Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)

We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a “filtration” procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×10⁹ independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with K_d values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.     

Translocated microbiome composition determines immunological outcome in treated HIV infection

1. Download : Download high-res image (252KB)
2. Download : Download full-size image

     

GM-CSF-DFF40: a novel humanized immunotoxin induces apoptosis in acute myeloid leukemia cells

DNA fragmentation factor 40 (DFF40) is an endonuclease that acts downstream in the apoptotic cascade. The objective of this study was to generate a novel humanized chimeric protein with human DFF40 fused with GM-CSF for targeting acute myeloid leukemia (AML) cells. cDNA cloning of human DFF40 and GM-CSF was done and the chimeric gene GM-CSF-DFF40 was generated by overlap extension PCR. The fusion protein was expressed in E.coli, purified, refolded and characterized. In vitro cytotoxicity was evaluated on various AML cell lines. Treated cell lines were screened for various morphological and biochemical changes that are characteristic of apoptosis, by different assays like annexin V-FITC staining, TUNEL assay, JC-1 staining and immunocytochemistry of pro-apoptotic proteins and caspases. Cell cycle analysis of treated cells was done to quantify the percentage of apoptotic cells. The chimeric protein was found to be cytotoxic to AML cells in a dose and time dependent manner. Morphological changes such as formation of apoptotic bodies were revealed by microscopic examination of treated cells after staining. Immunocytochemical staining demonstrated biochemical changes such as changes in mitochondrial membrane potential, mitochondrial co-localization of Bax, cytochrome c release, presence of activated caspase-3 and DNA fragmentation. FACS analysis proved the presence of apoptotic cells following treatment. The chimeric protein GM-CSF-DFF40 was found to mediate targeted killing of AML cells by inducing apoptosis. Thus, this chimeric construct can act as a prospective candidate for targeted therapy of AML and other malignancies where GM-CSF receptor expression is upregulated.     

Potential application of Bacillus subtilis SPB1 biosurfactants in laundry detergent formulations: Compatibility study with detergent ingredients and washing performance

Surfactants play a very important role in laundry and household cleaning products ingredients. In this research, the application of lipopeptide biosurfactants, produced by Bacillus subtilis SPB1, in the formulation of a washing powder was investigated. The SPB1 biosurfactant was mixed with sodium tripolyphosphate as a builder and sodium sulfate as filler. The efficiency of the formulated detergent composition with different washing conditions to remove a stain from cotton fabric was examined. The results showed that the formulated detergent was effective in oil removal, with optimal washing conditions of pH, temperature, striate and time of washing system of 7, 65°C, 1000 RPM and 60 min, respectively. A comparative study of different detergent compositions (biosurfactant‐based detergent, combined biosurfactant‐commercial detergent, and a commercial detergent) for the removal of oil and tea stains, proved that the bio‐scouring was more effective (>75%) in terms of the stain removal than the commercial powders (<60%). Moreover, the results demonstrated that the biosurfactant acts additively with a commercial detergent and enhances their performance from 33 to 45% in removing oil stain and from 57 to 64% in removing tea stain. As a conclusion, in addition to the low toxicity and the high biodegradability of the microbial biosurfactants, the results of this study have shown that the future use of this lipopeptide biosurfactant as laundry detergent additive is highly promising.     

Adipose tissue senescence is mediated by increased ATP content after a short‐term high‐fat diet exposure

In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin resistance are not fully elucidated. The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high‐fat diet (HFD, 1–10 weeks) in 5‐month‐old male C57BL/6J mice and the potential role of energy metabolism. We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence‐associated ß‐galactosidase activity and cyclin‐dependent kinase inhibitor 1A and 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors, and immune cells, together with adipocytes. WAT senescence was associated with higher glycolytic and mitochondrial activity leading to enhanced ATP content in HFD‐derived preadipocytes, as compared with chow diet‐derived preadipocytes. One‐month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while reducing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss. We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover a critical role for HFD‐induced elevated ATP as a local danger signal inducing WAT senescence. Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence, and metabolic abnormalities.     

Suppression of extraintestinal and intestinal Nippostrongylus brasiliensis-induced eosinophilia by Eimeria nieschulzi

Eosinophil responses in extraintestinal and intestinal tissues were examined in August and Sprague-Dawley rats infected with Nippostrongylus brasiliensis or Eimeria nieschulzi (or both), and in uninfected controls to test the hypothesis that E. nieschulzi suppresses the systemic N. brasiliensis-induced eosinophil response. Caudal vein blood, femoral bone marrow, bronchoalveolar lavage fluid, peritoneal lavage fluid, and duodenal and jejunal samples were collected on day 8 postinfection (PI) with E. nieschulzi, on day 16 PI of the N. brasiliensis infection, when these days coincided in the concurrently infected rats, and from uninfected controls. Differential white blood cell counts were made from blood smears and cytocentrifuged preparations, and duodenal and jejunal eosinophils per villus crypt unit were quantified. Eimeria nieschulzi significantly reduced N. brasiliensis-induced eosinophil levels in peripheral blood, lavage fluids, and duodenal and jejunal tissues in both rat strains. August and Sprague-Dawley rats monospecifically infected with N. brasiliensis and concurrently with both parasites demonstrated elevated eosinopoiesis compared with uninfected controls and rats infected with only E. nieschulzi; however, despite this, concurrently infected rats had a significantly greater level of eosinopoiesis than those infected with only the nematode. In addition, E. nieschulzi induced elevated neutrophil levels in both monospecifically and concurrently infected rats in all extraintestinal tissues examined in both rat strains, whereas lymphocyte counts decreased concomitantly. This study suggests that the intestinal coccidian E. nieschulzi has the ability to modulate the systemic inflammatory response to N. brasiliensis and that this is not a rat strain-specific phenomenon.     

Benthic foraminiferal paleoenvironmental reconstruction from the Upper Coniacian-Lower Campanian succession in north-western Tunisia

Quantitative analysis of benthic foraminifera is used to characterize the paleoenvironments of the Upper Coniacian-Lower Campanian succession in the Jbil section of north-western Tunisia. Foraminiferal parameters and benthic foraminiferal assemblages show that the studied section includes four distinct paleoenvironmental phases. From oldest to youngest, these are as follows: (1) an interval with a Praebulimina reussi assemblage with infaunal ratios as high as 96.1%. High abundances of P. reussi, reflecting an increase in organic matter flux to the seafloor (meso-to eutrophic) under oxygenated bottom-water conditions. (2) An interval characterized by a Gavelinella costulata assemblage with mixed infaunal/epifaunal foraminifera with higher Fisher's alpha values (ranging from 4 to 15.2), reflecting mesotrophic conditions in an outer shelf environment. (3) An interval with a Gaudryina laevigata assemblage indicative of a middle to outer shelf environment; there is a considerable increase in infaunal agglutinated foraminifera, as well as a relatively abundant and moderately diversified oxic/suboxic foraminifera. (4) The final interval occurs in the lower Campanian (the Globotruncana ventricosa Zone) and includes a Bolivinoides decoratus assemblage reflecting an outer shelf to upper bathyal environment. It contains a higher planktonic percentage and biodiversity with a slight increase in dysoxic species; the mixed infaunal/epifaunal content (57.6 to 73.3%) reflects mesotrophic conditions. Four well-recognized major sea-level falls are matched by the dual signatures of eustatic sea-level changes. These are coincident with the results of this study, which represent the first documentation of these events in Tunisian faunal and paleoenvironmental changes, at the following boundaries: Coniacian/Santonian, intra-Santonian, Santonian/Campanian, and intra-early Campanian.