa

Several forest species are severely affected by Phytophthora cinnamomi. The contribution of this oomycete to forest decline and dieback has been broadly reported. In particular, it is consensual that it is the causal agent of ink disease in Castanea sativa. It has been associated with the severe decline of Quercus species, namely the Q. suber and Q. ilex dieback in Portugal and Spain, and has been responsible for the infection of numerous native species and crops. This pathogen persists in the soil or on plant material in the form of chlamydospores allowing the infection of living root tissues when environmental conditions are favorable. © Microscopy Society of America 2012.

Unusual reactivity of selenoboranes towards epoxides: New selective routes to beta- hydroxyselenides and allylalcohols

Selenoboranes react with terminal, α, β-di- and trisubstituted epoxides to produce β-hydroxyselenides (or olefins) in the two first cases and allyl alcohols in the last one. A very high stereodescrimination has been observed for α, β-bisubstituted epoxides: the cis epoxide being much more reactive.

Cystathionine beta-synthase variants : identification, characterization and modulation by thiol compounds

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014

Next-generation sequencing is highly sensitive for the detection of beta-catenin mutations in desmoid-type fibromatoses

Desmoid-type fibromatoses are locally aggressive and frequently recurrent tumours, and an accurate diagnosis is essential for patient management. The majority of sporadic lesions harbour beta-catenin (CTNNB1) mutations. We used next-generation sequencing to detect CTNNB1 mutations and to compare the sensitivity and specificity of next-generation sequencing with currently employed mutation detection techniques: mutation-specific restriction enzyme digestion and polymerase chain reaction amplification. DNA was extracted from formalin-fixed paraffin-embedded needle biopsy or resection tissue sections from 144 patients with sporadic desmoid-type fibromatoses, four patients with syndrome-related desmoid-type fibromatoses and 11 morphological mimics. Two primer pairs were designed for CTNNB1 mutation hotspots. Using ≥10 ng of DNA, libraries were generated by Fluidigm and sequenced on the Ion Torrent Personal Genome Machine. Next-generation sequencing had a sensitivity of 92.36 % (133/144, 95 % CIs: 86.74 to 96.12 %) and a specificity of 100 % for the detection of CTNNB1 mutations in desmoid-type fibromatoses-like spindle cell lesions. All mutations detected by mutation-specific restriction enzyme digestion were identified by next-generation sequencing. Next-generation sequencing identified additional mutations in 11 tumours that were not detected by mutation-specific restriction enzyme digestion, two of which have not been previously described. Next-generation sequencing is highly sensitive for the detection of CTNNB1 mutations. This multiplex assay has the advantage of detecting additional mutations compared to those detected by mutation-specific restriction enzyme digestion (sensitivity 82.41 %). The technology requires minimal DNA and is time- and cost-efficient.

The effect of melanocortin peptides on Interleukin 1-beta induced chondrocyte cell death and inflammation

ntroduction: Osteoarthritis (OA) is a degenerative joint disease affecting more than 8.5 million people in the UK. Disruption in the catabolic and anabolic balance, with the catabolic cytokine Interleukin 1 beta (IL-1β) being involved in the initiation and progression of OA (1). Melanocortin peptides (α-MSH and D[Trp8]-γ-MSH) exert their anti-inflammatory effects via activation of melanocortin receptors (MC), with both MC1 and MC3 being identified as promising candidates as novel targets for OA (2). This study aims to assess the chondroprotective and anti-inflammatory effects of the pan melanocortin receptor agonist α-MSH and MC3 agonist D[Trp8]-γ-MSH following IL-1β chondrocyte stimulation. Methods: RT-PCR/ Western Blot: Human C-20/A4 chondrocytic cell-line were cultured in 6 well plates (1x106 cells/well) and harvested to determine MC and IL-1β expression by RT-PCR, and Western Blot. Cell-Culture: Cells were cultured in 96 well plates (1x106 cells/well) and stimulated with H2O2 (0.3%), TNF-α (60 pg/ml) or IL-1β (0-5000pg/ml) for 0-72h and cell viability determined. Drug Treatment: In separate experiments cells were pre-treated with 3 μg/ml α-MSH (Sigma-Aldrich Inc. Poole, UK), or D[Trp8]-γ-MSH (Phoenix Pharmaceuticals, Karlsrhue, Germany) (all dissolved in PBS) for 30 minutes prior to IL-1β (5000pg/ml) stimulation for 6-24h. Analysis: Cell viability was determined by using the three cell viability assays; Alamar Blue, MTT and the Neutral Red (NR) assay. Cell-free supernatants were collected and analysed for Interleukin -6 (IL-6) and IL-8 release by ELISA. Data expressed as Mean ± SD of n=4-8 determination in quadruplicate. *p≤ 0.05 vs. control. Results: Both RT-PCR, and Western Blot showed MC1 and MC3 expression on C-20/A4 cells. Cell viability analysis: IL-1β stimulation led to a maximal cell death of 35% at 6h (Alamar Blue), and 40% and 75% with MTT and Neutral Red respectively at 24h compared to control. The three cell viability assays have different cellular uptake pathways, which accounts for the variations observed in cell viability in response to the concentration of IL-1β, and time. Cytokine analysis by ELISA: IL-1β (5000pg/ml) stimulation for 6 and 24h showed maximal IL-6 production 292.3 ±3.8 and 275.5 ±5.0 respectively, and IL-8 production 353.3 ±2.6 and 598.3 ±8.6 respectively. Pre-treatment of cells with α-MSH and D[Trp8]-γ-MSH caused significant reductions in both IL-6 and IL-8 respectively following IL-1β stimulation at 6h. Conclusion: MC1/3 are expressed on C-20/A4 cells, activation by melanocortin peptides led to an inhibition of IL-1β induced cell death and pro-inflammatory cytokine release.

