scholarly journals Accelerating Early Antituberculosis Drug Discovery by Creating Mycobacterial Indicator Strains That Predict Mode of Action

2018 ◽  
Vol 62 (7) ◽  
pp. e00083-18 ◽  
Author(s):  
Maikel Boot ◽  
Susanna Commandeur ◽  
Amit K. Subudhi ◽  
Meriem Bahira ◽  
Trever C. Smith ◽  
...  

ABSTRACT Due to the rise of drug-resistant forms of tuberculosis, there is an urgent need for novel antibiotics to effectively combat these cases and shorten treatment regimens. Recently, drug screens using whole-cell analyses have been shown to be successful. However, current high-throughput screens focus mostly on stricto sensu life/death screening that give little qualitative information. In doing so, promising compound scaffolds or nonoptimized compounds that fail to reach inhibitory concentrations are missed. To accelerate early tuberculosis (TB) drug discovery, we performed RNA sequencing on Mycobacterium tuberculosis and Mycobacterium marinum to map the stress responses that follow upon exposure to subinhibitory concentrations of antibiotics with known targets, ciprofloxacin, ethambutol, isoniazid, streptomycin, and rifampin. The resulting data set comprises the first overview of transcriptional stress responses of mycobacteria to different antibiotics. We show that antibiotics can be distinguished based on their specific transcriptional stress fingerprint. Notably, this fingerprint was more distinctive in M. marinum. We decided to use this to our advantage and continue with this model organism. A selection of diverse antibiotic stress genes was used to construct stress reporters. In total, three functional reporters were constructed to respond to DNA damage, cell wall damage, and ribosomal inhibition. Subsequently, these reporter strains were used to screen a small anti-TB compound library to predict the mode of action. In doing so, we identified the putative modes of action for three novel compounds, which confirms the utility of our approach.

2014 ◽  
Vol 58 (7) ◽  
pp. 3828-3836 ◽  
Author(s):  
Florian P. Maurer ◽  
Vera L. Bruderer ◽  
Claudia Ritter ◽  
Claudio Castelberg ◽  
Guido V. Bloemberg ◽  
...  

ABSTRACTAntibiotic therapy of infections caused by the emerging pathogenMycobacterium abscessusis challenging due to the organism's natural resistance toward most clinically available antimicrobials. We investigated the bactericidal activity of antibiotics commonly administered inM. abscessusinfections in order to better understand the poor therapeutic outcome. Time-kill curves were generated for clinicalM. abscessusisolates,Mycobacterium smegmatis, andEscherichia coliby using antibiotics commonly categorized as bactericidal (amikacin and moxifloxacin) or bacteriostatic (tigecycline and linezolid). In addition, the impact of aminoglycoside-modifying enzymes on the mode of action of substrate and nonsubstrate aminoglycosides was studied by usingM. smegmatisas a model organism. While amikacin and moxifloxacin were bactericidal againstE. coli, none of the tested compounds showed bactericidal activity againstM. abscessus. Further mechanistic investigations of the mode of action of aminoglycosides inM. smegmatisrevealed that the bactericidal activity of tobramycin and gentamicin was restored by disruption of the chromosomalaac(2′) gene in the mycobacterial genome. The lack of bactericidal antibiotics in currently recommended treatment regimens provides a reasonable explanation for the poor therapeutic outcome inM. abscessusinfection. Our findings suggest that chromosomally encoded drug-modifying enzymes play an important role in the lack of aminoglycoside bactericidal activity against rapidly growing mycobacteria.


2019 ◽  
Vol 63 (8) ◽  
Author(s):  
Matthew B. McNeil ◽  
Gregory M. Cook

ABSTRACT There is an urgent need for novel therapeutics to treat Mycobacterium tuberculosis infections. Genetic strategies for validating novel targets are available, yet their time-consuming nature limits their utility. Here, using MmpL3 as a model target, we report on the application of mycobacterial CRISPR interference for the rapid validation of target essentiality and compound mode of action. This strategy has the potential to rapidly accelerate tuberculosis drug discovery.


2015 ◽  
Vol 197 (13) ◽  
pp. 2160-2170 ◽  
Author(s):  
Jessica K. Kajfasz ◽  
Isamar Rivera-Ramos ◽  
Kathleen Scott-Anne ◽  
Stacy Gregoire ◽  
Jacqueline Abranches ◽  
...  

ABSTRACTThe SpxA1 and SpxA2 (formerly SpxA and SpxB) transcriptional regulators ofStreptococcus mutansare members of a highly conserved family of proteins found inFirmicutes, and they were previously shown to activate oxidative stress responses. In this study, we showed that SpxA1 exerts substantial positive regulatory influence over oxidative stress genes following exposure to H2O2, while SpxA2 appears to have a secondary regulatory role.In vitrotranscription (IVT) assays using purified SpxA1 and/or SpxA2 showed that SpxA1 and, less often, SpxA2 directly activate transcription of some of the major oxidative stress genes. Addition of equimolar concentrations of SpxA1 and SpxA2 to the IVT reactions neither enhanced transcription of the tested genes nor disrupted the dominant role of SpxA1. Substitution of a conserved glycine residue (G52) present in both Spx proteins by arginine (SpxG52R) resulted in strains that phenocopied the Δspxstrains. Moreover, addition of purified SpxA1G52Rcompletely failed to activate transcription ofahpC,sodA, andtpx, further confirming that the G52 residue is critical for Spx functionality.IMPORTANCEStreptococcus mutansis a pathogen associated with the formation of dental caries in humans. Within the oral cavity,S. mutansroutinely encounters oxidative stress. Our previous data revealed that two regulatory proteins, SpxA1 and SpxA2 (formerly SpxA and SpxB), bear high homology to the Spx regulator that has been characterized as a critical activator of oxidative stress genes inBacillus subtilis. In this report, we prove that Spx proteins ofS. mutansdirectly activate transcription of genes involved in the oxidative stress response, though SpxA1 appears to have a more dominant role than SpxA2. Therefore, the Spx regulators play a critical role in the ability ofS. mutansto thrive within the oral cavity.


2012 ◽  
Vol 80 (7) ◽  
pp. 2265-2275 ◽  
Author(s):  
Jessica K. Kajfasz ◽  
Jorge E. Mendoza ◽  
Anthony O. Gaca ◽  
James H. Miller ◽  
Kristy A. Koselny ◽  
...  

ABSTRACTThe ability to cope with endogenous or host-generated reactive oxygen species is considered a key virulence attribute of the opportunistic pathogenEnterococcus faecalis, a leading cause of hospital-acquired infections. In this study, we usedin silicoand mutational analyses to identify and characterize the role of the Spx global regulator in oxidative stress tolerance and virulence inE. faecalis. While the Δspxstrain grew as well as the wild-type strain under anaerobic conditions, the mutant strain exhibited impaired growth under aerobic conditions and was highly sensitive to oxidative stress agents. Thespxmutant strain was also sensitive to a variety of other stressful conditions, including antibiotic stress and killing by the mouse-derived macrophage cell line J774. Using a murine model of foreign body-associated peritonitis, we demonstrated that the ability of the Δspxstrain to colonize the peritoneum and disseminate in the bloodstream was significantly reduced compared to that of the parent strain. Transcriptional analysis revealed that a large number of known oxidative stress genes are under positive control by Spx. Collectively, our results show that Spx is a major stress gene regulator and is implicated in the pathophysiology ofE. faecalis. The relationship of Spx to other oxidative stress regulators is also discussed.


2020 ◽  
Vol 47 (3) ◽  
pp. 547-560 ◽  
Author(s):  
Darush Yazdanfar ◽  
Peter Öhman

PurposeThe purpose of this study is to empirically investigate determinants of financial distress among small and medium-sized enterprises (SMEs) during the global financial crisis and post-crisis periods.Design/methodology/approachSeveral statistical methods, including multiple binary logistic regression, were used to analyse a longitudinal cross-sectional panel data set of 3,865 Swedish SMEs operating in five industries over the 2008–2015 period.FindingsThe results suggest that financial distress is influenced by macroeconomic conditions (i.e. the global financial crisis) and, in particular, by various firm-specific characteristics (i.e. performance, financial leverage and financial distress in previous year). However, firm size and industry affiliation have no significant relationship with financial distress.Research limitationsDue to data availability, this study is limited to a sample of Swedish SMEs in five industries covering eight years. Further research could examine the generalizability of these findings by investigating other firms operating in other industries and other countries.Originality/valueThis study is the first to examine determinants of financial distress among SMEs operating in Sweden using data from a large-scale longitudinal cross-sectional database.


2017 ◽  
Vol 55 (4) ◽  
pp. 376-389 ◽  
Author(s):  
Alice Huguet ◽  
Caitlin C. Farrell ◽  
Julie A. Marsh

Purpose The use of data for instructional improvement is prevalent in today’s educational landscape, yet policies calling for data use may result in significant variation at the school level. The purpose of this paper is to focus on tools and routines as mechanisms of principal influence on data-use professional learning communities (PLCs). Design/methodology/approach Data were collected through a comparative case study of two low-income, low-performing schools in one district. The data set included interview and focus group transcripts, observation field notes and documents, and was iteratively coded. Findings The two principals in the study employed tools and routines differently to influence ways that teachers interacted with data in their PLCs. Teachers who were given leeway to co-construct data-use tools found them to be more beneficial to their work. Findings also suggest that teachers’ data use may benefit from more flexibility in their day-to-day PLC routines. Research limitations/implications Closer examination of how tools are designed and time is spent in data-use PLCs may help the authors further understand the influence of the principal’s role. Originality/value Previous research has demonstrated that data use can improve teacher instruction, yet the varied implementation of data-use PLCs in this district illustrates that not all students have an equal opportunity to learn from teachers who meaningfully engage with data.


2017 ◽  
Vol 37 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Haluk Ay ◽  
Anthony Luscher ◽  
Carolyn Sommerich

Purpose The purpose of this study is to design and develop a testing device to simulate interaction between human hand–arm dynamics, right-angle (RA) computer-controlled power torque tools and joint-tightening task-related variables. Design/methodology/approach The testing rig can simulate a variety of tools, tasks and operator conditions. The device includes custom data-acquisition electronics and graphical user interface-based software. The simulation of the human hand–arm dynamics is based on the rig’s four-bar mechanism-based design and mechanical components that provide adjustable stiffness (via pneumatic cylinder) and mass (via plates) and non-adjustable damping. The stiffness and mass values used are based on an experimentally validated hand–arm model that includes a database of model parameters. This database is with respect to gender and working posture, corresponding to experienced tool operators from a prior study. Findings The rig measures tool handle force and displacement responses simultaneously. Peak force and displacement coefficients of determination (R2) between rig estimations and human testing measurements were 0.98 and 0.85, respectively, for the same set of tools, tasks and operator conditions. The rig also provides predicted tool operator acceptability ratings, using a data set from a prior study of discomfort in experienced operators during torque tool use. Research limitations/implications Deviations from linearity may influence handle force and displacement measurements. Stiction (Coulomb friction) in the overall rig, as well as in the air cylinder piston, is neglected. The rig’s mechanical damping is not adjustable, despite the fact that human hand–arm damping varies with respect to gender and working posture. Deviations from these assumptions may affect the correlation of the handle force and displacement measurements with those of human testing for the same tool, task and operator conditions. Practical implications This test rig will allow the rapid assessment of the ergonomic performance of DC torque tools, saving considerable time in lineside applications and reducing the risk of worker injury. DC torque tools are an extremely effective way of increasing production rate and improving torque accuracy. Being a complex dynamic system, however, the performance of DC torque tools varies in each application. Changes in worker mass, damping and stiffness, as well as joint stiffness and tool program, make each application unique. This test rig models all of these factors and allows quick assessment. Social implications The use of this tool test rig will help to identify and understand risk factors that contribute to musculoskeletal disorders (MSDs) associated with the use of torque tools. Tool operators are subjected to large impulsive handle reaction forces, as joint torque builds up while tightening a fastener. Repeated exposure to such forces is associated with muscle soreness, fatigue and physical stress which are also risk factors for upper extremity injuries (MSDs; e.g. tendinosis, myofascial pain). Eccentric exercise exertions are known to cause damage to muscle tissue in untrained individuals and affect subsequent performance. Originality/value The rig provides a novel means for quantitative, repeatable dynamic evaluation of RA powered torque tools and objective selection of tightening programs. Compared to current static tool assessment methods, dynamic testing provides a more realistic tool assessment relative to the tool operator’s experience. This may lead to improvements in tool or controller design and reduction in associated musculoskeletal discomfort in operators.


2019 ◽  
Vol 11 (22) ◽  
pp. 2919-2973 ◽  
Author(s):  
Li-Wen Xia ◽  
Meng-Yu Ba ◽  
Wei Liu ◽  
Weyland Cheng ◽  
Chao-Ping Hu ◽  
...  

Current traditional drugs such as enzyme inhibitors and receptor agonists/antagonists present inherent limitations due to occupancy-driven pharmacology as the mode of action. Proteolysis targeting chimeras (PROTACs) are composed of an E3 ligand, a connecting linker and a target protein ligand, and are an attractive approach to specifically knockdown-targeted proteins utilizing an event-driven mode of action. The length, hydrophilicity and rigidity of connecting linkers play important role in creating a successful PROTAC. Some PROTACs with a triazole linker have displayed promising anticancer activity. This review provides an overview of PROTACs with a triazole scaffold and discusses its structure–activity relationship. Important milestones in the development of PROTACs are addressed and a critical analysis of this drug discovery strategy is also presented.


Aging Cell ◽  
2021 ◽  
Vol 20 (2) ◽  
Author(s):  
Yang Cheng ◽  
Andrew Pitoniak ◽  
Julia Wang ◽  
Dirk Bohmann

2019 ◽  
Vol 36 (4) ◽  
pp. 569-586
Author(s):  
Ricardo Puziol Oliveira ◽  
Jorge Alberto Achcar

Purpose The purpose of this paper is to provide a new method to estimate the reliability of series system by using a discrete bivariate distribution. This problem is of great interest in industrial and engineering applications. Design/methodology/approach The authors considered the Basu–Dhar bivariate geometric distribution and a Bayesian approach with application to a simulated data set and an engineering data set. Findings From the obtained results of this study, the authors observe that the discrete Basu–Dhar bivariate probability distribution could be a good alternative in the analysis of series system structures with accurate inference results for the reliability of the system under a Bayesian approach. Originality/value System reliability studies usually assume independent lifetimes for the components (series, parallel or complex system structures) in the estimation of the reliability of the system. This assumption in general is not reasonable in many engineering applications, since it is possible that the presence of some dependence structure between the lifetimes of the components could affect the evaluation of the reliability of the system.


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