Differential Expression of Proteins in an Atypical Presentation of Autoimmune Lymphoproliferative Syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is a rare disease defined as a defect in the lymphocyte apoptotic pathway. Currently, the diagnosis of ALPS is based on clinical aspects, defective lymphocyte apoptosis and mutations in Fas, FasL and Casp 10 genes. Despite this, ALPS has been misdiagnosed. The aim of this work was to go one step further in the knowledge of the disease, through a molecular and proteomic analysis of peripheral blood mononuclear cells (PBMCs) from two children, a 13-year-old girl and a 6-year-old boy, called patient 1 and patient 2, respectively, with clinical data supporting the diagnosis of ALPS. Fas, FasL and Casp10 genes from both patients were sequenced, and a sample of the total proteins from patient 1 was analyzed by label-free proteomics. Pathway analysis of deregulated proteins from PBMCs was performed on the STRING and PANTHER bioinformatics databases. A mutation resulting in an in-frame premature stop codon and protein truncation was detected in the Fas gene from patient 2. From patient 1, the proteomic analysis showed differences in the level of expression of proteins involved in, among other processes, cell cycle, regulation of cell cycle arrest and immune response. Noticeably, the most down-regulated protein is an important regulator of the cell cycle process. This could be an explanation of the disease in patient 1.     

Optimization of the material flow in a manufacturing plant by use of artificial bee colony algorithm

To survive in today’s competitive global market, companies must perform strategic changes in order to increase productivity, eliminating wasted materials, time, and effort. This study will examine how to optimize the time and effort required to supply raw material to different production lines in a manufacturing plant in Juarez, Mexico by minimizing the distance an operator must travel to distribute material from a warehouse to a set of different production lines with corresponding demand. The core focus of this study is similar to that of the Vehicle Routing Problem in that it is treated as a combinatorial optimization problem. The artificial bee colony algorithm is applied in order to find the optimal distribution of material with the aim of establishing a standard time for this duty by examining how this is applied in a local manufacturing plant. Results show that using this approach may be convenient to set standard times in the selected company.     

CFD Modeling and Analysis of The Flow,Heat Transfer and Mass Transfer in a Blast Furnace Hearth

Understanding the complex phenomena in the BF hearth is essential to increasing furnace productivity and to extending furnace campaign. Numerical modeling provides a cost‐effective tool to obtain such knowledge. We have developed several continuum‐based mathematical/numerical models to simulate the flow, heat transfer and mass transfer in the lower part of BF and in the hearth. These models have generated an improved insight into the mechanisms for liquid drainage efficiency, lining erosion and wall protection in BF hearth under operational conditions. The current paper provides an overview of these studies, as well as dealing with three specific aspects: (a) Gas flow and pressure on the liquid surface, and its effect on the drainage characteristics; (b) Flow and temperature distributions of liquid iron in the hearth, and the temperature distribution in the refractories; and (c), Titania injection to form Ti(C,N)‐rich scaffolds on the hearth refractory surface, to protect the hearth from erosion.     

Outbreak of pneumonia in a long-term care facility: antecedent human parainfluenza virus 1 infection may predispose to bacterial pneumonia

OBJECTIVES: To determine the causes of an outbreak of lobar pneumonia. DESIGN: Matched (1:2) case-control study. SETTING: A 70-bed chronic care facility for older people. PARTICIPANTS: Residents of the facility. RESULTS: Ten residents developed pneumonia over a 10-day period. Two residents died. One case-patient had Streptococcus pneumoniae bacteremia; another had polymerase chain reaction evidence of S. pneumoniae infection. No other etiologic agent was identified. Only four of 10 case-patients had received routine diagnostic cultures of blood or sputum before the administration of antibiotics. Symptoms of upper respiratory illness (URI) among residents before the pneumonia outbreak corresponded with elevation of antibodies to human parainfluenza virus 1 (HPIV1). In a matched case-control study, six of 10 case-patients, compared with five of 20 controls, had symptoms of URI during the preceding month (matched odds ratio (MOR) = 4.5, 95% CI = 0.8-33). Nine case-patients had serum available, and five of these had both serologic evidence of recent HPIV1 infection and recent URI, compared with two of 18 controls (MOR = 9.0, 95% CI = 1.2-208). Only three residents had documentation of pneumococcal vaccination. CONCLUSIONS: Noninfluenza viral infections may play a role in the pathogenesis of some bacterial pneumonias. S. pneumoniae was the cause of at least two pneumonias; lack of preantibiotic cultures may have interfered with isolation of S. pneumoniae in others. Recent HPIV1 infection was epidemiologically linked to subsequently developing pneumonia. Spread of HPIV1 in the facility may have contributed to increased susceptibility to S. pneumoniae and, potentially, to other bacterial pathogens.     

