Developing Microelectrode Arrays for the Point-of-Care Multiplex Detection of Metabolites.

DNA-aptamer-functionalized electrode arrays can provide an intriguing method for detecting pathogen-derived exometabolites. This work addresses the limitations of previous aptamer-based pathogen detection methods by introducing a novel surface design that bridges the gap between initial efforts in this area and the demands of a point-of-care device. Specifically, the use of a diblock copolymer coating on a high-density microelectrode array and Cu-mediated cross coupling reactions that allow for the exclusive functionalization of that coating by any electrode or set of electrodes in the array provides a device that is stable for 1 year and compatible with the multiplex detection of small-molecule targets. The new chemistry developed allows one to take advantage of a large number of electrodes in the array with one experiment described herein capitalizing on the use of 960 individually addressable electrodes.

Spatial Visualization of A-to-I Editing in Cells Using Endonuclease V Immunostaining Assay (EndoVIA).

Adenosine-to-inosine (A-to-I) editing is one of the most widespread post-transcriptional RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Varying across tissue types, A-to-I editing is essential for numerous biological functions, and dysregulation leads to autoimmune and neurological disorders, as well as cancer. Recent evidence has also revealed a link between RNA localization and A-to-I editing, yet understanding of the mechanisms underlying this relationship and its biological impact remains limited. Current methods rely primarily on in vitro characterization of extracted RNA that ultimately erases subcellular localization and cell-to-cell heterogeneity. To address these challenges, we have repurposed endonuclease V (EndoV), a magnesium-dependent ribonuclease that cleaves inosine bases in edited RNA, to selectively bind and detect A-to-I edited RNA in cells. The work herein introduces an endonuclease V immunostaining assay (EndoVIA), a workflow that provides spatial visualization of edited transcripts, enables rapid quantification of overall inosine abundance, and maps the landscape of A-to-I editing within the transcriptome at the nanoscopic level.

Spatial visualization of A-to-I Editing in cells using Endonuclease V Immunostaining Assay (EndoVIA).

Adenosine-to-Inosine (A-to-I) editing is one of the most widespread post-transcriptional RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Varying across tissue types, A-to-I editing is essential for numerous biological functions and dysregulation leads to autoimmune and neurological disorders, as well as cancer. Recent evidence has also revealed a link between RNA localization and A-to-I editing, yet understanding of the mechanisms underlying this relationship and its biological impact remains limited. Current methods rely primarily on in vitro characterization of extracted RNA that ultimately erases subcellular localization and cell-to-cell heterogeneity. To address these challenges, we have repurposed Endonuclease V (EndoV), a magnesium dependent ribonuclease that cleaves inosine bases in edited RNA, to selectively bind and detect A-to-I edited RNA in cells. The work herein introduces Endonuclease V Immunostaining Assay (EndoVIA), a workflow that provides spatial visualization of edited transcripts, enables rapid quantification of overall inosine abundance, and maps the landscape of A-to-I editing within the transcriptome at the nanoscopic level.

Untangling Macropore Formation and Current Facilitation in P2X7.

Macropore formation and current facilitation are intriguing phenomena associated with ATP-gated P2X7 receptors (P2X7). Macropores are large pores formed in the cell membrane that allow the passage of large molecules. The precise mechanisms underlying macropore formation remain poorly understood, but recent evidence suggests two alternative pathways: a direct entry through the P2X7 pore itself, and an indirect pathway triggered by P2X7 activation involving additional proteins, such as TMEM16F channel/scramblase. On the other hand, current facilitation refers to the progressive increase in current amplitude and activation kinetics observed with prolonged or repetitive exposure to ATP. Various mechanisms, including the activation of chloride channels and intrinsic properties of P2X7, have been proposed to explain this phenomenon. In this comprehensive review, we present an in-depth overview of P2X7 current facilitation and macropore formation, highlighting new findings and proposing mechanistic models that may offer fresh insights into these untangled processes.

Dyspnea-induced Limitation (DYSLIM), a new self-administered concise questionnaire to evaluate dyspnea-related activity limitation in chronic respiratory diseases.

