FTIR micro-spectroscopy is a sensitive, non-destructive and label-free method offering diffraction-limited resolution with high signal-to-noise ratios when combined with a synchrotron radiation source. The vibrational signature of individual cells was used to validate an alternative strategy for reprogramming induced pluripotent stem cells generated from amniocytes. The iPSC lines PB09 and PB10, were reprogrammed from the same amniocyte cell line using respectively the Oct54, Sox2, Lin28, and Nanog and the Oct4 and Sox2 transcription factor cocktail. We show that cells reprogrammed by the two different sets of transfection factors have similar spectral signatures after reprogramming, except for a small subpopulation of cells in one of the cell lines. Mapping HeLa cells at subcellular resolution, we show that the Golgi apparatus, the cytoplasm and the nucleus have a specific spectral signature. The CH(3):CH(2) ratio is the highest in the nucleus and the lowest in the Golgi apparatus/endoplasmic reticulum, in agreement with the membrane composition of these organelles. This is confirmed by specific staining of the organelles with fluorescent dyes. Subcellular differentiation of cell compartments is also demonstrated in living cells.

International audienceCrystal formation in kidney tissue is increasingly recognized as a major cause of severe or acute renal failure. Kidney biopsies are currently performed and analyzed using different staining procedures. Unfortunately, none of these techniques are able to distinguish the different Ca phosphates (e.g., amorphous or nanostructured Ca phosphate apatite, octacalcium phosphate, brushite…) or Ca oxalates (whewellite and weddellite). Moreover, the crystal’s morphology, a structural parameter proven as a major information to the clinician regarding kidney stones, is not taken into account. Such major limitations call for a different research approach, based on physicochemical techniques. Here we propose classical observations through field-emission scanning electron microscopy experiments combined with energy dispersive spectroscopy as well as measurements through Raman and μFourier transform Infra-Red Spectroscopy. If necessary, in the case of microcrystals, observations using cutting edge technology such as Synchrotron Radiation (SR) – FTIR or SR-UV visible spectroscopy can be subsequently performed on the same sample. Taken together this set of diagnostic tools will help clinicians gather information regarding the nature and the spatial distribution at the subcellular scale of different chemical phases present in kidney biopsies as well as on the crystal morphology and therefore obtain more precise diagnosis

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Geobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene encoding an inducible nitrilase was located and cloned using degenerate primers coding for well-conserved nitrilase sequences, coupled with inverse PCR. The nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da, and the purified functional enzyme had an apparent molecular weight of approximately 600 kDa by size exclusion chromatography. Similar to several plant nitrilases and some bacterial nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. The ratios of acid to amide produced from the substrates we tested are significantly different to those reported for other enzymes, and this has implications for our understanding of the mechanism of the nitrilases which may assist with rational design of these enzymes. Electron microscopy and image classification showed complexes having crescent-like, "c-shaped", circular and "figure-8" shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively.