Anti-SpCas9 IgY Polyclonal Antibodies Production for CRISPR Research Use.

The CRISPR/Cas adaptative immune system has been harnessed as an RNA-guided, programmable genome editing tool, allowing for diverse biotechnological applications. The implementation of the system relies on the ability to detect the Cas9 protein in biological samples. This task is facilitated by employing antibodies, which exhibit several advantageous features and applications in the context of tropical neglected diseases. This study reports a one-month immunization scheme with the Cas9 protein fromStreptococcus pyogenes to produce IgY polyclonal antibodies (anti-SpCas9), which can be rapidly isolated by combining yolk de-lipidation with protein salting out using pectin and ammonium sulfate, respectively. Immunodetection assays indicate that the antibodies are highly sensitive, specific, and useful for detecting the SpCas9 protein in promastigotes ofLeishmania braziliensisexpressing exogenous SpCas9. Thus, the simple method for producing anti-SpCas9 IgY antibodies will accelerate CRISPR/Cas-based studies in Leishmania spp. This approach serves as a valuable research tool in this parasite model and holds the potential for wide application in various other biological samples, promoting the implementation of the system. In fact, a bioinformatics approach based on the identification of antigenic determinants in the SpCas9 protein suggests the possibility of using the anti-SpCas9 IgY antibodies in applications such as Prime and Base editing.

Influence of physical and chemical properties of HTSXT-FTIR samples on the quality of prediction models developed to determine absolute concentrations of total proteins, carbohydrates and triglycerides: a preliminary study on the determination of their absolute concentrations in fresh microalgal biomass.

Absolute concentrations of total macromolecules (triglycerides, proteins and carbohydrates) in microorganisms can be rapidly measured by FTIR spectroscopy, but caution is needed to avoid non-specific experimental bias. Here, we assess the limits within which this approach can be used on model solutions of macromolecules of interest. We used the Bruker HTSXT-FTIR system. Our results show that the solid deposits obtained after the sampling procedure present physical and chemical properties that influence the quality of the absolute concentration prediction models (univariate and multivariate). The accuracy of the models was degraded by a factor of 2 or 3 outside the recommended concentration interval of 0.5-35 µg spot(-1). Change occurred notably in the sample hydrogen bond network, which could, however, be controlled using an internal probe (pseudohalide anion). We also demonstrate that for aqueous solutions, accurate prediction of total carbohydrate quantities (in glucose equivalent) could not be made unless a constant amount of protein was added to the model solution (BSA). The results of the prediction model for more complex solutions, here with two components: glucose and BSA, were very encouraging, suggesting that this FTIR approach could be used as a rapid quantification method for mixtures of molecules of interest, provided the limits of use of the HTSXT-FTIR method are precisely known and respected. This last finding opens the way to direct quantification of total molecules of interest in more complex matrices.

Unravelling the matrix effect of fresh sampled cells for in vivo unbiased FTIR determination of the absolute concentration of total lipid content of microalgae.

Over the past years, the substitution of the classical biochemical quantification techniques by Fourier transform infrared (FTIR) spectroscopy has been widely studied on microalgae because of its tremendous application potential for bioprocess monitoring. In the present work, mandatory aspects that have never been approached by FTIR end-users working onto fresh biomass were assessed. We demonstrated first that fresh cells' FTIR spectra main characteristics could be severely and unspecifically altered when the properties of the sampled biomass were not monitored. Microscopy indicated that important cell reorganization could occur when diminishing the cells density of the sample. Molecular probing approach suggested that such a modification could provoke an alteration of the hydrogen-bonding network of the sample. The sample heterogeneity was found to impact also the shape and intensity of the recorded FTIR bands, participating then to a matrix effect uncharacterized until now. In the second part of our study, we selected FTIR spectra not influenced by this matrix effect and the corresponding accurate calibration data obtained by the whole cell analytical procedure to elaborate an optimized total lipid quantification PLS-R model. Results demonstrated that our strategy could provide a small volume sampling (1 mL of fresh culture), rapid (within minutes), robust (physiological condition independent), and accurate (as accurate as the reference method could be) FTIR absolute quantification method to determine the fresh microalgae intracellular total lipid content. To validate our unbiased FTIR approach, a photobioprocess monitoring pipeline was developed and allowed assessing the effect of light attenuation on total lipid production by the marine microalga Nannochloropsis oculata.

Green improved processes to extract bioactive phenolic compounds from brown macroalgae using Sargassum muticum as model.

A comparative study between "alternative" extraction processes such as centrifugal partition extraction (CPE), supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) and classical solid/liquid used in the laboratory are currently focusing on the efficiency (selectivity and productivity) to obtain bioactive phenolic compounds from the phaeophyte Sargassum muticum model. The choice of the best process was based on several measurements: (i) the total phenolic content measured by the colorimetric Folin-Ciocalteu assay, (ii) radical scavenger and antioxidant activities assessed by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, and the ß-carotene bleaching method and finally (iii) the method productivity. Irrespective of the solvent used in the processes, alternative methods are always sharply more effective than classical ones. With the exception of SFE which does not allow extracting the totality of the active phenolic compounds, two of the other extraction methods were particularly promising. First, CPE afforded the most important yields in concentrated phenolic compounds (PC) (22.90±0.65% DW) also displaying the best activities (0.52±0.02 and 0.58±0.19 mg/mL for IC50 and AAC700, respectively). Secondly, PLE using an EtOH:water mixture 75:25 (v/v) allowed a good PC extraction (10.18±0.25% DW) with huge efficiency. Despite a lesser activity of the extracts (0.77±0.01 and 1.59±0.15 mg/mL for IC50 and AAC700, respectively) PLE is a green process and potentially complies European norms requirements for the prospective valorization of phenolic compounds from S. muticum in Brittany.