The human fibroblast and human immune interferon genes and their expression in homologous and heterologous cells

The genetic information coding for human fibroblast interferon (IFN-beta) has been cloned both as a DNA copy (cDNA) and as a genomic clone. Human IFN-beta is made as a precursor and consists of a signal sequence 21 amino acid residues long followed by the mature protein 166 amino acids long. A single site for glycosylation is present. The human IFN-beta gene does not contain introns. Transfection of monkey cells with a chimeric SV40 derivative containing the human IFN-beta cDNA clone under control of the late SV40 promoter leads to secretion of high levels of IFN-beta. When a genomic clone is used in the same vector, IFN-beta synthesis can be further enhanced up to 30-fold by treatment with poly(rI) . poly(rC); this shows that a cis-active control element is present in the clone. An efficient expression system in Escherichia coli was worked out based on a plasmid containing the promoter PL of bacteriophage lambda, which is regulated by a temperature-sensitive repressor. This promoter is followed by a segment derived from bacteriophage MS2 that contains the ribosome-binding site of the replicase gene. The latter, however, is replaced by the human IFN-beta gene. Upon induction, high levels (about 5 x 10(9) IU 1(-1)) of IFN-beta are synthesized by the bacteria; this corresponds to about 2% of the total bacterial protein. The human immune (type II) interferon (IFN-gamma) gene has similarly been cloned. Partly purified mRNA derived from human spleen cells that had been induced with staphylococcal enterotoxin A was used as starting material. A full-length cDNA clone was sequenced. The total cDNA sequence is about 1150 nucleotides long; it contains a single open reading frame coding for 166 amino acids, the first 20 of which constitute the transmembrane signal. There are two sites for glycosylation. The amino acid sequence is quite different from that of IFN-alpha or IFN-beta, although a few similarities can be noted. The untranslated 3'-terminal region is about 550 nucleotides long. The IFN-gamma gene was expressed in monkey cells, again by using the SV40-derived vector, and the secreted product was characterized as true human IFN-gamma. A genomic clone in the form of a bacteriophage lambda derivative was also obtained. The IFN-gamma gene extends over at least 5 kilobases and contains at least two introns.     

Leptin receptor activation depends on critical cysteine residues in its fibronectin type III subdomains

The leptin receptor (LR) complex is composed of a single subunit belonging to the class I cytokine receptor family and exists as a preformed complex. The extracellular portion contains two cytokine receptor homology (CRH) domains, separated by an Ig-like domain and followed by two membrane-proximal fibronectin type III (FNIII) domains. The mechanisms underlying ligand-induced receptor activation are still poorly understood. LRs can exist as disulfide-linked dimers at the cell surface, even in the absence of leptin. We evaluated the role of the two unpaired cysteine residues (Cys-672 and Cys-751) in the FNIII domains in receptor clustering, leptin binding, and biological activity. Although mutation of cysteine on position 751 to serine has hardly any effect on ligand binding and receptor activation, the C672S mutant exhibits a marked reduction in STAT3-dependent signaling. The double mutant was completely devoid of biological activity, although leptin binding remained unaffected. Mutation of both residues resulted in complete loss of disulfide bridge formation of FNIII domains in solution. In contrast, no difference was observed in ligand-independent oligomerization of the membrane-bound receptor, suggesting a role for cysteines in the CRH2 domain in formation of the preformed LR complex. We propose a model wherein leptin-induced clustering of two preformed dimers forms the activated LR complex. Disulfide bridge formation involving Cys-672 and Cys-751 may be necessary for JAK activation and hence signaling.     

Morphological characterization of a human glioma cell l ine

A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells presented their surfaces dominated by microvilli.     

How formalin affects the outcome of routine and special stains

The discovery of formaldehyde for preserving tissue structures produced a new dimension in microscopy. Preserving structure and morphology became important; therefore, identifying a proper fixing agent for particular structures, chemical entities, and tissues, also became important. The methods for demonstrating tissue structures evolved and were implemented with careful observation and documentation of the results and outcomes. Formalin was incorporated into many techniques, and provided helpful results in many cases and hindrances in others. The effects of formalin on the outcomes of routine and special staining techniques are reported here.     

