Visible Light Control over the Cytolytic Activity of a Toxic Pore-Forming Protein.

Enabling control over the bioactivity of proteins with light, along with the principles of photopharmacology, has the potential to generate safe and targeted medical treatments. Installing light sensitivity in a protein can be achieved through its covalent modification with a molecular photoswitch. The general challenge in this approach is the need for the use of low energy visible light for the regulation of bioactivity.

Evidence of Cytolysin A nanopore incorporation in mammalian cells assessed by a graphical user interface.

Technologies capable of assessing cellular metabolites with high precision and temporal resolution are currently limited. Recent developments in the field of nanopore sensors allow the non-stochastic quantification of metabolites, where a nanopore is acting as an electrical transducer for selective substrate binding proteins (SBPs). Here we show that incorporation of the pore-forming toxin Cytolysin A (ClyA) into the plasma membrane of Chinese hamster ovary cells (CHO-K1) results in the appearance of single-channel conductance amenable to multiplexed automated patch-clamp (APC) electrophysiology.

Protein Sizing with 15 nm Conical Biological Nanopore YaxAB.

Nanopores are promising single-molecule tools for the electrical identification and sequencing of biomolecules. However, the characterization of proteins, especially in real-time and in complex biological samples, is complicated by the sheer variety of sizes and shapes in the proteome. Here, we introduce a large biological nanopore, YaxAB for folded protein analysis.

Specific Detection of Proteins by a Nanobody-Functionalized Nanopore Sensor.

Nanopores are label-free single-molecule analytical tools that show great potential for stochastic sensing of proteins. Here, we described a ClyA nanopore functionalized with different nanobodies through a 5-6 nm DNA linker at its periphery. Ty1, 2Rs15d, 2Rb17c, and nb22 nanobodies were employed to specifically recognize the large protein SARS-CoV-2 Spike, a medium-sized HER2 receptor, and the small protein murine urokinase-type plasminogen activator (muPA), respectively.

PlyAB Nanopores Detect Single Amino Acid Differences in Folded Haemoglobin from Blood.

The real-time identification of protein biomarkers is crucial for the development of point-of-care and portable devices. Here, we use a PlyAB biological nanopore to detect haemoglobin (Hb) variants. Adult haemoglobin (HbA) and sickle cell anaemia haemoglobin (HbS), which differ by just one amino acid, were distinguished in a mixture with more than 97 % accuracy based on individual blockades.

Automated Electrical Quantification of Vitamin B1 in a Bodily Fluid using an Engineered Nanopore Sensor.

The ability to measure the concentration of metabolites in biological samples is important, both in the clinic and for home diagnostics. Here we present a nanopore-based biosensor and automated data analysis for quantification of thiamine in urine in less than a minute, without the need for recalibration. For this we use the Cytolysin A nanopore and equip it with an engineered periplasmic thiamine binding protein (TbpA).

The Manipulation of the Internal Hydrophobicity of FraC Nanopores Augments Peptide Capture and Recognition.

The detection of analytes and the sequencing of DNA using biological nanopores have seen major advances over recent years. The analysis of proteins and peptides with nanopores, however, is complicated by the complex physicochemical structure of polypeptides and the lack of understanding of the mechanism of capture and recognition of polypeptides by nanopores. In this work, we show that introducing aromatic amino acids at precise positions within the lumen of α-helical fragaceatoxin C (FraC) nanopores increased the capture frequency of peptides and largely improved the discrimination among peptides of similar size.

Strategies for enzymological studies and measurements of biological molecules with the cytolysin A nanopore.

Pore-forming toxins are used in a variety of biotechnological applications. Typically, individual membrane proteins are reconstituted in artificial lipid bilayers where they form water-filled nanoscale apertures (nanopores). When a voltage is applied, the ionic current passing through a nanopore can be used for example to sequence biopolymers, identify molecules, or to study chemical or enzymatic reactions at the single-molecule level.

Towards remote monitoring in pediatric care and clinical trials-Tolerability, repeatability and reference values of candidate digital endpoints derived from physical activity, heart rate and sleep in healthy children.

Background: Digital devices and wearables allow for the measurement of a wide range of health-related parameters in a non-invasive manner, which may be particularly valuable in pediatrics. Incorporation of such parameters in clinical trials or care as digital endpoint could reduce the burden for children and their parents but requires clinical validation in the target population. This study aims to determine the tolerability, repeatability, and reference values of novel digital endpoints in healthy children.

Preparation of Cytolysin A (ClyA) Nanopores.

The ionic currents passing through nanopores can be used to sequence DNA and identify molecules at the single-molecule level. Recently, researchers have started using nanopores for the detection and analysis of proteins, providing a new platform for single-molecule enzymology studies and more efficient biomolecular sensing applications. For this approach, the homo-oligomeric Cytolysin A (ClyA) nanopore has been demonstrated as a powerful tool.

Preparation of Fragaceatoxin C (FraC) Nanopores.

Biological nanopores are an emerging class of biosensors with high-end precision owing to their reproducible fabrication at the nanometer scale. Most notably, nanopore-based DNA sequencing applications are currently being commercialized, while nanopore-based proteomics may become a reality in the near future.Although membrane proteins often prove to be difficult to purify, we describe a straightforward protocol for the preparation of Fragaceatoxin C (FraC) nanopores, which may have applications for DNA analysis and nanopore-based proteomics.

Current Blockades of Proteins inside Nanopores for Real-Time Metabolome Analysis.

Biological nanopores are emerging as powerful and low-cost sensors for real-time analysis of biological samples. Proteins can be incorporated inside the nanopore, and ligand binding to the protein adaptor yields changes in nanopore conductance. In order to understand the origin of these conductance changes and develop sensors for detecting metabolites, we tested the signal originating from 13 different protein adaptors.

Improvement of primary stability in ACL reconstruction by mesh augmentation of an established method of free tendon graft fixation. A biomechanical study on a porcine model.

Purpose: The aim of the present study was to compare primary stability in ACL reconstruction and ultimate load to failure of a mesh augmented hamstring tendon graft fixed with two cross pins to established hamstrings and bone-patellar-tendon-bone (BTB) graft fixation methods.

Methods: Forty fresh porcine femora were divided into four groups: (A): BTB graft fixed with two RigidFix® pins, (B): hamstring tendon graft fixed with a Milagro® interference screw, (C): hamstring tendon graft fixed with two RigidFix® pins, and (D): hamstring tendon graft augmented with Ultrapro® mesh fixed with two RigidFix® pins. Each graft underwent cyclic loading in tension and load to failure.

Primary stability of anterior lumbar stabilization: interdependence of implant type and endplate retention or removal.

This is a comparative in vitro biomechanical study of the primary stability of an anterior lumbar interbody stabilization. The objective was to compare the stability of a interbody stabilizing titanium cage with and without the retention of the bordering vertebral endplates, as well as to compare the titanium cage with a tricalcium phosphate block when the endplates are removed. An adequate blood supply is critical for interbody fusion, which suggests surgical treatment of the bordering endplates.

Comparative biomechanical testing of anterior and posterior stabilization procedures.

Study Design: This is a comparative in vitro biomechanical study in a calf lumbar spine model.

Objectives: The objective was to compare the primary stability of an anterior instrumentation, an intercorporal cage in combination with an anterior instrumentation, and a posterior instrumentation for monosegmental spondylodesis.

Summary Of Background Data: Spondylodesis can be achieved through a posterior lumbar fusion, posterior lumbar intercorporal fusion, or an anterior lumbar intercorporal fusion.

[Biomechanical testing of different ventral fixation devices on the bovine lumbar spine].

Aim: The primary stability of ventral fixation systems has been improved over the past years and special implants supporting minimally invasive procedures have been developed. The aim of this study was to analyze the primary stability of several of these implants.

Methods: Thirty (30) lumbar segments from 5 to 7-month-old calves were harvested and tested on a pure moment apparatus (PMA) as well as a modified materials testing machine (MTS) according to the European standard recommendations of Wilke et al.