Platelets induce Pai-1 mRNA expression in monocytes through TGF-BETA

Introduction: Plasminogen activator inhibitor type-1 (PAI-1) is a physiological modulator of fibrinolysis. High plasma PAI-1 is associated with the 4G/5G promoter polymorphism and with increased cardiovascular risk. Here we explored the role of platelets in regulating expression of the PAI-1 gene in monocytes. Methods: Blood from PAI-1 4G/5G genotyped volunteers (n=6) was incubated with the platelet GPVI-specific agonist, cross-linked collagen related peptide (CRP-XL), in the presence or absence of Mab 9E1 that blocks the binding of P-selectin to PSGL1. Monocytes were isolated by +ve selection on CD14 beads and monocyte PAI-1 mRNA expression was measured by real-time PCR. Results: Activation of platelets with CRP-XL resulted in platelets binding to >70% of monocytes and was accompanied by >5000-fold induction of PAI-1 mRNA, peaking at 4hrs. PAI-1 expression was independent of the 4G/5G genotype. Blocking the binding of platelets to monocytes enhanced PAI-1 induction (p<0.05 at 4 hrs). Incubation of isolated monocytes with the releasate from CRP-XL stimulated platelets also led to PAI-1 mRNA expression. The platelet secretome contains >100 different proteins. To identify the soluble factor(s) responsible for induction of PAI-1, neutralizing antibodies to likely candidates were added to monocytes incubated with the platelet releasate. Anti- TGF-beta inhibited platelet releasate-mediated PAI-1 mRNA induction by >80%. Monocyte PAI-1 was also induced by stimulation of PSGL-1 with a P-selectin-Fc chimera, in the absence of platelets, which was also blocked by the TGF-beta antibody. Conclusions: These results suggest that platelets induce PAI-1 mRNA in monocytes predominantly via TGF-beta, released from both platelets, and monocytes via activation by PSGL-1 signalling.This stimulation is independent of 4G/5G genotype

Desenvolvimento de uma tecnologia de produção de iogurtes funcionais com leite de cabra contendo fibra Beta vulgaris e frutos de Vaccinium cylindraceum

Dissertação de Mestrado, Tecnologia e Segurança Alimentar, 02 de Junho de 2014, Universidade dos Açores.

Evolution of Drug Resistance: Insight on TEM -Lactamases Structure and Activity and Beta-Lactam Antibiotics

Extended-spectrum β-lactamases (ESBLs) prevalence was studied in the north of Portugal, among 193 clinical isolates belonging to citizens in a district in the boundaries between this country and Spain from a total of 7529 clinical strains. In the present study we recovered some members of Enterobacteriaceae family, producing ESBL enzymes, including Escherichia coli (67.9%), Klebsiella pneumoniae (30.6%), Klebsiella oxytoca (0.5%), Enterobacter aerogenes (0.5%), and Citrobacter freundii (0.5%). β-lactamases genes blaTEM, blaSHV, and blaCTX-M were screened by polymerase chain reaction (PCR) and sequencing approaches. TEM enzymes were among the most prevalent types (40.9%) followed by CTX-M (37.3%) and SHV (23.3%). Among our sample of 193 ESBL-producing strains 99.0% were resistant to the fourth-generation cephalosporin cefepime. Of the 193 isolates 81.3% presented transferable plasmids harboring genes. Clonal studies were performed by PCR for the enterobacterial repetitive intragenic consensus (ERIC) sequences. This study reports a high diversity of genetic patterns. Ten clusters were found for E. coli isolates and five clusters for K. pneumoniae strains by means of ERIC analysis. In conclusion, in this country, the most prevalent type is still the TEM-type, but CTX-M is growing rapidly.

T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.

Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulin-secreting beta cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time- and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet beta cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.