BACKGROUND AND AIM: Few questionnaires are available for routine assessment of dyspnea. The study aimed to design a self-administered questionnaire assessing the impact of chronic dyspnea on daily activities, named DYSLIM (Dyspnea-induced Limitation). METHODS: The development followed 4 steps: 1: selection of relevant activities and related questions (focus groups); 2: clinical study: internal and concurrent validity vs. modified Medical Research Council (mMRC), Baseline Dyspnea Index (BDI) and Saint George Respiratory Questionnaire (SGRQ); 3: item reduction; 4: responsiveness. Eighteen activities (from eating to climbing stairs) were considered with 5 modalities for each: doing the task slowly, taking breaks, seeking assistance, changing habits, and activity avoidance. Each modality was graded from 5 (never) to 1 (very often). Validation study included 194 patients: COPD (FEV1 ≥ 50% pred: n = 40; FEV1 < 50% pred: n = 65); cystic fibrosis (n = 30), interstitial lung disease (n = 30), pulmonary hypertension (n = 29). Responsiveness was evaluated by post-pulmonary rehabilitation data in 52 COPD patients. RESULTS: Acceptability was high and short term (7 days) reproducibility was satisfactory (Kappa mostly above 0.7). Concurrent validity was high vs. mMRC (Spearman correlation coefficient, r = 0.71), BDI (r = - 0.75) and SGRQ (r = - 0.79). The reduced questionnaire with 8 activities (from cleaning to climbing stairs) and 3 modalities (slowly, seeking help, changing habits) showed a comparable validity and was chosen as the final short version. Effect size of rehabilitation was good for both the full (0.57) and short (0.51) versions. A significant correlation was also found between changes of SGRQ and DYSLIM post rehabilitation: r = - 0.68 and r = - 0.60 for full and reduced questionnaires, respectively. CONCLUSION: The DYSLIM questionnaire appears promising for the evaluation of dyspnea-induced limitations in chronic respiratory diseases and seems suitable for use in various contexts.

Keratoconus and the Impact of Treatment on Patients' Quality of Life: A Qualitative Study.

INTRODUCTION: Keratoconus has a significant impact on patients' quality of life (QoL), from diagnosis to the advanced stages of the disease. The aim of this research was to identify domains of QoL affected by this disease and its treatment. METHODS: Phone interviews were conducted using a semi-structured interview guide, with patients with keratoconus stratified according to their current treatment. A board of keratoconus experts helped identify the guide's main themes. RESULTS: Thirty-five patients (rigid contact lenses, n = 9; cross-linking, n = 9; corneal ring implants, n = 8; and corneal transplantation, n = 9) were interviewed by qualitative researchers. Phone interviews revealed several QoL domains affected by the disease and its treatments: "psychological", "social life", "professional life", "financial costs" and "student life". All domains were impacted, independently of the treatment history. Few differences were found between treatment regimens and keratoconus stages. Qualitative analysis enabled the development of a conceptual framework based on Wilson and Cleary's model for patient outcomes common to all patients. This conceptual model describes the relationship between patients' characteristics, their symptoms, their environment, their functional visual impairment and the impact on their QoL. CONCLUSIONS: These qualitative findings supported the generation of a questionnaire to evaluate the impact of keratoconus and its treatment on patients' QoL. Cognitive debriefings confirmed its content validity. The questionnaire is applicable for all stages of keratoconus and treatments and may help tracking change over time in regular clinical settings. Psychometric validation is yet to be performed before its use in research and clinical practices.

Optical control of PIEZO1 channels.

PIEZO proteins are unusually large, mechanically-activated trimeric ion channels. The central pore features structural similarities with the pore of other trimeric ion channels, including purinergic P2X receptors, for which optical control of channel gating has been previously achieved with photoswitchable azobenzenes. Extension of these chemical optogenetics methods to mechanically-activated ion channels would provide tools for specific manipulation of pore activity alternative to non-specific mechanical stimulations. Here we report a light-gated mouse PIEZO1 channel, in which an azobenzene-based photoswitch covalently tethered to an engineered cysteine, Y2464C, localized at the extracellular apex of the transmembrane helix 38, rapidly triggers channel gating upon 365-nm-light irradiation. We provide evidence that this light-gated channel recapitulates mechanically-activated PIEZO1 functional properties, and show that light-induced molecular motions are similar to those evoked mechanically. These results push the limits of azobenzene-based methods to unusually large ion channels and provide a simple stimulation means to specifically interrogate PIEZO1 function.

Tracking the Message: Applying Single Molecule Localization Microscopy to Cellular RNA Imaging.

RNA function is increasingly appreciated to be more complex than merely communicating between DNA sequence and protein structure. RNA localization has emerged as a key contributor to the intricate roles RNA plays in the cell, and the link between dysregulated spatiotemporal localization and disease warrants an exploration beyond sequence and structure. However, the tools needed to visualize RNA with precise resolution are lacking in comparison to methods available for studying proteins. In the past decade, many techniques have been developed for imaging RNA, and in parallel super resolution and single-molecule techniques have enabled imaging of single molecules in cells. Of these methods, single molecule localization microscopy (SMLM) has shown significant promise for probing RNA localization. In this review, we highlight current approaches that allow super resolution imaging of specific RNA transcripts and summarize challenges and future opportunities for developing innovative RNA labeling methods that leverage the power of SMLM.

