Targeting Lipoprotein Biogenesis: Considerations towards Antimicrobials

Decades have passed without approval of a new antibiotic class. Several companies have recently halted related discovery efforts because of multiple obstacles. One promising route under research is to target the lipoprotein maturation pathway in light of major recent findings and the virulence roles of lipoproteins. To support the future design of selective drugs, considerations and priority-setting are established for the main lipoprotein processing enzymes (Lgt, LspA, and Lnt) based on microbiology, biochemistry, structural biology, chemical design, and pharmacology. Although not all bacterial species will be similarly impacted by drug candidates, several advantages make LspA a top target to pursue in the development of novel antibiotics effective against bacteria that are resistant to existing drugs

In situ Image Processing and Data Binning Strategy for Particle Engineering Applications

Crystallization control can be improved through real-time monitoring technologies. Here, a workflow is demonstrated on rapid batch cooling crystallization of L-glutamic acid. First, in situ images were generated using video microscopy sensors and analyzed, by employing a single, rapid macro code to extract particle data descriptors. A binning procedure (over time) was performed, where every data point represented the counts of particles within a specific size or shape range per 100 images. This binning method was found more informative in tracking of the populations compared to whole image averages or individual particle datapoints. This study provides a step-by-step guide towards improving mechanistic modeling, control via feedback, automation, and continuous manufacturing for Industry 4.0

Rapid analysis of magnesium in infant formula powder using laser-induced breakdown spectroscopy

Laser-induced breakdown spectroscopy (LIBS) was investigated to determine magnesium (Mg) content in infant formula powder. To predict Mg content in the range established by the Codex Alimentarius, a partial least squares regression (PLSR) model was developed using a calibration data set (n = 30) based on full cross-validation and validated using an independent validation data set (n = 21). The prediction model performance was evaluated using the regression coefficients of determination (Rcv2 = 0.94 and Rp2 = 0.85) with the root mean square errors on cross-validation and prediction (RMSECV = 60 mg kg−1 and RMSEP = 80 mg kg−1). The limit of detection (150 mg kg−1) was also calculated. In addition, LIBS successfully predicted the Mg content distributed within a pellet. This study demonstrated that LIBS is suitable as a rapid reagent-free method for the quantification of Mg in powdered infant formula and can provide spatial information with acceptable accuracy

Non-invasive 3D and 360° optical imaging of micro-particles

Scanning electron microscopy and X-ray microtomography are useful methods for high resolution shape imaging. Visible microscopy is also common, however, developing a low-cost and customizable system for surface and shape investigation of optically active particles is challenging. In this work, we demonstrate an assembly offering good light sensitivity, flexibility of illumination and contrasts from varying angles. The design was applied, together with recent programs for focus-stacking, to analyze crystals of taurine, L-glutamic acid, acetylsalicylic acid, and copper sulfate, along with digital 3D-360° modelling of phosphorescent [Ru(bpy)3]Cl2 and strontium aluminate particles. We further tested the approach for real time monitoring of size, shape and texture analysis of fat filled milk particles and acid whey powders. The findings show proof of concept for detailed feature imaging of particles directly from the process environment

The N-acyltransferase Lnt: Structure-function insights from recent simultaneous studies

Bacterial lipoproteins have been researched for decades due to their roles in a large number of biological functions. There were no structures of their main three membrane processing enzymes, until 2016 for Lgt and LspA, and then 2017 for Lnt with not one but three simultaneous, independent publications. We have analyzed the recent findings for this apolipoprotein N-acyltransferase Lnt, with comparisons between the novel structures, and with soluble nitrilases, to determine the significance of unique features in terms of substrate's recognition and binding mechanism influenced by exclusive residues, two transmembrane helices, and a flexible loop

Adaptive Background Correction of Crystal Image Datasets:Towards Automated Process Control

Improving the data descriptor calculation of crystal’s physical properties requires sophisticated imaging techniques and algorithms. It has been possible to construct 2D population balance models benefiting from characteristic measurements of both crystal’s length and width, compared to the single representative sizes used in 1D models. Our aim is to ameliorate the procedure of determining shape (and not only size) factors, in an automated fashion and directly from the process, for implementation in future models. Here, approaches suitable for real-time applications were employed including engineered imaging sensors and adaptive algorithms. We described the latter in detail for varying 2D image datasets. Their basic concept is similar. Each is applicable to an entire dataset, thus demonstrating efficacy for a variety of particle environments. While the challenge of particle segmentation for higher concentrations was not scrutinized here, this approach reduced processing time, steps and supervision, for the benefit of certain applications requiring process monitoring and automation

The Carboxy-Terminal αN Helix of the Archaeal XerA Tyrosine Recombinase Is a Molecular Switch to Control Site-Specific Recombination

International audienceTyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding. Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers