Novel Sealing Technique for Practical Liquid-Core Photonic Crystal Fibers

In this letter, we describe a simple and effective technique to prevent evaporation in liquid-core photonic crystal fibers (PCFs). The technique consists of using a micropipette to deploy a micro-droplet of an ultraviolet curable polymer adhesive in both core inputs. After it is cured, the adhesive creates sealing polymer plugs with quite satisfactory insertion loss (overall optical transmission of about 15%). Processed fibers remained liquid-filled for at least six weeks. From a practical point of view, we conducted a supercontinuum generation experiment in a water-core PCF to demonstrate a 120-minute spectral width stability and the ability to withstand at least 3-mW average power at the sealed fiber input. Similar experiments carried out with nonsealed fibers produced supercontinuum spectra lasting no longer than 10 minutes, with average powers kept below 0.5 mW to avoid thermally induced evaporation.

Promoter regions of Plasmodium vivax are poorly or not recognized by Plasmodium falciparum

Abstract Background Heterologous promoter analysis in Plasmodium has revealed the existence of conserved cis regulatory elements as promoters from different species can drive expression of reporter genes in heterologous transfection assays. Here, the functional characterization of different Plasmodium vivax promoters in Plasmodium falciparum using luciferase as the reporter gene is presented. Methods Luciferase reporter plasmids harboring the upstream regions of the msp1, dhfr, and vir3 genes as well as the full-length intergenic regions of the vir23/24 and ef-1α genes of P. vivax were constructed and transiently transfected in P. falciparum. Results Only the constructs with the full-length intergenic regions of the vir23/24 and ef-1α genes were recognized by the P. falciparum transcription machinery albeit to values approximately two orders of magnitude lower than those reported by luc plasmids harbouring promoter regions from P. falciparum and Plasmodium berghei. A bioinformatics approach allowed the identification of a motif (GCATAT) in the ef-1α intergenic region that is conserved in five Plasmodium species but is degenerate (GCANAN) in P. vivax. Mutations of this motif in the P. berghei ef-1α promoter region decreased reporter expression indicating it is active in gene expression in Plasmodium. Conclusion Together, this data indicates that promoter regions of P. vivax are poorly or not recognized by the P. falciparum transcription machinery suggesting the existence of P. vivax-specific transcription regulatory elements.

Thermo and mechanical cycling and veneering method do not influence Y-TZP core/veneer interface bond strength

Objectives: The purpose of this study was to evaluate the influence of thermal and mechanical cycling and veneering technique on the shear bond strength of Y-TZP (yttrium oxide partially stabilized tetragonal zirconia polycrystal) core–veneer interfaces. Materials and methods: Cylindrical Y-TZP specimens were veneered either by layering (n = 20) or by pressing technique (n = 20). A metal ceramic group (CoCr) was used as control (n = 20). Ten specimens for each group were thermal and mechanical cycled and then all samples were subjected to shear bond strength in a universal testing machine with a 0.5 mm/min crosshead speed. Mean shear bond strength (MPa) was analysed with a 2-way analysis of variance and Tukey’s test ( p < 0.05). Failure mode was determined using stereomicroscopy and scanning electron microscopy (SEM). Results: Thermal and mechanical cycling had no influence on the shear bond strength for all groups. The CoCr group presented the highest bond strength value ( p < 0.05) (34.72 7.05 MPa). There was no significant difference between Y-TZP veneered by layering (22.46 2.08 MPa) or pressing (23.58 2.1 MPa) technique. Failure modes were predominantly adhesive for CoCr group, and cohesive within veneer for Y-TZP groups. Conclusions: Thermal and mechanical cycling, as well as the veneering technique does not affect Y-TZP core–veneer bond strength. Clinical significance: Different methods of veneering Y-TZP restorations would not influence the clinical performance of the core/veneer interfaces.

The promoter of filamentation (POF1) protein from Saccharomyces cerevisiae is an ATPase involved in the protein quality control process

Background: The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene. Results: Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo. Conclusions: Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

Extracytoplasmic function (ECF) sigma factor σF is involved in Caulobacter crescentus response to heavy metal stress

