Identification of mycolic acid forms using surface-enhanced Raman scattering as a fast detection method for tuberculosis

Background: Tuberculosis (TB) is the ninth leading cause of death worldwide and the leading cause from a single infectious agent, based on the WHO Global Tuberculosis Report in 2017. TB causes massive health care burdens in many parts of the world, specifically in the resource constrained developing world. Most deaths from TB could be prevented with cost effective early diagnosis and appropriate treatment. Purpose: Conventional TB detection methods are either too slow as it takes a few weeks for diagnosis or they lack the specificity and accuracy. Thus the objective of this study was to develop a fast and efficient detection for TB using surface enhanced Raman scattering (SERS) technique. Methods: SERS spectra for different forms of mycolic acids (MAs) that are both synthetic origin and actual extracts from the mycobacteria species were obtained by label-free direct detection mode. Similarly, we collected SERS spectra for γ-irradiated whole bacteria (WB). Measurements were done using silver (Ag) coated silicon nanopillar (Ag SNP) as SERS substrate. Results: We report the SERS based detection of MA, which is a biomarker for mycobacteria species including Mycobacterium tuberculosis. For the first time, we also establish the SERS spectral characterization of the three major forms of MA – αMA, methoxy-MA, and keto-MA, in bacterial extracts and also in γ-irradiated WB. We validated our findings by mass spectrometry. SERS detection of these three forms of MA could be useful in differentiating pathogenic and nonpathogenic Mycobacterium spp. Conclusions: We have demonstrated the direct detection of three major forms of MA – αMA, methoxy-MA, and keto-MA, in two different types of MA extracts from MTB bacteria, namely delipidated MA and undelipidated MA and finally in γ-irradiated WB. In the near future, this study could pave the way for a fast and efficient detection method for TB, which is of high clinical significance

Establishment of a human cell-based in vitro battery to assess developmental neurotoxicity hazard of chemicals

Developmental neurotoxicity (DNT) is a major safety concern for all chemicals of the human exposome. However, DNT data from animal studies are available for only a small percentage of manufactured compounds. Test methods with a higher throughput than current regulatory guideline methods, and with improved human relevance are urgently needed. We therefore explored the feasibility of DNT hazard assessment based on new approach methods (NAMs). An in vitro battery (IVB) was assembled from ten individual NAMs that had been developed during the past years to probe effects of chemicals on various fundamental neurodevelopmental processes. All assays used human neural cells at different developmental stages. This allowed us to assess disturbances of: (i) proliferation of neural progenitor cells (NPC); (ii) migration of neural crest cells, radial glia cells, neurons and oligodendrocytes; (iii) differentiation of NPC into neurons and oligodendrocytes; and (iv) neurite outgrowth of peripheral and central neurons. In parallel, cytotoxicity measures were obtained. The feasibility of concentration-dependent screening and of a reliable biostatistical processing of the complex multi-dimensional data was explored with a set of 120 test compounds, containing subsets of pre-defined positive and negative DNT compounds. The battery provided alerts (hit or borderline) for 24 of 28 known toxicants (82% sensitivity), and for none of the 17 negative controls. Based on the results from this screen project, strategies were developed on how IVB data may be used in the context of risk assessment scenarios employing integrated approaches for testing and assessment (IATA).European Food Safety Authority (EFSA-Q-2018-00308), the Danish Environmental Protection Agency (EPA), Denmark, under the grant number MST-667-00205, the State Ministry of Baden-Wuerttemberg, Germany, for Economic Affairs, Labour and Tourism (NAM-Accept), the project CERST (Center for Alternatives to Animal Testing) of the Ministry for culture and science of the State of North-Rhine Westphalia, Germany (file number 233–1.08.03.03- 121972/131–1.08.03.03–121972), the European Chemical Industry Council Long-Range Research Initiative (Cefic LRI) under the project name AIMT11 and the BMBF (NeuroTool). It has also received funding from the European Union's Horizon 2020 research and innovation program under grant agreements No. 964537 (RISK-HUNT3R), No. 964518 (ToxFree), No. 101057014 (PARC) and No. 825759 (ENDpoiNTs)

13C labeling experiments at metabolic nonstationary conditions: An exploratory study

<p>Abstract</p> <p>Background</p> <p>Stimulus Response Experiments to unravel the regulatory properties of metabolic networks are becoming more and more popular. However, their ability to determine enzyme kinetic parameters has proven to be limited with the presently available data. In metabolic flux analysis, the use of ¹³C labeled substrates together with isotopomer modeling solved the problem of underdetermined networks and increased the accuracy of flux estimations significantly.</p> <p>Results</p> <p>In this contribution, the idea of increasing the information content of the dynamic experiment by adding ¹³C labeling is analyzed. For this purpose a small example network is studied by simulation and statistical methods. Different scenarios regarding available measurements are analyzed and compared to a non-labeled reference experiment. Sensitivity analysis revealed a specific influence of the kinetic parameters on the labeling measurements. Statistical methods based on parameter sensitivities and different measurement models are applied to assess the information gain of the labeled stimulus response experiment.</p> <p>Conclusion</p> <p>It was found that the use of a (specifically) labeled substrate will significantly increase the parameter estimation accuracy. An overall information gain of about a factor of six is observed for the example network. The information gain is achieved from the specific influence of the kinetic parameters towards the labeling measurements. This also leads to a significant decrease in correlation of the kinetic parameters compared to an experiment without ¹³C-labeled substrate.</p

A Novel Signaling Network Essential for Regulating Pseudomonas aeruginosa Biofilm Development

The important human pathogen Pseudomonas aeruginosa has been linked to numerous biofilm-related chronic infections. Here, we demonstrate that biofilm formation following the transition to the surface attached lifestyle is regulated by three previously undescribed two-component systems: BfiSR (PA4196-4197) harboring an RpoD-like domain, an OmpR-like BfmSR (PA4101-4102), and MifSR (PA5511-5512) belonging to the family of NtrC-like transcriptional regulators. These two-component systems become sequentially phosphorylated during biofilm formation. Inactivation of bfiS, bfmR, and mifR arrested biofilm formation at the transition to the irreversible attachment, maturation-1 and -2 stages, respectively, as indicated by analyses of biofilm architecture, and protein and phosphoprotein patterns. Moreover, discontinuation of bfiS, bfmR, and mifR expression in established biofilms resulted in the collapse of biofilms to an earlier developmental stage, indicating a requirement for these regulatory systems for the development and maintenance of normal biofilm architecture. Interestingly, inactivation did not affect planktonic growth, motility, polysaccharide production, or initial attachment. Further, we demonstrate the interdependency of this two-component systems network with GacS (PA0928), which was found to play a dual role in biofilm formation. This work describes a novel signal transduction network regulating committed biofilm developmental steps following attachment, in which phosphorelays and two sigma factor-dependent response regulators appear to be key components of the regulatory machinery that coordinates gene expression during P. aeruginosa biofilm development in response to environmental cues

Two-component signal transduction in Corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets

In bacteria, adaptation to changing environmental conditions is often mediated by two-component signal transduction systems. In the prototypical case, a specific stimulus is sensed by a membrane-bound histidine kinase and triggers autophosphorylation of a histidine residue. Subsequently, the phosphoryl group is transferred to an aspartate residue of the cognate response regulator, which then becomes active and mediates a specific response, usually by activating and/or repressing a set of target genes. In this review, we summarize the current knowledge on two-component signal transduction in Corynebacterium glutamicum. This Gram-positive soil bacterium is used for the large-scale biotechnological production of amino acids and can also be applied for the synthesis of a wide variety of other products, such as organic acids, biofuels, or proteins. Therefore, C. glutamicum has become an important model organism in industrial biotechnology and in systems biology. The type strain ATCC 13032 possesses 13 two-component systems and the role of five has been elucidated in recent years. They are involved in citrate utilization (CitAB), osmoregulation and cell wall homeostasis (MtrAB), adaptation to phosphate starvation (PhoSR), adaptation to copper stress (CopSR), and heme homeostasis (HrrSA). As C. glutamicum does not only face changing conditions in its natural environment, but also during cultivation in industrial bioreactors of up to 500 m3 volume, adaptability can also be crucial for good performance in biotechnological production processes. Detailed knowledge on two-component signal transduction and regulatory networks therefore will contribute to both the application and the systemic understanding of C. glutamicum and related species

Identification of mycolic acid forms using surface-enhanced Raman scattering as a fast detection method for tuberculosis

Jayakumar Perumal,1 US Dinishm,1 Anne K Bendt,2 Agne Kazakeviciute,1,3 Chit Yaw Fu,1 Irvine Lian Hao Ong,4 Malini Olivo1 1Laboratory of Bio-optical Imaging, Singapore Bioimaging Consortium, Agency for Science, Technology, and Research (A*STAR), Singapore; 2Singapore Lipidomics Incubator, Life Sciences Institute, National University of Singapore, Singapore; 3Department of Statistical Science, University College London, London, UK; 4Matralix Pte Ltd, Singapore Background: Tuberculosis (TB) is the ninth leading cause of death worldwide and the leading cause from a single infectious agent, based on the WHO Global Tuberculosis Report in 2017. TB causes massive health care burdens in many parts of the world, specifically in the resource constrained developing world. Most deaths from TB could be prevented with cost effective early diagnosis and appropriate treatment.Purpose: Conventional TB detection methods are either too slow as it takes a few weeks for diagnosis or they lack the specificity and accuracy. Thus the objective of this study was to develop a fast and efficient detection for TB using surface enhanced Raman scattering (SERS) technique.Methods: SERS spectra for different forms of mycolic acids (MAs) that are both synthetic origin and actual extracts from the mycobacteria species were obtained by label-free direct detection mode. Similarly, we collected SERS spectra for &gamma;-irradiated whole bacteria (WB). Measurements were done using silver (Ag) coated silicon nanopillar (Ag SNP) as SERS substrate.Results: We report the SERS based detection of MA, which is a biomarker for mycobacteria species including Mycobacterium tuberculosis. For the first time, we also establish the SERS spectral characterization of the three major forms of MA &ndash; &alpha;MA, methoxy-MA, and keto-MA, in bacterial extracts and also in &gamma;-irradiated WB. We validated our findings by mass spectrometry. SERS detection of these three forms of MA could be useful in differentiating pathogenic and nonpathogenic Mycobacterium spp.Conclusions: We have demonstrated the direct detection of three major forms of MA &ndash; &alpha;MA, methoxy-MA, and keto-MA, in two different types of MA extracts from MTB bacteria, namely delipidated MA and undelipidated MA and finally in &gamma;-irradiated WB. In the near future, this study could pave the way for a fast and efficient detection method for TB, which is of high clinical significance. Keywords: Mycobacterium tuberculosis, MTB, nontuberculosis mycobacteria, NTM, mycolic acid, MA, SERS, silver-coated silicon nanopillars, Ag SNPs, liquid chromatography mass spectrometry, LC-M

Lessons from the Singapore cohorts showcase symposium—open call for collaborations

10.1007/s10654-023-00999-1European Journal of Epidemiolog

Umbilical Cord Plasma Lysophospholipids and Triacylglycerols Associated with Birthweight Percentiles

10.3390/nu16020274Nutrients16227