Molecular Biology

Introducción: La obtención de cultivos celulares epiteliales corneales humanos podría ser de gran utilidad tanto para la investigación básica como para su aplicación clínica en el tratamiento de diversas enfermedades corneales. Material y Métodos: Muestras de limbo esclerocorneal procedentes de donante humano cadáver se pusieron en cultivo con medios específicos para células epiteliales con distintos factores de crecimiento. En la mitad de los cultivos se utilizaron células alimentadoras 3T3. Resultados: Se obtuvieron cultivos primarios de células epiteliales corneales con un alto grado de proliferación, sin importar si el crecimiento se dio sobre células 3T3 o no. La tasa de contaminación de los cultivos fue menor en aquellos donde sí se encontraban estas células alimentadoras. Discusión: La obtención de células epiteliales corneales es posible en el laboratorio a partir de pequeñas biopsias del limbo esclerocorneal tras su expansión en cultivo, utilizando una técnica de cultivo estándar, fácilmente reproducible. Palabras clave: limbo esclerocorneal, ingeniería tisular, epitelio corneal, cultivos celulares. El limbo esclerocorneal es una zona de transición entre la córnea y la esclerótica. Histológicamente, el epitelio del limbo es la zona que separa el epitelio columnar conjuntival del epitelio escamoso estratificado

X&Y chromosomal aneuploidies are among the most common human whole-chromosomal copy number changes, but the population-based incidence and prevalence in the childbearing population is unclear. This retrospective analysis of prospectively collected data leveraged a routine non-invasive prenatal test (NIPT) using parental genotyping to estimate the population-based incidence of X&Y chromosome variations in this population referred for NIPT (generally due to advanced maternal age). From 141,916 women and 29,336 men, 119 X&Y chromosomal abnormalities (prevalence: 1 in 1,439) were identified. Maternal findings include: 43 cases of 45,X (40 mosaic); 30 cases of 47,XXX (12 mosaic); 3 cases of 46,XX uniparental disomy; 2 cases of 46,XY/46, XX; 23 cases of mosaicism of unknown type; 2 cases of 47,XX,i(X)(q10). Paternal findings include: 2 cases of 47,XXY (1 mosaic); 10 cases of 47,XYY (1 mosaic); 4 partial Y deletions. Single chromosome aneuploidy was present in one of every 1,439 individuals considered in this study, showing 47,XXX; 47,XX,i(X)(q10); 47,XYY; 47,XXY, partial Y deletions, and a high level of mosaicism for 45,X. This expands significantly our understanding of X&Y chromosomal variations and fertility issues, and is critical for families and adults affected by

A coupled analysis of the reaction kinetics and microstructural changes of Fe 2 O 3 /Al 2 O 3 oxygen carriers during redox reaction cycles for chemical looping systems was conducted. The microstructural changes in the oxygen carrier particles were investigated using an image analysis method with cross-sectional backscattered electron images, and microstructural information such as the particle size, porosity, and two-phase boundary between the iron oxide and pores was obtained. The microstructural changes and the degradation kinetics during redox cycles were investigated under various operating conditions (reaction temperatures, reduction times, oxygen partial pressures during the oxidation process, and weight ratios of the oxygen carriers). The degradation coefficient and the reaction enthalpy of the particles in the oxidation process are linearly related, implying that increasing the local temperature of the oxygen carriers causes the coarsening of iron oxide particles via sintering and microstructural changes. Our analysis contributes to the design of highly stable oxygen carrier particles and the improvement of the operating conditions for chemical looping systems.

A coupled analysis of the reaction kinetics and microstructural changes of Fe 2 O 3 /Al 2 O 3 oxygen carriers during redox reaction cycles for chemical looping systems was conducted. The microstructural changes in the oxygen carrier particles were investigated using an image analysis method with cross-sectional backscattered electron images, and microstructural information such as the particle size, porosity, and two-phase boundary between the iron oxide and pores was obtained. The microstructural changes and the degradation kinetics during redox cycles were investigated under various operating conditions (reaction temperatures, reduction times, oxygen partial pressures during the oxidation process, and weight ratios of the oxygen carriers). The degradation coefficient and the reaction enthalpy of the particles in the oxidation process are linearly related, implying that increasing the local temperature of the oxygen carriers causes the coarsening of iron oxide particles via sintering and microstructural changes. Our analysis contributes to the design of highly stable oxygen carrier particles and the improvement of the operating conditions for chemical looping systems.