Background The α-proteobacterium Caulobacter crescentus inhabits low-nutrient environments and can tolerate certain levels of heavy metals in these sites. It has been reported that C. crescentus responds to exposure to various heavy metals by altering the expression of a large number of genes. Results In this work, we show that the ECF sigma factor σF is one of the regulatory proteins involved in the control of the transcriptional response to chromium and cadmium. Microarray experiments indicate that σF controls eight genes during chromium stress, most of which were previously described as induced by heavy metals. Surprisingly, σF itself is not strongly auto-regulated under metal stress conditions. Interestingly, σF-dependent genes are not induced in the presence of agents that generate reactive oxygen species. Promoter analyses revealed that a conserved σF-dependent sequence is located upstream of all genes of the σF regulon. In addition, we show that the second gene in the sigF operon acts as a negative regulator of σF function, and the encoded protein has been named NrsF (Negative regulator of sigma F). Substitution of two conserved cysteine residues (C131 and C181) in NrsF affects its ability to maintain the expression of σF-dependent genes at basal levels. Furthermore, we show that σF is released into the cytoplasm during chromium stress and in cells carrying point mutations in both conserved cysteines of the protein NrsF. Conclusion A possible mechanism for induction of the σF-dependent genes by chromium and cadmium is the inactivation of the putative anti-sigma factor NrsF, leading to the release of σF to bind RNA polymerase core and drive transcription of its regulon.

Association of genetic variants in the promoter region of genes encoding p22phox (CYBA) and glutamate cysteine ligase catalytic subunit (GCLC) and renal disease in patients with type 1 diabetes mellitus

Background Oxidative stress is recognized as a major pathogenic factor of cellular damage caused by hyperglycemia. NOX/NADPH oxidases generate reactive oxygen species and NOX1, NOX2 and NOX4 isoforms are expressed in kidney and require association with subunit p22phox (encoded by the CYBA gene). Increased expression of p22phox was described in animal models of diabetic nephropathy. In the opposite direction, glutathione is one of the main endogenous antioxidants whose plasmatic concentrations were reported to be reduced in diabetes patients. The aim of the present investigation was to test whether functional single nucleotide polymorphisms (SNPs) in genes involved in the generation of NADPH-dependent O2•- (-675 T → A in CYBA, unregistered) and in glutathione metabolism (-129 C → T in GCLC [rs17883901] and -65 T → C in GPX3 [rs8177412]) confer susceptibility to renal disease in type 1 diabetes patients. Methods 401 patients were sorted into two groups according to the presence (n = 104) or absence (n = 196) of overt diabetic nephropathy or according to glomerular filtration rate (GFR) estimated by Modification of Diet in Renal Disease (MDRD) equation: ≥ 60 mL (n = 265) or < 60 mL/min/1.73 m2 (n = 136) and were genotyped. Results No differences were found in the frequency of genotypes between diabetic and non-diabetic subjects. The frequency of GFR < 60 mL/min was significantly lower in the group of patients carrying CYBA genotypes T/A+A/A (18.7%) than in the group carrying the T/T genotype (35.3%) (P = 0.0143) and the frequency of GFR < 60 mL/min was significantly higher in the group of patients carrying GCLC genotypes C/T+T/T (47.1%) than in the group carrying the C/C genotype (31.1%) (p = 0.0082). Logistic regression analysis identified the presence of at least one A allele of the CYBA SNP as an independent protection factor against decreased GFR (OR = 0.38, CI95% 0.14-0.88, p = 0.0354) and the presence of at least one T allele of the GCLC rs17883901 SNP as an independent risk factor for decreased GFR (OR = 2.40, CI95% 1.27-4.56, p = 0.0068). Conclusions The functional SNPs CYBA -675 T → A and GCLC rs17883901, probably associated with cellular redox imbalances, modulate the risk for renal disease in the studied population of type 1 diabetes patients and require validation in additional cohorts.

Increased interleukin-10 and interferon-γ levels in Plasmodium vivax malaria suggest a reciprocal regulation which is not altered by IL-10 gene promoter polymorphism

Background In human malaria, the naturally-acquired immune response can result in either the elimination of the parasite or a persistent response mediated by cytokines that leads to immunopathology. The cytokines are responsible for all the symptoms, pathological alterations and the outcome of the infection depends on the reciprocal regulation of the pro and anti-inflammatory cytokines. IL-10 and IFN-gamma are able to mediate this process and their production can be affected by single nucleotide polymorphisms (SNPs) on gene of these cytokines. In this study, the relationship between cytokine IL-10/IFN-gamma levels, parasitaemia, and their gene polymorphisms was examined and the participation of pro-inflammatory and regulatory balance during a natural immune response in Plasmodium vivax-infected individuals was observed. Methods The serum levels of the cytokines IL-4, IL-12, IFN-gamma and IL-10 from 132 patients were evaluated by indirect enzyme-linked immunosorbent assays (ELISA). The polymorphism at position +874 of the IFN-gamma gene was identified by allele-specific polymerase chain reaction (ASO-PCR) method, and the polymorphism at position -1082 of the IL-10 gene was analysed by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism). Results The levels of a pro- (IFN-gamma) and an anti-inflammatory cytokine (IL-10) were significantly higher in P. vivax-infected individuals as compared to healthy controls. The IFN-gamma levels in primoinfected patients were significantly higher than in patients who had suffered only one and more than one previous episode. The mutant alleles of both IFN-gamma and IL-10 genes were more frequent than the wild allele. In the case of the IFNG+874 polymorphism (IFN-gamma) the frequencies of the mutant (A) and wild (T) alleles were 70.13% and 29.87%, respectively. Similar frequencies were recorded in IL-10-1082, with the mutant (A) allele returning a frequency of 70.78%, and the wild (G) allele a frequency of 29.22%. The frequencies of the alleles associated with reduced production of both IFN-gamma and IL-10 were high, but this effect was only observed in the production of IFN-gamma. Conclusions This study has shown evidence of reciprocal regulation of the levels of IL-10 and IFN-gamma cytokines in P. vivax malaria, which is not altered by the presence of polymorphism in the IL-10 gene.

