Effects of Low Intensity Focused Ultrasound on Liposomes Containing Channel proteins.

The ability to reversibly and non-invasively modulate region-specific brain activity in vivo suggests Low Intensity Focused Ultrasound (LIFU) as potential therapeutics for neurological dysfunctions such as epilepsy and Parkinson's disease. While in vivo studies provide evidence of the bioeffects of LIFU on neuronal activity, they merely hint at potential mechanisms but do not fully explain how this technology achieves these effects. One potential hypothesis is that LIFU produces local membrane depolarization by mechanically perturbing the neuronal cell membrane, or activating channels or other proteins embedded in the membrane. Proteins that sense mechanical perturbations of the membrane, such as those gated by membrane tension, are prime candidates for activating in response to LIFU and thus leading to the neurological responses that have been measured. Here we use the bacterial mechanosensitive channel MscL, which has been purified and reconstituted in liposomes, to determine how LIFU may affect the activation of this membrane-tension gated channel. Two bacterial voltage-gated channels, KvAP and NaK2K F92A channels were also studied. Surprisingly, the results suggest that ultrasound modulation and membrane perturbation does not induce channel gating, but rather induces pore formation at the membrane protein-lipid interface. However, in vesicles with high MscL mechanosensitive channel concentrations, apparent decreases in pore formation are observed, suggesting that this membrane-tension-sensitive protein may serve to increase the elasticity of the membrane, presumably because of expansion of the channel in the plane of the membrane independent of channel gating

Swelling-Activated Ca2+ Channels Trigger Ca2+ Signals in Merkel Cells

Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute to touch reception in the Merkel cell-neurite complex

Effects of Low Intensity Focused Ultrasound on Liposomes Containing Channel proteins.

The ability to reversibly and non-invasively modulate region-specific brain activity in vivo suggests Low Intensity Focused Ultrasound (LIFU) as potential therapeutics for neurological dysfunctions such as epilepsy and Parkinson's disease. While in vivo studies provide evidence of the bioeffects of LIFU on neuronal activity, they merely hint at potential mechanisms but do not fully explain how this technology achieves these effects. One potential hypothesis is that LIFU produces local membrane depolarization by mechanically perturbing the neuronal cell membrane, or activating channels or other proteins embedded in the membrane. Proteins that sense mechanical perturbations of the membrane, such as those gated by membrane tension, are prime candidates for activating in response to LIFU and thus leading to the neurological responses that have been measured. Here we use the bacterial mechanosensitive channel MscL, which has been purified and reconstituted in liposomes, to determine how LIFU may affect the activation of this membrane-tension gated channel. Two bacterial voltage-gated channels, KvAP and NaK2K F92A channels were also studied. Surprisingly, the results suggest that ultrasound modulation and membrane perturbation does not induce channel gating, but rather induces pore formation at the membrane protein-lipid interface. However, in vesicles with high MscL mechanosensitive channel concentrations, apparent decreases in pore formation are observed, suggesting that this membrane-tension-sensitive protein may serve to increase the elasticity of the membrane, presumably because of expansion of the channel in the plane of the membrane independent of channel gating

Human Fecal Bile Acid Analysis after Investigational Microbiota-Based Live Biotherapeutic Delivery for Recurrent <i>Clostridioides difficile</i> Infection

Microbiome-based therapeutics are increasingly evaluated as a strategy to reduce recurrent Clostridioides difficile infection (rCDI), with proposed mechanisms including restoration of the microbiota and microbiota-mediated functions, such as bile acid (BA) metabolism. This study reports a quantitative and sensitive assay for targeted metabolomic assessment, and the application of the assay to profile BA composition in a Phase 2 trial of the investigational microbiota-based live biotherapeutic RBX2660 for reduction of rCDI. A liquid chromatography tandem mass spectrometry method was developed to extract and quantify 35 BAs from 113 participant stool samples from 27 RBX2660-treated rCDI participants in the double-blinded, placebo-controlled clinical trial. The results demonstrate a high-confidence assay as represented by sensitivity, linearity, accuracy, and precision. Furthermore, the assay enabled the observation of primary BAs as the dominant BA species at baseline in stool samples from clinical trial participants, consistent with the expected loss of commensals after broad-spectrum antibiotic treatment. After RBX2660 administration, there was a significant drop in primary BAs concurrent with increased secondary BAs that sustained through 24 months post-RBX2660. Taken together, we describe a robust assay that demonstrates altered BA metabolism in rCDI patients treated with RBX2660 administration

Introductory Statistics

Introductory Statistics follows scope and sequence requirements of a one-semester introduction to statistics course and is geared toward students majoring in fields other than math or engineering. The text assumes some knowledge of intermediate algebra and focuses on statistics application over theory. Introductory Statistics includes innovative practical applications that make the text relevant and accessible, as well as collaborative exercises, technology integration problems, and statistics labs