Differential screening of phage-ab libraries by oligonucleotide microarray technology

A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag) in the phagemid coding for each phage-displayed antibody fragment (phage-Ab) present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection) and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.     

Adsorption of ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase on calcium oxalate

We used ethylenediaminetetraacetic acid dianhydride (EDTAD) to modify oxalate decarboxylase (OXDC) to improve its adsorption on calcium oxalate stones. The modified sites were identified by Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and the adsorption mechanism of the EDTAD-modified OXDC on calcium oxalate (CaOx) was investigated. We investigated adsorption time, initial enzyme concentration, temperature and solution pH on the adsorption process. Data were analyzed using kinetics, thermodynamics and isotherm adsorption models. UPLC-MS showed that EDTAD was attached to OXDC covalently and suggested that the chemical modification occurred at both the free amino of the side chain and the α-NH₂ of the peptide. The adsorption capacity of the EDTAD-OXDC on calcium oxalate was 53.37% greater than that of OXDC at the initial enzyme concentration of 5 mg/ml, pH = 7.0, at 37° C. The modified enzyme (EDTAD-OXDC) demonstrated improved oxalate degradation activity at pH 4.5?6.0. Kinetic data fitting analysis suggested a pseudo second order kinetic model. Estimates of the thermodynamic parameters including ΔG⁰, ΔH⁰ and ΔS⁰ of the adsorption process showed it to be feasible, spontaneous and endothermic. Isotherm data fitting analysis indicated that the adsorption process is reduced to monolayer adsorption at a low enzyme concentration and to multilayer adsorption at a high enzyme concentration. It may be possible to apply OXDC to degradation of calcium oxalate stones.     

Chromosomal Instability and Telomere Length Variations during the Life Span of Human Fibroblast Clones

Growth characteristics, karyotype changes, and telomere length variations were analyzed during the life span of 12 anchorage-independent clones isolated from a xeroderma pigmentosum fibroblast strain. After an initial period of comparable active growth, all the clones showed a decline in the growth rate and finally entered a phase of replicative senescence; however, the number of population doublings and the time required to enter senescence varied among the clones. Repeated cytogenetic analyses during culture propagation showed the appearance of chromosome anomalies, mainly telomeric association (tas) and unbalanced translocations. In all the clones the percentage of abnormal mitoses increased with culture passage, but reached different levels (from less than 10% to about 100%). This finding indicates that the replicative block may be associated with differently altered cytogenetic patterns. Specific chromosome arms (5p, 16q, 19q, and 20q) were preferentially involved intas,suggesting that alterations in chromosome ends may occur which predispose to fusion. In some clones it was possible to demonstrate the origin of marker chromosomes from the evolution oftas.Telomere length analysis by Southern blotting on DNA samples prepared from 7 clones and from the parental cell lines showed that the terminal restriction fragment (TRF) profiles were homogeneous in senescent parental cells and in the clones during the last part of their life in culture, regardless of the degree of karyotype abnormalities. The homogeneity of the TRF profiles supports the hypothesis of a critical telomere length at senescence.     

Phenotypic characteristics and tendency to apoptosis of peripheral blood mononuclear cells from HIV+ long term non progressors

The aim of this study was to analyze (i) phenotype, (ii) in vitro spontaneous and induced apoptosis, (iii) glutathione (GSH) intracellular content and (iv) inhibitors of apoptosis of potential therapeutical use in peripheral blood mononuclear cells (PBMC) from HIV+ long term non progressors (LTNP), in comparison with progressors (HIV+P) and seronegative controls (HIV-). Three groups of subjects were studied: 15 HIV+P (patients losing >150 CD4+/year), 9 LTNP (subjects infected by HIV for at least 7 years without clinical and immunological signs of progression, with a mean of 898 CD4+/microL) and 18 HIV-. All subjects were living in a large community for former drug addicts, and were matched for age and sex. We used flow cytometry for analyzing PBMC phenotype and apoptosis; high performance liquid chromatography for measuring intracellular GSH content. PBMC phenotype of LTNP shared characteristics with those of both HIV- and HIV+P. Indeed, LTNP showed a normal number CD4+ cells (an inclusion criteria), but significantly increased numbers of CD8+ lymphocytes, activated T cells, CD19+, CD5+ B lymphocytes and CD57+ cells, as well as a decrease in CD19+, CD5- B lymphocytes and CD16+ cells. In LTNP, spontaneous apoptosis was similar to that of HIV- and significantly lower than that of HIV+P. Adding interleukin-2 (IL-2) or nicotinamide (NAM) significantly decreased spontaneous apoptosis in LTNP and HIV+P. Pokeweed mitogen-induced apoptosis was also similar in LTNP and HIV-, but significantly lower than that of HIV+P. In HIV+P, but also in LTNP, spontaneous apoptosis was inversely correlated to the absolute number and percentage of CD4+ cells and directly correlated to the number and percentage of activated T cells present in peripheral blood. GSH intracellular content was greatly decreased in PBMC from HIV+P and slightly, but significantly, reduced in LTNP. Adding 2-deoxy-D-ribose, an agent provoking apoptosis through GSH depletion, to quiescent PBMC resulted in similar levels of massive cell death in the three groups. This phenomenon was equally prevented in the three groups by N-acetyl-cysteine but not by IL-2. A complex immunological situation seems to occur in LTNP. Indeed, PBMC from LTNP are characterized by a normal in vitro tendency to undergo apoptosis despite the presence of a strong activation of their immune system, unexpectedly similar to that of HIV+P. Our data suggest that NAM and IL-2 are possible candidates for reducing spontaneous apoptosis in HIV infection.     