USO DO DUBLIN CORE NA DESCRIÇÃO DE OBRAS RARAS NA WEB: A COLEÇÃO DA BIBLIOTECA BRASILIANA DIGITAL

Acessível ao público desde junho de 2009, a Biblioteca Brasiliana Digital, da Universidade de São Paulo tem por objetivo facultar para a pesquisa, a maior Brasiliana custodiada por uma universidade. Sua intenção é disponibilizar virtualmente parte do acervo da Universidade oferecendo-se como um instrumento útil e funcional para a pesquisa e o estudo dos temas e cultura brasileiros, além de oferecer um modelo tecnológico de gestão que possa ser difundido a outras coleções, acervos e instituições. Este trabalho apresenta os resultado da implantação de um esquema de metadados baseado no formato Dublin Core, para a descrição de obras raras e especiais na web. Especificamente, apresenta os procedimentos e processos de descrição de conteúdos das diversas tipologias documentais (livros, periódicos, gravuras etc.) e formatos digitais (pdf, jpeg entre outros). Palavras-Chave: Bibliotecas digitais; Metadados; Dublin Core.

The intronic long noncoding RNA ANRASSF1 recruits PRC2 to the RASSF1A promoter, reducing the expression of RASSF1A and increasing cell proliferation

The down-regulation of the tumor-suppressor gene RASSF1A has been shown to increase cell proliferation in several tumors. RASSF1A expression is regulated through epigenetic events involving the polycomb repressive complex 2 (PRC2); however, the molecular mechanisms modulating the recruitment of this epigenetic modifier to the RASSF1 locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand on the RASSF1 gene locus in several cell lines and tissues and binds PRC2. ANRASSF1 is transcribed through RNA polymerase II and is 5'-capped and polyadenylated; it exhibits nuclear localization and has a shorter half-life compared with other lncRNAs that bind PRC2. ANRASSF1 endogenous expression is higher in breast and prostate tumor cell lines compared with non-tumor, and an opposite pattern is observed for RASSF1A. ANRASSF1 ectopic overexpression reduces RASSF1A abundance and increases the proliferation of HeLa cells, whereas ANRASSF1 silencing causes the opposite effects. These changes in ANRASSF1 levels do not affect the RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase in both PRC2 occupancy and histone H3K27me3 repressive marks, specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression was detected on PRC2 occupancy and histone H3K27me3 at the promoter regions of RASSF1C and the four other neighboring genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrated that ANRASSF1 forms an RNA/DNA hybrid and recruits PRC2 to the RASSF1A promoter. Together, these results demonstrate a novel mechanism of epigenetic repression of the RASSF1A tumor suppressor gene involving antisense unspliced lncRNA, in which ANRASSF1 selectively represses the expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome might contribute to a location-specific epigenetic modulation of genes.

Photoluminescence of core-shell nanoparticles made from yttrium stabilized zirconia powder grain coated with alumina

A YSZ@Al2O3 nanocomposite was obtained by Al2O3 coating on the surface of yttrium stabilized zirconia via a polymeric precursor method. The resulting core–shell structures were characterized by X-ray diffraction, scanning electron microscopy, transmission electronic microscopy and PL spectra. The TEM micrographs clearly show a homogeneous Al2O3 shell around the ZrO2 core. The observed PL is related to surface–interface defects. Such novel technologies can, in principle, explore materials which are not available in the bulk single crystal form but their figure-of-merit is dramatically dependent on the surface–interface defect states.