␤-alanine (Ala) betaine, an osmoprotectant suitable under saline and hypoxic environments, is found in most members of the halophytic plant family Plumbaginaceae. In Limonium latifolium (Plumbaginaceae), it is synthesized via methylation of ␤-Ala by the action of a trifunctional S-adenosyl l-methionine (Ado-Met): ␤-Ala N-methyltransferase (NMTase). Peptide sequences from purified ␤-Ala NMTase were used to design primers for reverse transcriptase-PCR, and several cDNA clones were isolated. The 5Ј end of the cDNA was cloned using a 5Ј-rapid amplification of cDNA ends protocol. A 500-bp cDNA was used as a probe to screen a -gt10 L. latifolium leaf cDNA library. Partial cDNA clones represented two groups, NMTase A and NMTase B, differing only in their 3Ј-untranslated regions. The full-length NMTase A cDNA was 1,414 bp and included a 1128-bp open reading frame and a 119-bp 5Ј-untranslated region. The deduced amino acid sequence of 375 residues had motifs known to be involved in the binding of Ado-Met. The NMTase mRNA was expressed in L. latifolium leaves but was absent in Limonium sinuatum, a member of the genus that lacks the synthetic pathway for ␤-Ala betaine. NMTase mRNA expression was high in young and mature leaves and was enhanced by light. NMTase cDNA was expressed in yeast (Saccharomyces cerevisiae) under the control of a galactose-inducible promoter. Protein extracts of galactose-induced recombinant yeast had Ado-Met-specific NMTase activities that were highly specific to ␤-Ala, N-methyl ␤-Ala, and N,N-dimethyl ␤-Ala as methyl acceptors. NMTase activities were not detectable in comparable protein extracts of yeast, transformed with vector control. The NMTase protein sequence shared homology with plant caffeic acid O-methyltransferases and related enzymes. Phylogenetic analyses suggested that ␤-Ala NMTase represents a novel family of N-methyltransferases that are evolutionarily related to O-methyltransferases.

Traditional materials from new sourcesconflicts in analytical methods for calcium carbonate. Calcium carbonate, (E170) is a common food and pharmaceutical additive/ingredient. In addition to a source of calcium, the carbonate has uses including as a colour, acidity regulator and bulking agent. Globally, a range of regulatory agencies and pharmacopoeia control the analyses and specification of additives in food, supplements, pharmaceutical substances and excipients. Accordingly, a range of specifications and analyses exist for calcium carbonate depending on the application and market of the product. In this contribution, we analyse calcium carbonates from geological, synthetic and biogenic sources, focussing on acid insoluble impurities, a test required by current monographs. Analysis of calcium carbonate from different origins may require modification of existing tests to comply with regulatory bodies, due to the variation of impurities specific to the source of the material. We suggest an analytical approach involving centrifugation that improves analytical efficiency (up to 85% time reduction), especially for calcium carbonate of biological origin.

New methods for peptide modification are in high demand in drug discovery, chemical biology and materials chemistry; methods that modify natural peptides are particularly attractive. A Pd-catalyzed, C-H functionalization protocol for the olefination of phenylalanine residues in peptides is reported, which is compatible with common amino acid protecting groups, and the scope of the styrene reaction partner is broad. Bidentate coordination of the peptide to the catalyst appears crucial for the success of the reaction.

We evaluated post-germination seedling vigor in indica and japonica rice under submergence based on shoot elongation and recovery after submergence. The japonica cultivars showed more vigorous shoot elongation than indica cultivars under submergence in the dark, whereas an opposite response was observed under the light-dark submergence. Both Nipponbare and FR13A that are respectively known as submergence intolerant and tolerant cultivars showed vigorous shoot growth under submergence and high recovery after the stress, irrespective of the light/dark conditions. The results show that vigorous shoot growth enables rice seedlings to escape and survive submergence stress. RT-PCR analysis showed that expression of SUB1A, which is known as a key gene controlling submergence tolerance, was induced in elongating seedlings of FR13A and other indica cultivars including floating rice under submergence with light illumination. Our results suggest that light-induced SUB1A gene expression does no...

The dimeric integral membrane protein nicotinamide nucleotide transhydrogenase is required for cellular regeneration of NADPH in mitochondria and prokaryotes, for detoxification and biosynthesis purposes. Under physiological conditions, transhydrogenase couples the reversible reduction of NADP C by NADH to an inward proton translocation across the membrane. Here, we present crystal structures of the NAD(H)-binding domain I of transhydrogenase from Escherichia coli, in the absence as well as in the presence of oxidized and reduced substrate. The structures were determined at 1.9-2.0 A ˚resolution. Overall, the structures are highly similar to the crystal structure of a previously published NAD(H)-binding domain, from Rhodospirillum rubrum transhydrogenase. However, this particular domain is unique, since it is covalently connected to the integralmembrane part of transhydrogenase. Comparative studies between the structures of the two species reveal extensively differing surface properties and point to the possible importance of a rigid peptide (PAPP) in the connecting linker for conformational coupling. Further, the kinetic analysis of a deletion mutant, from which the protruding b-hairpin was removed, indicates that this structural element is important for catalytic activity, but not for domain I:domain III interaction or dimer formation. Taken together, these results have important implications for the enzyme mechanism of the large group of transhydrogenases, including mammalian enzymes, which contain a connecting linker between domains I and II.

