Core-Shell Imprinted Particles for Adenovirus Binding

Virus-imprinted polymers were synthesized via surface imprinting strategies to produce core-shell imprinted particles selective for human adenovirus type 5. High binding affinity of the target virus towards the resulting imprinted layer was confirmed and unspecific binding was reduced in presence of blocking agents, i.e., via bovine serum albumin and skim milk in combination with Tween 20. In addition, the imprinted materials were applied for adenovirus extraction from cell culture supernatants. High levels of virus binding with negligible binding of matrix proteins confirmed the suitability of these materials for binding and extraction of the target virus from complex matrices.

Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification

Colony forming unit (CFU) determination by agar plating is still regarded as the gold standard for biofilm quantification despite being time- and resource-consuming. Here, we propose an adaption of the high-throughput Start-Growth-Time (SGT) method from planktonic to biofilm analysis, which indirectly quantifies CFU/mL numbers by evaluating regrowth curves of detached biofilms. For validation, the effect of dalbavancin, rifampicin and gentamicin against mature biofilms of Staphylococcus aureus and Enterococcus faecium was measured by accessing different features of the viability status of the cell, i.e., the cultivability (conventional agar plating), growth behavior (SGT) and metabolic activity (resazurin assay). SGT correlated well with the resazurin assay for all tested antibiotics, but only for gentamicin and rifampicin with conventional agar plating. Dalbavancin treatment-derived growth curves showed a compared to untreated controls significantly slower increase with reduced cell doubling times and reduced metabolic rate, but no change in CFU numbers was observed by conventional agar plating. Here, unspecific binding of dalbavancin to the biofilm interfered with the SGT methodology since the renewed release of dalbavancin during detachment of the biofilms led to an unintended antimicrobial effect. The application of the SGT method for anti-biofilm testing is therefore not suited for antibiotics which stick to the biofilm and/or to the bacterial cell wall. Importantly, the same applies for the well-established resazurin method for anti-biofilm testing. However, for antibiotics which do not bind to the biofilm as seen for gentamicin and rifampicin, the SGT method presents a much less labor-intensive method suited for high-throughput screening of anti-biofilm compounds.

Assessing the Potential of Molecular Imaging for Myelin Quantification in Organotypic Cultures

Ex vivo models for the noninvasive study of myelin-related diseases represent an essential tool to understand the mechanisms of diseases and develop therapies against them. Herein, we assessed the potential of multimodal imaging traceable myelin-targeting liposomes to quantify myelin in organotypic cultures. Methods: MRI testing was used to image mouse cerebellar tissue sections and organotypic cultures. Demyelination was induced by lysolecithin treatment. Myelin-targeting liposomes were synthetized and characterized, and their capacity to quantify myelin was tested by fluorescence imaging. Results: Imaging of freshly excised tissue sections ranging from 300 µm to 1 mm in thickness was achieved with good contrast between white (WM) and gray matter (GM) using T2w MRI. The typical loss of stiffness, WM structures, and thickness of organotypic cultures required the use of diffusion-weighted methods. Designed myelin-targeting liposomes allowed for semiquantitative detection by fluorescence, but the specificity for myelin was not consistent between assays due to the unspecific binding of liposomes. Conclusions: With respect to the sensitivity, imaging of brain tissue sections and organotypic cultures by MRI is feasible, and myelin-targeting nanosystems are a promising solution to quantify myelin ex vivo. With respect to specificity, fine tuning of the probe is required. Lipid-based systems may not be suitable for this goal, due to unspecific binding to tissues.

Detection of MOG-IgG in Clinical Samples by Live Cell-Based Assays: Performance of Immunofluorescence Microscopy and Flow Cytometry

