Multispectral in-line hologram reconstruction with aberration compensation applied to Gram-stained bacteria microscopy.

In multispectral digital in-line holographic microscopy (DIHM), aberrations of the optical system affect the repeatability of the reconstruction of transmittance, phase and morphology of the objects of interest. Here we address this issue first by model fitting calibration using transparent beads inserted in the sample. This step estimates the aberrations of the optical system as a function of the lateral position in the field of view and at each wavelength. Second, we use a regularized inverse problem approach (IPA) to reconstruct the transmittance and phase of objects of interest. Our method accounts for shift-variant chromatic and geometrical aberrations in the forward model. The multi-wavelength holograms are jointly reconstructed by favouring the colocalization of the object edges. The method is applied to the case of bacteria imaging in Gram-stained blood smears. It shows our methodology evaluates aberrations with good repeatability. This improves the repeatability of the reconstructions and delivers more contrasted spectral signatures in transmittance and phase, which could benefit applications of microscopy, such as the analysis and classification of stained bacteria.

Single-cell scattering and auto-fluorescence-based fast antibiotic susceptibility testing for gram-negative and gram-positive bacteria.

In this study, we assess the scattering of light and auto-fluorescence from single bacterial cells to address the challenge of fast (<2 h), label-free phenotypic antimicrobial susceptibility testing (AST). Label-free flow cytometry is used for monitoring both the respiration-related auto-fluorescence in two different fluorescence channels corresponding to FAD and NADH, and the morphological and structural information contained in the light scattered by individual bacteria during incubation with or without antibiotic. Large multi-parameter data are analyzed using dimensionality reduction methods, based either on a combination of 2D binning and Principal Component Analysis, or with a one-class Support Vector Machine approach, with the objective to predict the Susceptible or Resistant phenotype of the strain. For the first time, both Escherichia coli (Gram-negative) and Staphylococcus epidermidis (Gram-positive) isolates were tested with a label-free approach, and, in the presence of two groups of bactericidal antibiotic molecules, aminoglycosides and beta-lactams. Our results support the feasibility of label-free AST in less than 2 h and suggest that single cell auto-fluorescence adds value to the Susceptible/Resistant phenotyping over single-cell scattering alone, in particular for the mecA+ Staphylococcus (i.e., resistant) strains treated with oxacillin.

hCAF1/CNOT7 regulates interferon signalling by targeting STAT1.

Stringent regulation of the interferon (IFN) signalling pathway is essential for maintaining the immune response to pathogens and tumours. The transcription factor STAT1 is a crucial mediator of this response. Here, we show that hCAF1/CNOT7 regulates class I and II IFN pathways at different crucial steps. In resting cells, hCAF1 can control STAT1 trafficking by interacting with the latent form of STAT1 in the cytoplasm. IFN treatment induces STAT1 release, suggesting that hCAF1 may shield cytoplasmic STAT1 from undesirable stimulation. Consistently, hCAF1 silencing enhances STAT1 basal promoter occupancy associated with increased expression of a subset of STAT1-regulated genes. Consequently, hCAF1 knockdown cells exhibit an increased protection against viral infection and reduced viral replication. Furthermore, hCAF1 participates in the extinction of the IFN signal, through its deadenylase activity, by speeding up the degradation of some STAT1-regulated mRNAs. Since abnormal and unbalanced JAK/STAT activation is associated with immune disorders and cancer, hCAF1 could play a major role in innate immunity and oncogenesis, contributing to tumour escape.

hCAF1, a new regulator of PRMT1-dependent arginine methylation.

Protein arginine methylation is an emergent post-translational modification involved in a growing number of cellular processes, including transcriptional regulation, cell signaling, RNA processing and DNA repair. Although protein arginine methyltransferase 1 (PRMT1) is the major arginine methyltransferase in mammals, little is known about the regulation of its activity, except for the regulation induced by interaction with the antiproliferative protein BTG1 (B-cell translocation gene 1). Since the protein hCAF1 (CCR4-associated factor 1) was described to interact with BTG1, we investigated a functional link between hCAF1 and PRMT1. By co-immunoprecipitation and immunofluorescence experiments we demonstrated that endogenous hCAF1 and PRMT1 interact in vivo and colocalize in nuclear speckles, a sub-nuclear compartment enriched in small nuclear ribonucleoproteins and splicing factors. In vitro methylation assays indicated that hCAF1 is not a substrate for PRMT1-mediated methylation, but it regulates PRMT1 activity in a substrate-dependent manner. Moreover, small interfering RNA (siRNA)-mediated silencing of hCAF1 in MCF-7 cells significantly modulates the methylation of endogenous PRMT1 substrates. Finally, we demonstrated that in vitro and in the cellular context, hCAF1 regulates the methylation of Sam68 and histone H4, two PRMT1 substrates. Since hCAF1 and PRMT1 have been involved in the regulation of transcription and RNA metabolism, we speculate that hCAF1 and PRMT1 could contribute to the crosstalk between transcription and RNA processing.