The it-1 gene is often activated by proviral insertion in mouse mammary tumors. Direct evidence for the normal function of this gene and its role in tumorigenesis has therefore been lacking. To examine possible biological effects of int-i activa- tion in in vitro cell systems, we have constructed recombinant molecules of genomic int-1 DNA, transcriptionally activated by retroviral promoters. Transfection of these constructs in- to cuboidal RAC311C mammary cells leads to morphological transformation of the cells and rapid tumorigenicity.

Over evolutionary time, pathogen challenge shapes the immune phenotype of the host to better respond to an incipient threat. The extent and direction of this selection pressure depend on the local pathogen composition, which is in turn determined by biotic and abiotic features of the environment. However, little is known about adaptation to local pathogen threats in wild animals. The Gentoo penguin (Pygoscelis papua) is a species complex that lends itself to the study of immune adaptation because of its circumpolar distribution over a large latitudinal range, with little or no admixture between different clades. In this study, we examine the diversity in a key family of innate immune genes—the Toll-like receptors (TLRs)—across the range of the Gentoo penguin. The three TLRs that we investigated present varying levels of diversity, with TLR4 and TLR5 greatly exceeding the diversity of TLR7. We present evidence of positive selection in TLR4 and TLR5, which points to pathogen-driven ad...

Several neurodegenerative diseases, like Alzheimer's (AD), are characterized by amyloid fibrillar deposition of misfolded proteins, and this feature can be exploited for both diagnosis and therapy design. In this paper, structural modifications of curcumin scaffold were examined in order to improve its bioavailability and stability in physiological conditions, as well as its ability to interfere with β-amyloid fibrils and aggregates. The acid-base behaviour of curcumin derivatives, their pharmacokinetic stability in physiological conditions, and in vitro ability to interfere with Aβ fibrils at different incubation time were investigated. The mechanisms governing these phenomena have been studied at atomic level by means of molecular docking and dynamic simulations. Finally, biological activity of selected curcuminoids has been investigated in vitro to evaluate their safety and efficiency in oxidative stress protection on hippocampal HT-22 mouse cells. Two aromatic rings, π-conju...

The heterotrimeric NF-Y complex is a pioneer factor that binds to CCAAT-genes and regulates their transcription. NF-Y cooperates with multiple transcription factors and co-regulators in order to positively or negatively influence gene transcription. The recruitment of NF-Y to CCAAT box is significantly enriched in cancer-associated gene promoters loci and positively correlates with malignancy. NF-Y subunits, in particular the DNA-binding subunit NF-YA and the histone-fold subunit NF-YC, appear overexpressed in specific types of cancer. Here we demonstrate that NF-Y subunits expression is finely regulated through transcriptional and post-translational mechanisms thus allowing control over basal expression levels. NF-Y negatively regulates the transcription of the genes encoding for its subunits. DNA pull-down/affinity purification assay coupled with Mass Spectrometry identified putative co-regulators, such as Lamin A, involved in NF-YA gene transcription level. We also evidentiate ho...

A mitochondrial RNA splice defect in the first intron of the COB gene (bI1) can be suppressed by a dominant nuclear mutation SUP-IO1. Starting with a gene bank of yeast nuclear DNA from a SUP-101 suppressor strain cloned in the YEpl3 plasmid, we have isolated a recombinant plasmid which exerts a suppressor activity similar to the SUP-IO1 allele. The N3(2) insert of this plasmid contains an open reading frame (ORF) of 1014 bp which is transcribed to a 12 S RNA. Deletion of the 5' end of this ORF and its upstream sequences abolishes the suppressor activity. The N3(2) insert thus carries a functional gene (called MRS3) which can suppress a mitochondrial splice defect. The chromosomal equivalent of the cloned gene has been mapped to chromosome 10. Disruption of this chromosomal gene has no phenotypic effect on wild-type cells.