Molecular basis oh herpes simplex virus entry into the cell and retargeting of the viral tropism for the design of oncolytic herpesviruses

Herpes simplex virus 1 (HSV-1) infects oral epitelial cells, then spreads to the nerve endings and estabilishes latency in sensory ganglia, from where it may, or may not reactivate. Diseases caused by virus reactivation include mild diseases such as muco-cutaneous lesions, and more severe, and even life-threatening encephalitis, or systemic infections affecting diverse organs. Herpes simplex virus represents the most comprehensive example of virus receptor interaction in Herpesviridae family, and the prototype virus encoding multipartite entry genes. In fact, it encodes 11-12 glycoproteins and a number of additional membrane proteins: five of these proteins play key roles in virus entry into subsceptible cells. Thus, glycoprotein B (gB) and glycoprotein C (gC) interact with heparan sulfate proteoglycan to enable initial attachment to cell surfaces. In the next step, in the entry cascade, gD binds a specific surface receptor such as nectin1 or HVEM. The interaction of glycoprotein D with the receptor alters the conformation of gD to enable the activation of gB, glycoprotein H, and glycoprotein L, a trio of glycoproteins that execute the fusion of the viral envelope with the plasma membrane. In this thesis, I described two distinct projects: I. The retargeting of viral tropism for the design of oncolytic Herpesviruses: • capable of infecting cells through the human epitelial growth factor receptor 2 (HER2), overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis; • detargeted from its natural receptors, HVEM and nectin1. To this end, we inserted a ligand to HER2 in gD. Because HER2 has no natural ligand, the selected ligand was a single chain antibody (scFv) derived from MAb4D5 (monoclonal antibody to HER2), herein designated scHER2. All recombinant viruses were targeted to HER2 receptor, but only two viruses (R-LM113 and R-LM249) were completely detargeted from HVEM and nectin1. To engineer R-LM113, we removed a large portion at the N-terminus of gD (from aa 6 to aa 38) and inserted scHER2 sequence plus 9-aa serine-glycine flexible linker at position 39. On the other hand, to engineer R-LM249, we replaced the Ig-folded core of gD (from aa 61 to aa 218) with scHER2 flanked by Ser-Gly linkers. In summary, these results provide evidence that: i. gD can tolerate an insert almost as big as gD itself; ii. the Ig-like domain of gD can be removed; iii. the large portion at the N-terminus of gD (from aa 6 to aa 38) can be removed without loss of key function; iv. R-LM113 and R-LM249 recombinants are ready to be assayed in animal models of mammary and ovary tumour. This finding and the avaibility of a large number of scFv greatly increase the collection of potential receptors to which HSV can be redirected. II. The production and purification of recombinant truncated form of the heterodimer gHgL. We cloned a stable insect cell line expressing a soluble form of gH in complex with gL under the control of a metalloprotein inducible promoter and purified the heterodimer by means of ONE-STrEP-tag system by IBA. With respect to biological function, the purified heterodimer is capable: • of reacting to antibodies that recognize conformation dependent epitopes and neutralize virion infectivity; • of binding a variety cells at cell surface. No doubt, the availability of biological active purified gHgL heterodimer, in sufficient quantities, will speed up the efforts to solve its crystal structure and makes it feasible to identify more clearly whether gHgL has a cellular partner, and what is the role of this interaction on virus entry.

A core calculus for the analysis and implementation of biologically inspired languages

The application of Concurrency Theory to Systems Biology is in its earliest stage of progress. The metaphor of cells as computing systems by Regev and Shapiro opened the employment of concurrent languages for the modelling of biological systems. Their peculiar characteristics led to the design of many bio-inspired formalisms which achieve higher faithfulness and specificity. In this thesis we present pi@, an extremely simple and conservative extension of the pi-calculus representing a keystone in this respect, thanks to its expressiveness capabilities. The pi@ calculus is obtained by the addition of polyadic synchronisation and priority to the pi-calculus, in order to achieve compartment semantics and atomicity of complex operations respectively. In its direct application to biological modelling, the stochastic variant of the calculus, Spi@, is shown able to model consistently several phenomena such as formation of molecular complexes, hierarchical subdivision of the system into compartments, inter-compartment reactions, dynamic reorganisation of compartment structure consistent with volume variation. The pivotal role of pi@ is evidenced by its capability of encoding in a compositional way several bio-inspired formalisms, so that it represents the optimal core of a framework for the analysis and implementation of bio-inspired languages. In this respect, the encodings of BioAmbients, Brane Calculi and a variant of P Systems in pi@ are formalised. The conciseness of their translation in pi@ allows their indirect comparison by means of their encodings. Furthermore it provides a ready-to-run implementation of minimal effort whose correctness is granted by the correctness of the respective encoding functions. Further important results of general validity are stated on the expressive power of priority. Several impossibility results are described, which clearly state the superior expressiveness of prioritised languages and the problems arising in the attempt of providing their parallel implementation. To this aim, a new setting in distributed computing (the last man standing problem) is singled out and exploited to prove the impossibility of providing a purely parallel implementation of priority by means of point-to-point or broadcast communication.