Perceived Unmet Needs in Patients Living With Advanced Bladder Cancer and Their Caregivers: Infodemiology Study Using Data From Social Media in the United States.

BACKGROUND: Locally advanced or metastatic bladder cancer (BC), which is generally termed advanced BC (aBC), has a very poor prognosis, and in addition to its physical symptoms, it is associated with emotional and social challenges. However, few studies have assessed the unmet needs and burden of aBC from patient and caregiver perspectives. Infodemiology, that is, epidemiology based on internet health-related content, can help obtain more insights on patients' and caregivers' experiences with aBC. OBJECTIVE: The study aimed to identify the main discussion themes and the unmet needs of patients with aBC and their caregivers through a mixed methods analysis of social media posts. METHODS: Social media posts were collected between January 2015 and April 2021 from US geolocalized sites using specific keywords for aBC. Automatic natural language processing (regular expressions and machine learning) methods were used to filter out irrelevant content and identify verbatim posts from patients and caregivers. The verbatim posts were analyzed to identify main discussion themes using biterm topic modeling. Difficulties or unmet needs were further explored using qualitative research methods by 2 independent annotators until saturation of concepts. RESULTS: A total of 688 posts from 262 patients and 1214 posts from 679 caregivers discussing aBC were identified. Analysis of 340 randomly selected patient posts and 423 randomly selected caregiver posts uncovered 33 unique unmet need categories among patients and 36 among caregivers. The main unmet patient needs were related to challenges regarding adverse events (AEs; 28/95, 29%) and the psychological impact of aBC (20/95, 21%). Other patient unmet needs identified were prognosis or diagnosis errors (9/95, 9%) and the need for better management of aBC symptoms (9/95, 9%). The main unmet caregiver needs were related to the psychological impacts of aBC (46/177, 26.0%), the need for support groups and to share experiences between peers (28/177, 15.8%), and the fear and management of patient AEs (22/177, 12.4%). CONCLUSIONS: The combination of manual and automatic methods allowed the extraction and analysis of several hundreds of social media posts from patients with aBC and their caregivers. The results highlighted the emotional burden of cancer for both patients and caregivers. Additional studies on patients with aBC and their caregivers are required to quantitatively explore the impact of this disease on quality of life.

Development of the SPUR tool: a profiling instrument for patient treatment behavior.

BACKGROUND: Long-term treatment adherence is a worldwide concern, with nonadherence resulting from a complex interplay of behaviors and health beliefs. Determining an individual's risk of nonadherence and identifying the drivers of that risk are crucial for the development of successful interventions for improving adherence. Here, we describe the development of a new tool assessing a comprehensive set of characteristics predictive of patients' treatment adherence based on the Social, Psychological, Usage and Rational (SPUR) adherence framework. Concepts from existing self-reporting tools of adherence-related behaviors were identified following a targeted MEDLINE literature review and a subset of these concepts were then selected for inclusion in the new tool. SPUR tool items, simultaneously generated in US English and in French, were tested iteratively through two rounds of cognitive interviews with US and French patients taking long-term treatments for chronic diseases. The pilot SPUR tool, resulting from the qualitative analysis of patients' responses, was then adapted to other cultural settings (China and the UK) and subjected to further rounds of cognitive testing. RESULTS: The literature review identified 27 relevant instruments, from which 49 concepts were included in the SPUR tool (Social: 6, Psychological: 13, Usage: 11, Rational: 19). Feedback from US and French patients suffering from diabetes, multiple sclerosis, or breast cancer (n = 14 for the first round; n = 16 for the second round) indicated that the SPUR tool was well accepted and consistently understood. Minor modifications were implemented, resulting in the retention of 45 items (Social: 5, Psychological: 14, Usage: 10, Rational: 16). Results from the cognitive interviews conducted in China (15 patients per round suffering from diabetes, breast cancer or chronic obstructive pulmonary disease) and the UK (15 patients suffering from diabetes) confirmed the validity of the tool content, with no notable differences being identified across countries or chronic conditions. CONCLUSION: Our qualitative analyses indicated that the pilot SPUR tool is a promising model that may help clinicians and health systems to predict patient treatment behavior. Further steps using quantitative methods are needed to confirm its predictive validity and other psychometric properties.