Résumé -Saprochaete suaveolens est une levure non conventionnelle (LNC) appréciable pour sa haute productivité en composés organiques volatils (COVs), tant d'un point de vue quantitatif que qualitatif. Cette souche est notamment très intéressante pour sa production d'esters α-insaturés rarement retrouvés dans le bouquet aromatique des souches levuriennes. Le métabolisme de cette souche peut ainsi être considéré comme un modèle très prometteur pour la production de COVs par les levures. Cette étude visait donc à l'optimisation de la production de COVs par la S. suaveolens par une méthode de mutagenèse aléatoire. Ainsi, après avoir mis au point une méthode de mutagenèse UV adaptée à la souche de S. suaveolens, une méthode rapide de sélection des mutants d'intérêts a été élaborée. Cette méthode a permis de sélectionner 9 souches mutantes parmi plus de 15 000 clones irradiés. Les analyses de COVs produits par ces 9 souches ont mis en exergue un mutant produisant 8 fois plus de COVs que la souche sauvage de S. suaveolens. Les analyses biochimiques ont montré que cette surproduction résultait de la perte d'activité de l'Enoyl-CoA hydratase et d'une augmentation de l'activité de l'acyl-CoA hydrolase chez le mutant.

Adverse health effects caused by heavy metal toxicity, particularly lead, commonly manifest in children due to their higher absorption rate and frequent exposure from play areas such as school grounds. The aim of this study is to: (1) determine lead levels in soil and plant samples and compare to US EPA acceptable limits; and (2) assess health risk by determining if an association exists between the occurrence of physical and neurological symptoms of lead poisoning among the pre-school children in Manila (exposed group) and pre-school children in Bataan (unexposed group). Using flame absorption spectroscopy, it was found that lead levels in plant species and soil samples ranged from 0.11-0.16 ppm and 1.12-2.59, respectively. These were still within the limits set by US EPA at 300 ppm (plants) and 400 ppm (soil). However, if accidentally ingested, the amount absorbed would exceed serum safe level of 10 mcg/dl, leading to possible toxicity. On the other hand, the cross-sectional surve...

A monoclonal antibody was used to characterize a 135-kDa surface-located membrane protein (Lmp1) generally present in Mycoplasma hominis strains. The monoclonal antibody, 552, was applied to identify the corresponding gene in an expression library of M. hominis PG21 DNA. The M. hominis PG21 lmp1 gene was sequenced, and its gene product was characterized with the goal of elucidating the structure and function of Lmp1. A total of 7,196 bp in the lmp1 region was sequenced. An open reading frame of 4,032 bp, encoding a protein of 1,344 amino acids with a calculated molecular weight of 147,000, was identified. Analysis of the deduced amino acid sequence predicted a hydrophilic protein with a basic pI (10.0). The N-terminal 24 amino acids were a typical leader sequence. Downstream from the first 726 nucleotides, six similar direct repeats of 471 nucleotides were found. In repeat 7, a single-base substitution, C-->A, gave rise to the stop codon of lmp1. Thus, the C-terminal 945 amino ac...

Follistatin (FST) is a cysteine-rich autocrine glycoprotein and plays an important role in mammalian prenatal and postnatal development. FST binds to and inhibit myostatin (MSTN), a potent negative regulator of skeletal muscle growth, and FST abundance enhances muscle growth in animals via inhibition of MSTN activity. The objective of this study was to produce biologically active, four chicken FST-type proteins in an Escherichia coli expression system. Gibson assembly cloning method was used to insert the DNA fragments of four FST-type proteins, designated as FST288, NDFSD1/2, NDFSD1, and NDFSD1/1, into pMALc5x vector downstream of the maltose-binding protein (MBP) gene, and the plasmids containing the inserts were eventually transformed into Shuffle E. coli strain for protein expression. We observed a soluble expression of the four MBP-fused FST-type proteins, and the proteins could be easily purified by the combination of amylose and heparin resin affinity chromatography. MBP-fuse...

Background: Sequence similarity searching is an important and challenging task in molecular biology and next-generation sequencing should further strengthen the need for faster algorithms to process such vast amounts of data. At the same time, the internal architecture of current microprocessors is tending towards more parallelism, leading to the use of chips with two, four and more cores integrated on the same die. The main purpose of this work was to design an effective algorithm to fit with the parallel capabilities of modern microprocessors. A parallel algorithm for comparing large genomic banks and targeting middle-range computers has been developed and implemented in PLAST software. The algorithm exploits two key parallel features of existing and future microprocessors: the SIMD programming model (SSE instruction set) and the multithreading concept (multicore). Compared to multithreaded BLAST software, tests performed on an 8-processor server have shown speedup ranging from 3 to 6 with a similar level of accuracy. A parallel algorithmic approach driven by the knowledge of the internal microprocessor architecture allows significant speedup to be obtained while preserving standard sensitivity for similarity search problems.

Franklin's scientific legacy is rooted in her pioneering use of X-ray crystallography-a technique that not only visualizes molecular structure but captures resonant interference patterns created by coherent waveforms interacting with organized matter. Her most iconic contribution, Photo 51, revealed the double-helical nature of DNA, a structure now understood not just as a biochemical code but a vibrational lattice carrying harmonic information-what ORT terms bioresonant geometry. 1.2 The Tobacco Mosaic Virus: Viral Architecture as Field Harmonics Franklin's later work on the Tobacco Mosaic Virus (TMV) demonstrated that even RNA-based organisms exhibit helical symmetry, a key principle in resonance field coherence. She identified that TMV is a single strand of RNA arranged helically within a cylindrical protein shell, confirming that even simple pathogens adhere to harmonic organization.

Five isostructural lanthanide-organic coordination frameworks with a unique 3-D 5-connected (4(7).6(3))(4(3).6(5).8(2)) network, namely, [Ln(phen)(L)]n (Ln = Dy for 1, Gd for 2, Ho for 3, Er for 4, and Tb for 5), have been prepared based on bridging 5-hydroxyisophthalic acid (H3L) and chelating 1,10-phenanthroline (phen) coligand. Significantly, the Dy(III) complex 1 is an organized array of single-molecular magnets (SMMs), with frequency-dependent out-of-phase ac susceptibility signals and magnetization hysteresis at 4 K. Further analysis of the magnetic results can reveal that the SMM behavior of 1 should arise from the smaller ferromagnetic interaction between the Dy(III) ions. Complex 1 was also characterized by X-ray absorption spectra, which give the clear X-ray magnetic circular dichroism signal.

This synthesis expands the role of ghrelin in taste cell function into a broad Omniological Resonance Theory (ORT) context, postulating that peripheral sensory reception is modulated by dynamic field harmonics linked to endocrine-emotive flux. Ghrelin's influence on sodium and sour tastant response reflects a local resonance adaptation mechanism, where ghrelin acts as a biochemical phase-locking agent between somatic oscillations and cognitive pattern recognition. This synthesis interlinks with ORT modules including field nutrition, tastememory circuits, neurosomatic feedback loops, and resonance-based cognition.

Identifying animal species used in osseous industry production is crucial for reconstructing humananimal interactions in ancient societies. However, bone artefact manufacture often involves intensive modifications to raw materials that hamper taxonomic identifications. Here, for the first time in central Eurasia, we taxonomically assess bone objects stored in museum collections, recovered from Late Neolithic to Iron Age contexts in Armenia, using a minimally invasive peptide mass fingerprinting technique, also known as Zooarchaeology by Mass Spectrometry (ZooMS). Our pilot study shows remarkable collagen preservation in the bone artefacts, demonstrating the rich potential of ZooMS for examining legacy collections. The successful ZooMS screening provided taxonomic identification for 86% of the artefacts, offering insights into species selection for bone manufacturing, as well as broader socioeconomic developments and interregional links. Our study underscores the utility of minimally invasive proteomic techniques, enabling the preservation of cultural and historical artefacts while addressing limitations of studying museum collections. 1234567890():,; 1234567890():,;

Oxidation of DNA by reactive oxygen species (ROS) yields 8-oxo-7,8-dihydroguanosine (8-oxodG) as primary oxidation product, which can lead to downstream G to T transversion mutations. DNA mutations are nonrandom, and mutations at specific codons are associated with specific cancers, as widely documented for the p53 tumor suppressor gene. Here, we present the first direct LC-MS/MS study (without isotopic labeling or hydrolysis) of primary oxidation sites of p53 exon 7. We oxidized a 32 base pair (bp) double-stranded (ds) oligonucleotide representing exon 7 of the p53 gene. Oxidized oligonucleotides were cut by a restriction endonuclease to provide small strands and enable positions and amounts of 8-oxodG to be determined directly by LC-MS/MS. Oxidation sites on the oligonucleotide generated by two oxidants, catechol/Cu 2+ / NADPH and Fenton's reagent, were located and compared. Guanines in codons 243, 244, 245, and 248 were most frequently oxidized by catechol/Cu 2+ /NADPH with relative oxidation of 5.6, 7.2, 2.6, and 10.7%, respectively. Fenton's reagent oxidations were more specific for guanines in codons 243 (20.3%) and 248 (10.4%). Modeling of docking of oxidizing species on the dsoligonucleotide were consistent with the experimental codon oxidation sites. Significantly, codons 244 and 248 are mutational "hotspots" in nonsmall cell and small cell lung cancers, supporting a possible role of oxidation in p53 mutations leading to lung cancer.

Sete amostras citopáticas do vírus da Diarréia Viral Bovina (BVDV) isoladas de casos clínicos e do sangue de bezerros de rebanhos com problemas reprodutivos foram analisadas. Todas as amostras caracterizadas possuíam uma mistura de vírus citopáticos (cp) e não-citopáticos (ncp), que foram clonados biologicamente, originando populações puras de vírus de cada biotipo. Os clones cp e ncp obtidos foram caracterizados antigenicamente com um painel de anticorpos monoclonais (MAbs) e quanto à expressão da proteína não-estrutural NS3. A análise de reconhecimento pelos MAbs revelou dois padrões de reatividade: 1. Em cinco casos, os vírus cp e ncp de uma mesma amostra mostraram-se antigenicamente muito semelhantes entre si, indicando tratar-se de verdadeiros "pares" de vírus, nos quais o vírus cp origina-se do ncp através de mutações ou recombinações; 2. Duas amostras, no entanto, continham vírus cp e ncp com diferenças antigênicas consideráveis entre si. A análise de polipeptídeos ...

in Paramecium au&a indicated that as fission age increased the amount of macronuclear DNA decreased. It was also found that the amount of RNA synthesis as determined by the in vivo incorporation of [aH]uridine decreased as the fission age increased. An alternative in situ assay of the DNA template activity determined by the RNA polymerase-catalyzed incorporation of rH]UTP is described. The DNA template activity of older cells was shown to be significantly lower on a per cell basis than that of younger cells. The majority of this reduction was shown to be due to the gradual loss of DNA template with an increase in fission age. The specific activity of the DNA template, however, does show a small but significant decrease as the fission age of the cell increases.

OBJETIVO: Verificar a correlação entre os polimorfismos dos genes MBL2, TGF-β1 e CD14 com a gravidade da doença pulmonar em pacientes com fibrose cística (FC), bem como correlacionar a presença dos alelos ΔF508 com a gravidade da doença naqueles pacientes. MÉTODOS: Estudo clínico-laboratorial, de corte transversal, com 105 pacientes fibrocísticos de um hospital universitário em 2005-2006. Foram analisados 202 doadores de sangue saudáveis como controles para a pesquisa dos polimorfismos no gene TGF-β1 e CD14. A análise de polimorfismos nos genes MBL2 e TGF-β1 no códon 10, posição +869, foi realizada pela técnica da PCR alelo-específica. A genotipagem do polimorfismo C-159T no gene CD14 foi realizada através de PCR e digestão enzimática. RESULTADOS: Dos 105 pacientes com FC avaliados, 67 apresentavam doença pulmonar grave segundo o escore de Shwachman. Os polimorfismos do gene MBL2 não foram associados com a gravidade da doença nos fibrocísticos. A análise do polimorfismo T869C no gen...

The objective of this study was to investigate the effect(s) of bovine herpesvirus 1 (BHV-1) infection on the expression of MHC class I molecules in murine fibroblasts. L-cells were infected with BHV-1 at a multiplicity of infection (m.o.i.) of 10 plaque forming units (PFU) per cell, and the expression of MHC class I molecules was analyzed by flow cytometry and immunoprecipitation. Temporal studies revealed a reduction in class I expression beginning at 8 h post infection (p.i.) which reached a maximum between 10 to 16 h p.i. The loss of class I expression was restored in the presence of phosphonoacetic acid (30 p-g/ml), a late herpesviral protein synthesis inhibitor. However, addition of cycloheximide, a total protein synthesis inhibitor (100 ixg/ml), did not result in any difference in class I expression between virus-infected and mock-infected cells. These results suggest that the reduced class I expression is a direct consequence of BHV-1 infection, and that the late viral gene product(s) may be involved in this process. Similar phenomena may occur in natural BHV-1 infection in cattle, and this may be one of the mechanisms of immune suppression by

The successful prophylactic treatment of hemophilia A by frequent infusions of plasma concentrates or recombinant factor VIII (hFVIII) indicates that gene therapy may be a potential alternative for the treatment of the disease. For efficient delivery and long-term expression of the hFVIII gene, a novel minimal adenovirus (mini-Ad) vector, MiniAdFVIII, has been developed. The vector is devoid of all viral genes and carries the full-length hFVIII cDNA under the control of the human 12.5-kb albumin promoter. The MiniAdFVIII vector was propagated with the assistance of an ancillary vector in 293 cells and was purified by CsCl banding. Sustained expression of hFVIII at physiologic levels (100-800 ng/mL) was achieved in mice after a single intravenous injection of MiniAdFVIII. The expressed hFVIII had a structure identical to that of recombinant hFVIII, as determined by Western blot analysis. The functionality of the protein was confirmed by the restoration of blood coagulation capacity i...