T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates

Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.     

Establishment of primary cell culture from stria vascularis explants. Morphological and functional characterization

To provide the prerequisite for long-term study of the inner ear related to structural and functional integrity, tissue of stria vascularis with spiral ligament was isolated from Wistar rat cochleas and cultured using the explant-culture technique. The following culture media were used: EMEM with Hepes buffer, hydrocortisone (400 ng/ml), transferrin (5 micrograms/ml). triiodothyronine (10(-9) M), cholera toxin (10(-10) M), insulin (5 micrograms/ml), and epidermal growth factor (10 ng/ml). To characterize the cells growing out from the explant, immunofluorescence with cytokeratin (cytokeratin 18) and ultrastructural examination with SEM and TEM were performed. The marginal cell function was investigated by expression of Na+, K(+)-ATPase antisera against beta 2 subunit of rat Na+, K(+)-ATPase and P-NPPase. We were able to maintain the cultured cells for 3 weeks or more. Monolayered marginal cells were observed beyond 14 days in vitro and the expression of cytokeratin 18 was especially enhanced. The cultured marginal cells were almost identical to in vivo cells both as regards ultrastructural features and Na+, K(+)-ATPase activity. The present results suggest that the primary explant culture technique is a reliable in vitro model of strial marginal cells. However, establishment of the cell line is needed for long-term study.     

The association rate constant for heme binding to globin is independent of protein structure

Rate constants for CO-heme binding to 35 different recombinant apomyoglobins and several other apoproteins were measured in an effort to understand the factors governing heme affinity and the velocity of the association reaction. Surprisingly, the rate constant for the binding of monomeric heme is approximately 1 x 10(8) M-1 s-1 regardless of the structure or overall affinity of the apoprotein for iron-porphyrin. Major differences between the proteins are reflected primarily in the rates of dissociation of the prosthetic group. Slow phases observed in the reaction of CO heme with excess apomyoglobin result from formation of nonspecific heme-protein complexes which must dissociate before heme can bind specifically in the heme pocket. Once the specific heme-globin complex is formed, the heme pocket rapidly collapses around the porphyrin, simultaneously forming the bond between the proximal His93 and the heme iron atom. The overall affinity of sperm whale apomyoglobin for hemin is approximately 1 x 10(14) M-1. Nonspecific hydrophobic interactions between the porphyrin and the apolar heme cavity account for a factor of 10(5)-10(7). Covalent bond formation between Fe3+ and His93(F8) provides an additional factor of 10(3)-10(4). Specific interactions with conserved amino acids in the heme pocket contribute the final factor of 10(3)-10(4).     

Novel NMDA antagonists: replacement of the pyridinium ring of 6,11-ethanobenzo[b]quinolizinium cations with heteroisoquinolinium cations

Replacement of the pyridinium ring of 6,11-ethanobenzo[b]quinolizinium cations with thiazolium (4a and 4b) and N-methylimidazolium (4c and 4d) resulted in equipotent compounds in the [3H]TCP binding assay. The corresponding N-methyl-1,2,4-triazolium analogs were less potent in this assay. The thiazolium derivative 4b, with a Ki = 2.9 nM, is being evaluated as a possible neuroprotective N-methyl-D-aspartic acid (NMDA) antagonist.     

Protein-nucleic acid interactions in bacteriophage phi 29 DNA replication

phi 29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the phi 29 DNA ends. Structure-function studies have been carried out in the phi 29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of phi 29 DNA polymerase contains the 3'-5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of phi 29 DNA replication. The structure of the protein p6-DNA complex has been determined, as well as the main signals at the phi 29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The phi 29 protein p5 is the single-stranded DNA binding (SSB) protein involved in phi 29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the phi 29 SSB-ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19,285-bp phi 29 DNA molecule by a factor of 4000 after 1 h of incubation at 30 degrees C. The infectivity of the in vitro amplified DNA was identical to that of phi 29 DNA obtained from virions.