Bacterial mechanosensitive channels: models for studying mechanosensory transduction

Significance: Sensations of touch and hearing are manifestations of mechanical contact and air pressure acting on touch receptors and hair cells of the inner ear, respectively. In bacteria, osmotic pressure exerts a significant mechanical force on their cellular membrane. Bacteria have evolved mechanosensitive (MS) channels to cope with excessive turgor pressure resulting from a hypo-osmotic shock. MS channel opening allows the expulsion of osmolytes and water, thereby restoring normal cellular turgor and preventing cell lysis. Recent Advances: As biological force-sensing systems, MS channels have been identified as the best examples of membrane proteins coupling molecular dynamics to cellular mechanics. The bacterial MS channel of large conductance (MscL) and MS channel of small conductance (MscS) have been subjected to extensive biophysical, biochemical, genetic, and structural analyses. These studies have established MscL and MscS as model systems for mechanosensory transduction. Critical Issues: In recent years, MS ion channels in mammalian cells have moved into focus of mechanotransduction research, accompanied by an increased awareness of the role they may play in the pathophysiology of diseases, including cardiac hypertrophy, muscular dystrophy, or Xerocytosis. Future Directions: A recent exciting development includes the molecular identification of Piezo proteins, which function as nonselective cation channels in mechanosensory transduction associated with senses of touch and pain. Since research on Piezo channels is very young, applying lessons learned from studies of bacterial MS channels to establishing the mechanism by which the Piezo channels are mechanically activated remains one of the future challenges toward a better understanding of the role that MS channels play in mechanobiology

Validation in Forensic Text Comparison: Issues and Opportunities

It has been argued in forensic science that the empirical validation of a forensic inference system or methodology should be performed by replicating the conditions of the case under investigation and using data relevant to the case. This study demonstrates that the above requirement for validation is also critical in forensic text comparison (FTC); otherwise, the trier-of-fact may be misled for their final decision. Two sets of simulated experiments are performed: one fulfilling the above validation requirement and the other overlooking it, using mismatch in topics as a case study. Likelihood ratios (LRs) are calculated via a Dirichlet-multinomial model, followed by logistic-regression calibration. The derived LRs are assessed by means of the log-likelihood-ratio cost, and they are visualized using Tippett plots. Following the experimental results, this paper also attempts to describe some of the essential research required in FTC by highlighting some central issues and challenges unique to textual evidence. Any deliberations on these issues and challenges will contribute to making a scientifically defensible and demonstrably reliable FTC available

Validation in Forensic Text Comparison: Issues and Opportunities

It has been argued in forensic science that the empirical validation of a forensic inference system or methodology should be performed by replicating the conditions of the case under investigation and using data relevant to the case. This study demonstrates that the above requirement for validation is also critical in forensic text comparison (FTC); otherwise, the trier-of-fact may be misled for their final decision. Two sets of simulated experiments are performed; one fulfilling the above validation requirement and the other overlooking it, using mismatch in topics as a case study. Likelihood ratios (LRs) are calculated via a Dirichlet-multinomial model, followed by logistic regression calibration. The derived LRs are assessed by means of the log-likelihood-ratio cost, and they are visualized using Tippett plots. Following the experimental results, this paper also attempts to describe some of the essential research required in FTC by highlighting some central issues and challenges unique to textual evidence. Any deliberations on these issues and challenges will contribute to making a scientifically defensible and demonstrably reliable FTC availabl

A high-throughput technique for screening novel antibacterial agents targeting bacterial mechanosensitive channels

Mechanosensitive Ion channels are essential for maintaining cellular homeostasis. It has been proposed that they function as osmotically activated emergency valves, guarding against membrane rupture by opening pores to release excessive turgor pressure. Interfering with their function suggests many novel mechanisms to complement conventional antimicrobials to kill or inhibit growth of bacterial pathogens. Typical of this class of mechanosensitive channel are the large conduction homopentameric bacterial channel, MscL (MW∼17kDa) and the small conduction homoheptameric bacterial channel, MscS (∼37kDa)

Transient potential gradients and impedance measures of tethered bilayer lipid membranes : pore-forming peptide insertion and the effect of electroporation

In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >∼150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa

Regulation of the membrane insertion and conductance activity of the metamorphic chloride intracellular channel protein CLIC1 by cholesterol.

The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer

Membrane Conductance of Lysteriolsin.

<p>Representative impedance spectroscopy recordings of (A-E) 2 µM Lysteriolysin and (F) buffer control, added to tethered membranes made from AM199 containing varying amounts of cholesterol (mol% chol) (A) 50, (B) 25, (C) 12.5, (D) 6.25, (E) 0, (F) 50; [n = 3].</p

Membrane Conductance of α-hemolysin.

<p>Representative impedance spectroscopy recordings of 50 nM α-hemolysin added to tethered membranes made from AM199 lipid containing varying amounts of cholesterol (mol% chol) (A) 0, (B) 10, (C) 20, (D) 30, (E) 40, (F) 50; [n = 3].</p

Insertion of CLIC1 into Lipid Monolayers.

<p>Representative area traces of Langmuir films held at a surface pressure of 20 mN/m with 0.036 µM CLIC1 (A-C) and (D, E) no CLIC, added beneath POPC monolayers containing cholesterol (mol% chol) (A) 16.7, (B) 9.0, (C) 0.0, (D) 16.7, (E) 0.</p