The objective of this study was to investigate the effect(s) of bovine herpesvirus 1 (BHV-1) infection on the expression of MHC class I molecules in murine fibroblasts. L-cells were infected with BHV-1 at a multiplicity of infection (m.o.i.) of 10 plaque forming units (PFU) per cell, and the expression of MHC class I molecules was analyzed by flow cytometry and immunoprecipitation. Temporal studies revealed a reduction in class I expression beginning at 8 h post infection (p.i.) which reached a maximum between 10 to 16 h p.i. The loss of class I expression was restored in the presence of phosphonoacetic acid (30 p-g/ml), a late herpesviral protein synthesis inhibitor. However, addition of cycloheximide, a total protein synthesis inhibitor (100 ixg/ml), did not result in any difference in class I expression between virus-infected and mock-infected cells. These results suggest that the reduced class I expression is a direct consequence of BHV-1 infection, and that the late viral gene product(s) may be involved in this process. Similar phenomena may occur in natural BHV-1 infection in cattle, and this may be one of the mechanisms of immune suppression by

Inflammatory bowel disease (IBD) is an inflammatory condition with mucosal ulceration, edema and hemorrhage of gastrointestinal tract. Curcumin has been shown to mitigate colitis in animal models. However, its usefulness is reduced due to poor pharmacokinetic behavior and low oral bioavailability. To address this, novel pH-sensitive hydrolyzed polyacrylamide-grafted-xanthan gum (PAAm-g-XG) nanoparticles (NPs) loaded with curcumin were prepared for colonic delivery. Optimized nanoparticles (CN20) were spherical, with an average size of 425 nm. A negligible amount of curcumin (≈8%) was released from CN20 NPs in pH 1.2 and 4.5 solutions. When the pH was increased to 7.2, curcumin release was comparatively faster than that observed with pH 1.2 and 4.5 collectively. In pH 6.8 solution, excellent release of curcumin was observed. Highest curcumin release was observed when rat caecal contents were incorporated in pH 6.8 solution, indicating microflora-dependent drug release property of NPs. In acetic acid-induced IBD in rats, curcumin NPs reduced myeloperoxidase and nitrite levels, prevented weight loss and attenuated colonic inflammation. Curcumin was better absorbed systemically in nanoparticulate form with increased C max (~3 fold) and AUC (~2.5 fold) than when delivered as free curcumin. We demonstrate successful development of grafted co-polymeric NPs containing drug suitable for colon targeting.

AID is critical for immunoglobulin class switch DNA recombination (CSR) and somatic hypermutation (SHM). Here we showed that AID expression was induced by the HoxC4 homeodomain transcription factor, which bound to a highly conserved HoxC4-Oct site in the Aicda promoter. This site functioned in synergy with a conserved Sp-NF-κB-binding site. HoxC4 was preferentially expressed in germinal center B cells and was upregulated by CD154:CD40 engagement, lipopolysaccharide and interleukin-4. HoxC4 deficiency resulted in impaired CSR and SHM, due to decreased AID expression and not other putative HoxC4-dependent activity. Enforced expression of AID in Hoxc4 -/-B cells fully restored CSR. Thus, HoxC4 directly activates the Aicda promoter, thereby inducing AID expression, CSR and SHM. CSR and SHM are critical for the maturation of antibody responses to foreign and selfantigens. CSR recombines switch (S) region DNA located upstream of constant heavy chain (C H ) region exons, thereby changing immunoglobulin (Ig) C H regions and endowing antibodies with new biological effector functions. SHM introduces mainly point mutations in Ig variable regions, thereby providing the structural substrate for selection of higher affinity antibody mutants by antigen. In spite of the recent advances made in the identification of some factors involved in CSR and SHM, the intimate mechanisms of these processes remain elusive. Both CSR and SHM require activation-induced cytidine deaminase (AID), which is expressed by activated B cells, mainly in germinal centers (GCs) of peripheral lymphoid organs1,2. AID initiates CSR and SHM by deaminating dC residues to yield dU:dG mispairs in DNA3-8. These dU:dG mispairs trigger DNA repair processes entailing introduction of mutations in V(D)J regions or DNA breaks, including doublestranded DNA breaks, which lead to non-classic non-homologous end-joining and CSR3,5,9-14. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Neonatal hypoxic-ischaemic encephalopathy (HIE) is major cause of neonatal mortality and morbidity. Therapeutic hypothermia is standard clinical care for moderate hypoxic-ischaemic (HI) brain injury, however it reduces the risk of death and disability only by 11% and 40% of the treated infants still develop disabilities. Thus it is necessary to develop supplementary therapies to complement therapeutic hypothermia in the treatment of neonatal HIE. The modified Rice-Vannucci model of HI in the neonatal mouse is well developed and widely applied with different periods of hypothermia used as neuroprotective strategy in combination with other agents. However, different studies use different periods, time of initiation and duration of hypothermia following HI, with subsequent varying degrees of neuroprotection. So far most rodent data is obtained using exposure to 5-6h of therapeutic hypothermia. Our aim was to compare the effect of exposure to three different short periods of hypothermia (1h, 1.5h and 2h) following HI insult in the postnatal day 7 C57/Bl6 mouse, and to determine the shortest period providing neuroprotection. Our data suggests that 1h and 1.5h of hypothermia delayed by 20min following a 60min exposure to 8%O 2 do not prove neuroprotective. However, 2h of hypothermia significantly reduced tissue loss, TUNEL+ cell death and microglia and astroglia activation. We also observed improved functional outcome 7 days after HI. We suggest that the minimal period of cooling necessary to provide moderate short term neuroprotection and appropriate for the development and testing of combined treatment is 2h.

Little is known about sex differences in healthy life expectancy among the oldest old, the fastest growing segment of the older population. This study examines sex differences in total, healthy and unhealthy life expectancy among nonagenarians. Methods: Longitudinal data of 884 older adults aged 90 and over participating in the Vitality 90+ study (Tampere, Finland) were used, including 2,501 observations (health or death states) from 5 measurement waves between 2001 and 2014. Using the MSM and ELECT packages in R, multistate survival models were performed to estimate the transition probabilities of older adults through the different health states and to calculate life expectancies. The analyses were done separately for two health indicators (disability and multimorbidity) to see whether patterns were consistent. Results: Women had higher total life expectancies than men (about 8 months), but also higher unhealthy life expectancies. Men had a higher disability-free life expectancy between the age of 90 and 95 compared to women. For multimorbidity, no sex differences in healthy life expectancy were found. This study showed that the male-female health-survival paradox remains at very old age. Women aged 90+ live longer than men, and spend more time in poor health.

Possest Calculus and Geometry constitute a unified philosophical-mathematical system that reframes empiricity, not as the outcome of inference, optimization, or representation, but as rhythmic persistence through reorganizational tension. Moving beyond filtration theory (Schimmelpfennig) and traditional analytic methods, Possest introduces a post-Newtonian, post-topological foundation based on survival-through-resonance. This synthesis formalizes the mathematical core of Possest, articulating key operators, geometric principles, algebraic functions, simulations, and applications within the broader framework of Omniological Resonance Theory (ORT).

The α3(V) chain is poorly characterized among type V collagen chains. Pro-α3(V) collagen is expressed in newly synthesized bone as well as in the superficial fascia of developing muscle. Present study examined the expression in a mouse model of wound healing. Real-time reverse transcriptase polymerase chain reaction and in situ hybridization revealed transient expression of pro-α3(V) chain at a lower level than other fibrillar collagen genes after injury. Immunohistochemistry showed a similar expression pattern in the injured skin. In addition, electron microscopy showed that pro-α3(V) chain was localized in the amorphous nonfibrillar region, but not in fine or dense fibrils. The pro-α3(V) chain co-localized with heparan sulfate, which appeared in the skin after injury and might bind via an acidic segment of the pro-α3(V) chain. The matrix containing the pro-α3(V) chain may therefore be needed for the initiation of wound healing.

Background: Acute myeloid leukemia, as most cancers, results from exposure to carcinogens and an impaired inherited individual capacity to eliminate xenobiotics. The present case-control study measures the relationship between glutathione S-transferase (GST) T1 and M1 null genotypes and the risk of acute myeloid leukemia. Methods: We identified the GSTT1 and GSTM1 genotypes by multiplex polymerase chain reaction in 129 acute myeloid leukemia patients and 129 controls. Results: Individuals that carried GSTT1 null had a risk of acute myeloid leukemia when compared to GSTT1 present carriers (OR: 2.80; 95% CI: 1.63-4.80, P=0.00036). However, GSTM1 null did not influence the risk for acute myeloid leukemia (OR: 1.20; 95% CI: 0.72-1.97, P=0.53). The combined GSTT1 null/GSTM1 present genotype showed an association with the risk for acute myeloid leukemia compared to those that carried both functional genotypes (OR: 8.85; 95% CI: 3.09-23.8, P=0.0001). The double null genotype also showed an association with the risk for acute myeloid leukemia (OR: 2.32, 95% CI: 1.15-4.66, P=0.019). Conclusion: Both GSTT1 null and GST double-null genotypes may be risk factors for acute myeloid leukemia. Further studies are needed to confirm these results.