Despite the emerging importance of reactive electrophilic drugs, deconvolution of their principal targets remains difficult. The lack of genetic tractability/interventions and reliance on secondary ...validation using other non-specific compounds frequently complicate the earmarking of individual binders as functionally- or phenotypically-sufficient pathway regulators. Using a redox-targeting approach to interrogate how on-target binding of pleiotropic electrophiles translates to a phenotypic output in vivo, we here systematically track the molecular components attributable to innate immune cell toxicity of the electrophilic-drug dimethyl fumarate (Tecfidera®). In a process largely independent of canonical Keap1/Nrf2-signaling, Keap1-specific modification triggers mitochondrial-targeted neutrophil/macrophage apoptosis. On-target Keap1-ligand-engagement is accompanied by dissociation of Wdr1 from Keap1 and subsequent coordination with cofilin, intercepting Bax. This phagocytic-specific cell-killing program is recapitulated by whole-animal administration of dimethyl fumarate, where individual depletions of the players identified above robustly suppress apoptosis.
Mechanical allodynia is a major symptom of neuropathic pain whereby innocuous touch evokes severe pain. Here we identify a population of peripheral sensory neurons expressing TrkB that are both ...necessary and sufficient for producing pain from light touch after nerve injury in mice. Mice in which TrkB-Cre-expressing neurons are ablated are less sensitive to the lightest touch under basal conditions, and fail to develop mechanical allodynia in a model of neuropathic pain. Moreover, selective optogenetic activation of these neurons after nerve injury evokes marked nociceptive behavior. Using a phototherapeutic approach based upon BDNF, the ligand for TrkB, we perform molecule-guided laser ablation of these neurons and achieve long-term retraction of TrkB-positive neurons from the skin and pronounced reversal of mechanical allodynia across multiple types of neuropathic pain. Thus we identify the peripheral neurons which transmit pain from light touch and uncover a novel pharmacological strategy for its treatment.
This Protocol Extension describes the adaptation of an existing Protocol detailing the use of targetable reactive electrophiles and oxidants, an on-demand redox targeting toolset in cultured cells. ...The adaptation described here is for use of reactive electrophiles and oxidants technologies in live zebrafish embryos (Z-REX). Zebrafish embryos expressing a Halo-tagged protein of interest (POI)-either ubiquitously or tissue specifically-are treated with a HaloTag-specific small-molecule probe housing a photocaged reactive electrophile (either natural electrophiles or synthetic electrophilic drug-like fragments). The reactive electrophile is then photouncaged at a user-defined time, enabling proximity-assisted electrophile-modification of the POI. Functional and phenotypic ramifications of POI-specific modification can then be monitored, by coupling to standard downstream assays, such as click chemistry-based POI-labeling and target-occupancy quantification; immunofluorescence or live imaging; RNA-sequencing and real-time quantitative polymerase chain reaction analyses of downstream-transcript modulations. Transient expression of requisite Halo-POI in zebrafish embryos is achieved by messenger RNA injection. Procedures associated with generation of transgenic zebrafish expressing a tissue-specific Halo-POI are also described. The Z-REX experiments can be completed in <1 week using standard techniques. To successfully execute Z-REX, researchers should have basic skills in fish husbandry, imaging and pathway analysis. Experience with protein or proteome manipulation is useful. This Protocol Extension is aimed at helping chemical biologists study precision redox events in a model organism and fish biologists perform redox chemical biology.
Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 ...displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5Low MuSCs and skews the stem cell pool toward MYF5High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles.
•The StemRep model represents a novel tool to study muscle stem cell heterogeneity•Differential expression of Myf5 identifies two subsets of muscle stem cells•Myf5Low muscle stem cells are deeply quiescent and have high self-renewal capacity•Aging leads to selective loss of deeply quiescent Myf5Low muscle stem cells
Ancel et al. introduce StemRep, a tool that enables real-time monitoring of Pax7 and Myf5 proteins in muscle stem cells. The authors describe a deeply quiescent muscle stem cell subset that is primarily lost with age. This model broadens the understanding of the role of Myf5 in regulating muscle stem cell quiescence and self-renewal in homeostatic, regenerating, and aged muscles.
This study is aimed at delineating the effectiveness of implementing flipped classroom learning models and analyzing the impact of this learning model on students’ listening ability. This study was ...conducted in the second year BBA class, at St. Mary’s Centenary Degree College, Secunderabad. The participants of this study were 45 students of general English Class. This study is designed as a descriptive qualitative research. The data obtained were based on observation, questionnaire and interview. Initially, observation techniques were used to gain information on how flipped classroom learning models were implemented. Secondly, questionnaires consisting of 15 open-ended statements were given to each participant to gain information about the impact of flipped classroom learning model toward students’ satisfaction. Finally, an interview was administered to strengthen the data of the questionnaire. The results of this study were that the flipped classroom learning model has been well - implemented in teaching the poem “As I grew Older” implicitly enhancing the listening skills. In addition, it was also found that this learning mode broke the physical limitation between students and lecturers. Another important result of this study was the improved motivational level and satisfaction from the end of the students and lecturers. Moreover, it was also found that flipped classrooms were difficult to implement in this classroom. However, it promoted self-directed learning and resulted in improved listening ability of the ESL learners once the learners were motivated.
In this present era with growing population, markets play an important role in providing the required and desired utilities to the people. For this it is important to enhance the Customer ...Relationship Management which can be achieved by segmenting the market utilities by various factors like weekly sales, demand and supply. In this research paper we discover the valuable information that is weekly sales and develop an efficient business strategy model to increase the profitability of the market through supply as well meet the demands of the customers. Our objectives are i) To find the top profitable products of the market across all the branches as well as their correlation with other products, ii) Understand the customer behavior according to the market flow and iii) Forecast the sales using ARIMA (Auto Regressive Moving Average). In this project, as we lack proper information about the customer identity, we use k means clustering which is an unsupervised learning model to cluster the customers according to the weekly sales behavior. Since we concentrate on the Weekly Sales to understand the market behavior, the best method to be applied on the dataset is the moving average technique. Here, we use ARIMA model to get the best results for forecasting as our time series data is considered to be stationary.
Pseudomonas aeruginosa
depends on its quorum sensing (QS) system for its virulence factors’ production and biofilm formation. Biofilms of
P. aeruginosa
on the surface of indwelling catheters are ...often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle
P. aeruginosa
infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by
Lactobacillus
species, against
P. aeruginosa
PAO1
.
Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of
P. aeruginosa
PAO1. Swarming motility, another important criterion for biofilm formation of
P. aeruginosa
PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with
P. aeruginosa
QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands
N
-butyryl-
l
-homoserine lactone (C
4
–HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS;
Pseudomonas
quinolone signal). Using an in vivo intraperitoneal catheter
-
associated medaka fish infection model, we proved that PLA inhibited the initial attachment of
P. aeruginosa
PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against
P. aeruginosa
.
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