Human antibodies against Myelin Oligodendrocyte Glycoprotein (MOG) from immunoglobulin-G subclasses (MOG-IgG) have been recently associated with a new subgroup of neurological autoimmune diseases with distinct clinical characteristics from multiple sclerosis and neuromyelitis optica spectrum disorders. The use of MOG-IgG as a biomarker is an essential tool to assist in the diagnosis and clinical prognosis. The cell-based assay (CBA) is a methodology that expresses high levels of natively folded human MOG protein in the cell membrane being the methodology most used for clinical MOG-IgG diagnosis. However, there is still no consensus about the best approach to perform CBA to improve the results. The CBA using flow cytometry (CBA-FC) is an automated technique with objective quantification, reducing the subject of human bias that occurred at CBA using immunofluorescence (CBA-IF). In this study, we compared the performance of CBA-IF and CBA-FC as an acquisition tool analysis. The sera of 104 patients diagnosed with inflammatory Central Nervous System diseases were tested in both CBA-IF and CBA-FC. We used the dilution of 1:128 for CBA-IF and three different dilutions (1:20, 1:100, and 1:640) for CBA-FC. The CBA-FC and CBA-IF results had 88.5% agreement between assays and the CBA-IF titers by endpoint-dilution correlated with the CBA-FC titers. The highest serum dilution resulted in an increased CBA-FC specificity, but there was a reduction in the CBA-FC sensitivity. Our study showed that CBA-FC can be used in clinical practice as a diagnostic technique for MOG-IgG. In addition, in some specific cases, the combination of both techniques could be used as a tool to discriminate unspecific binding and overcome single assay limitations.

Unspecific binding but specific disruption of the group I intron by the StpA chaperone

Chaperone proteins — the most disordered among all protein groups — help RNAs fold into their functional structure by destabilizing misfolded configurations or stabilizing the functional ones. But disentangling the mechanism underlying RNA chaperoning is challenging, mostly due to inherent disorder of the chaperones and the transient nature of their interactions with RNA. In particular, it is unclear how specific the interactions are and what role is played by amino acid charge and polarity patterns. Here, we address these questions in the RNA chaperone StpA. By adapting direct coupling analysis (DCA) to treat in tandem sequences written in two alphabets, nucleotides and amino acids, we could analyze StpA-RNA interactions and identify a two-pronged mechanism: StpA disrupts specific positions in the group I intron while globally and loosely binding to the entire structure. Moreover, the interaction is governed by the charge pattern: negatively charged regions in the destabilizing StpA N-terminal affect a few specific positions in the RNA, located in stems and in the pseudoknot. In contrast, positive regions in the C-terminal contain strongly coupled amino acids that promote non-specific or weakly-specific binding to the RNA. The present study opens new avenues to examine the functions of disordered proteins and to design disruptive proteins based on their charge patterns.

Multistable and dynamic CRISPRi-based synthetic circuits

Gene expression control based on CRISPRi (clustered regularly interspaced short palindromic repeats interference) has emerged as a powerful tool for creating synthetic gene circuits, both in prokaryotes and in eukaryotes; yet, its lack of cooperativity has been pointed out as a potential obstacle for dynamic or multistable circuit construction. Here we use CRISPRi to build prominent synthetic gene circuits in Escherichia coli. We report the first-ever CRISPRi oscillator (“CRISPRlator”), bistable network (toggle switch) and stripe pattern-forming incoherent feed-forward loop (IFFL). Our circuit designs, conceived to feature high predictability and orthogonality, as well as low metabolic burden and context-dependency, allowed us to achieve robust circuit behaviors. Mathematical modeling suggests that unspecific binding in CRISPRi is essential to establish multistability. Our work demonstrates the wide applicability of CRISPRi in synthetic circuits and paves the way for future efforts towards engineering more complex synthetic networks, boosted by the advantages of CRISPR technology.

SPME Extraction Methods of Doped Organochlorine Pesticides in Moroccan Water Samples

To investigate Organochlorine (OC) in Moroccan water samples, two different methods of extraction being the solid phase micro extraction (SPME) and the liquid-liquid extraction (LLE) are exploited. The gas chromatography with an electron capture detector (GC-ECD) and the gas chromatography coupled to a mass spectrum (GC-MS) are elaborated. In order to get the best extraction, different process parameters are cheeked and optimized. The relevant results are obtained using a time of unspecific binding properties of 30 minutes’ duration and mode of agitation for 30 min with agitation by a magnetic stirrer and a temperature of 30 °C. In particular, a comparative study between the results of SPME and LLE coupled to (GC-ECD) and (GC-MS), respectively, are given. Concretely, it has been found that the SPME extraction technique is a very useful method to analyze the pesticide residues in water samples controlled by local laboratories.