Wildtool,a flexible,first-line risk assessment system for wildlife-borne pathogens

We describe the prototype of an electronic tool for risk assessment with dynamic ranking of wildlife-borne pathogens in function of their need for surveillance. Data about pathogens, their hosts and occurrences are obtained from literature and are classified as qualitative scores under six main criteria with their sub-criteria, corresponding to the elements of a standard risk assessment. Pathogen-specific data are reviewed by experts. The information is processed per pathogen through an algorithm and through summing up of the values obtained by converting four-tiered qualitative sub-criteria scores to weighted five-tiered numerical values. For a consistent comparison between pathogens, the “unknown” sub-criteria scores are assigned a median value of 3, allowing preservation of the sub-criteria concerned and their weights for the risk assessment, but minimizing the effect of this score on the outcome. Irregular data availability is further accommodated by a different data processing for comprehensiveness and refinement requirements, which is realised by a respective first- and second-level ranking of pathogens, the latter using additional quantitative and qualitative data for the release assessment. Continuous data updates are necessary to reflect the current situation in the field. Output flexibility is implemented by the possibility to run queries based on the choice of a region, a specific target group susceptible to the pathogens and a set of weights for the sub-criteria.     

Algorithmic detection of the beginning and end of bipolar electrograms: Implications for novel methods to assess local activation time during atrial tachycardia

Activation mapping is required to effectively ablate atrial tachycardia (AT). Conventional tools to assess local activation time (LAT) are based upon the peak of the bipolar electrogram (B-EGM, LAT_Peak) and the maximal negative slope of the unipolar electrogram (U-EGM, LAT_Slope). Bipolar electrograms are influenced by wavefront direction, bipole orientation, and inter-electrode spacing causing ambiguity in peak detection, whereas unipolar electrograms are disturbed by the presence of far-field signals. We developed a new algorithm to detect the beginning and end of bipolar electrograms (t_begin and t_end). Then, we introduced new LAT methods related to the onset of B-EGMs (LAT_Onset), the center of mass of B-EGMs (LAT_CoM), and the slope of U-EGMs within a pre-defined window (LAT_Slope-hybrid).In total 3752 recordings from 31 AT patients were retrospectively analyzed. The signal-to-noise ratio (SNR) for B-EGMs was calculated to differentiate algorithmically high from low quality electrograms (HQ and LQ). In a subset of 328 B-EGMs, five experts validated the t_begin as determined by the algorithm by visual rating. The newly developed LAT methods were compared to the conventional LAT methods and to one another (Bland–Altman plots) in both HQ (n = 3003) and LQ EGMs (n = 749).The t_begin algorithm was accurate (deviation < ±10 ms) in 96 ± 4% of HQ and 91 ± 8% of LQ B-EGMs. BA plots revealed the following difference (bias) and variation in HQ and LQ EGMs respectively: (1) LAT_Onset vs. LAT_Peak: 27 ± 30 ms and 24 ± 62 ms; (2) LAT_CoM vs. LAT_Peak: 0 ± 16 ms and 2 ± 38 ms; (3) LAT_Slope-hybrid vs. LAT_Slope: 1 ± 32 ms and 15 ± 110 ms; (4) LAT_Onset vs. LAT_CoM: 22 ± 24 ms and 18 ± 22 ms; (5) LAT_Onset vs. LAT_Slope-hybrid: 16 ± 18 ms and 13 ± 22 ms; and (6) LAT_CoM vs. LAT_Slope-hybrid: 5 ± 20 ms and 4 ± 18 ms.In the present study, we introduced three new methods to assess local activation time in AT, based upon an algorithm detecting accurately the beginning and end of the B-EGM complex. BA analysis of the new methods showed similar variation in high and low quality EGMs, suggesting that they introduce less ambiguity than the conventional peak method. LAT_Onset consistently yielded an earlier activation moment. LAT_Slope-hybrid – by blanking far-field potentials – seems to be the optimal method for detection of the maximal negative slope in U-EGMs. Interestingly, LAT_CoM in B-EGMs coincided with the maximal negative slope in U-EGMs, suggesting its physiological sense and future use. The new LAT methods can be implemented in real-time mapping applications.     

Development of an anesthesia data warehouse: Preliminary results

Hospital information system manages patient's hospitalization information across different applications and databases. As statistics are performed with difficulties on these different databases, the university hospital of Lille developed an anesthesia data warehouse. This common structure stores data related to anesthesia procedures and patient hospital stay. In that way, the joint analysis on intervention's events and patient's outcome is possible. However, data quality remains one of the main issues in this kind of project. Indeed, errors in patient identifier result in difficulties to link data between the different sources. This problem will be approached in the next phase of the project.