Invasion Pattern of Herb Garlic Mustard (Alliaria petiolata) in High Quality Forests

The invasion of non-indigenous plant species poses a severe threat to native plant communities. Garlic mustard (Alliaria petiolata) is a naturalized European biennial herb that has spread rapidly through the eastern US and adjacent Canada. To determine garlic mustard rate of spread, eleven permanent plots (50×25 m) were located in seven high quality (relatively undisturbed) forests in the early stages of invasion. Garlic mustard presence was recorded within six 50×2 m permanent belt transects, and density and percent cover by age class were recorded in 36 permanent 1 m² quadrats, between 1989 and 1992, and again in 1997. Garlic mustard spread at an average rate of 5.4 m per year between 1989 and 1992, in all plots combined. Within individual plots rate of spread varied substantially, with location of the front increasing up to 36 m and decreasing as much as 18 m between years. While the front alternately advanced and retreated, over time garlic mustard consistently advanced through all forests. Rate of spread was influenced by establishment of satellite populations, and disturbance (wind-throw and flooding). The pattern of spread within plots was one of a ragged advancing front, supplemented by establishment of satellite populations 6–40 m distant from the front, which then coalesced with the main population. Garlic mustard presence between 1989 and 1997 increased significantly within all plots, and in each age class within each plot. The greatest increases occurred in plots where this plant was initially rarest. Garlic mustard cover and density varied nonsignificantly during the same time period. These results indicate that after garlic mustard invades a forest it becomes a permanent part of the community, annually increasing in presence but fluctuating in cover and density. Garlic mustard maintains a low profile under low disturbance conditions, but increases rapidly with periodic disturbance. This study monitored garlic mustard invasion in high quality relatively undisturbed forests, and may underestimate the rate of spread in low quality highly disturbed forests.     

Trunk muscle activity during stability ball and free weight exercises

The purpose of this investigation was to compare trunk muscle activity during stability ball and free weight exercises. Nine resistance-trained men participated in one testing session in which squats (SQ) and deadlifts (DL) were completed with loads of approximately 50, 70, 90, and 100% of one-repetition maximum (1RM). Isometric contractions during 3 stability ball exercises (quadruped (QP), pelvic thrust (PT), ball back extension (BE)) were also completed. During all exercises, average integrated electromyography (IEMG) from the rectus abdominus (RA), external oblique (EO), longissimus (L1) and multifidus (L5) was collected and analyzed. Results demonstrate that when expressed relative to 100% DL 1RM, muscle activity was 19.5 +/- 14.8% for L1 and 30.2 +/- 19.3% for L5 during QP, 31.4 +/- 13.4% for L1 and 37.6 +/- 12.4% for L5 during PT, and 44.2 +/- 22.8% for L1 and 45.5 +/- 21.6% for L5 during BE. IEMG of L1 during SQ and DL at 90 and 100% 1RM, and relative muscle activity of L5 during SQ and DL at 100% 1RM was significantly greater (P < or = 0.05) than in the stability ball exercises. Furthermore, relative muscle activity of L1 during DL at 50 and 70% 1RM was significantly greater than in QP and PT. No significant differences were observed in RA and EO during any of the exercises. In conclusion, activity of the trunk muscles during SQs and DLs is greater or equal to that which is produced during the stability ball exercises. It appears that stability ball exercises may not provide a sufficient stimulus for increasing muscular strength or hypertrophy; consequently, the role of stability ball exercises in strength and conditioning programs is questioned. SQs and DLs are recommended for increasing strength and hypertrophy of the back extensors.     

Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy

Renal fibrosis is the common histological feature of advanced glomerular and tubulointerstitial disease leading to end-stage renal disease (ESRD). However, specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persistent inflammation is a key event in the development of fibrosis, we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that, in the unilateral ureteral obstruction model of renal fibrosis, the B1R is overexpressed and that delayed treatment with an orally active nonpeptide B1R antagonist blocks macrophage infiltration, leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.     

Genephony: a knowledge management tool for genome-wide research

Background  

One of the consequences of the rapid and widespread adoption of high-throughput experimental technologies is an exponential increase of the amount of data produced by genome-wide experiments. Researchers increasingly need to handle very large volumes of heterogeneous data, including both the data generated by their own experiments and the data retrieved from publicly available repositories of genomic knowledge. Integration, exploration, manipulation and interpretation of data and information therefore need to become as automated as possible, since their scale and breadth are, in general, beyond the limits of what individual researchers and the basic data management tools in normal use can handle. This paper describes Genephony, a tool we are developing to address these challenges.