Detection of a target protein (GroEl2) in Mycobacterium tuberculosis using a derivative of 1,2,4-triazolethiols.

Herein, we identified a potent lead compound RRA2, within a series of 54 derivatives of 1,2,4-triazolethiols (exhibit good potency as an anti-mycobacterial agents) against intracellular Mycobacterium tuberculosis (Mtb). Compound RRA2 showed significant mycobactericidal activity against active stage Mycobacterium bovis BCG and Mtb with minimum inhibitory concentration (MIC) values of 2.3 and 2.0 µg/mL, respectively. At MIC value, RRA2 compound yielded 0.82 log reduction of colony-forming unit (cfu) against non-replicating Mtb. Furthermore, RRA2 compound was selected for further target identification due to the presence of alkyne group, showing higher selectivity index (> 66.66 ± 0.22, in non-replicating stage). Using "click" chemistry, we synthesized the biotin linker-RRA2 conjugate, purified with HPLC method and confirmed the conjugation of biotin linker-RRA2 complex by HR-MS analysis. Furthermore, we successfully pulled down and identified a specific target protein GroEl2, from Mtb whole-cell extract. Furthermore, computational molecular modeling indicated RRA2 could interact with GroEl2, which explains the structure-activity relationship observed in this study. GroEL-2 identified a potent and specific target protein for RRA 2 compound in whole cell extract of Mtb H37Ra.

Viability determination of Ascaris ova in raw wastewater: a comparative evaluation of culture-based, BacLight Live/Dead staining and PMA-qPCR methods.

Accurate evaluation of viable Ascaris ova in wastewater is the key to mitigating Ascaris reinfections in endemic regions. In this study, the viability of Ascaris ova in raw wastewater was determined using three different detection methods: culture-based, BacLight Live/Dead staining and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR). Furthermore, comparative assessment of viability utilising the aforementioned detection methods was performed using seeded experiments in wastewater. The percentage of viability was: culture-based (82%), BacLight Live/Dead staining (87%) and PMA-qPCR (85%) respectively. Despite the fact that no statistical difference was shown in the viability determination among the three methods, PMA-qPCR-based viability determination would be preferable over the other two methods for evaluating potential public health risks with A. suum ova due to its accuracy, being least subjective and its rapid reaction time.

Self-Assembled Functional Nanostructure of Plasmid DNA with Ionic Liquid [Bmim][PF6]: Enhanced Efficiency in Bacterial Gene Transformation.

The electrostatic interaction between the negatively charged phosphate groups of plasmid DNA and the cationic part of hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]), initiates spontaneous self-assembly to form the functional nanostructures made up of DNA and ionic liquid (IL). These functional nanostructures were demonstrated as promising synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells. In particular, the functional nanostructures that were made up of 1 µL of IL ([Bmim][PF6]) and 1 µg of plasmid DNA can increase the transformation efficiency by 300-400% in microbial systems, without showing any toxicity for E. coli DH5α cells. (31)P nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron (XPS) spectroscopic analysis revealed that the electrostatic interaction between negatively charged phosphate oxygen and cationic Bmim(+) tends to initiate the self-assembly process. Thermogravimetric analysis of the DNA-IL functional nanostructures showed that these nanostructures consist of ∼16 wt % ionic liquid, which is considered to provide the stability to the plasmid DNA that eventually enhanced the transformation efficiency.

Charge-switchable gold nanoparticles for enhanced enzymatic thermostability.

This study illustrates a facile strategy for efficient immobilization of enzymes on a metal nanoparticle surface. The strategy proposed here enables the enzymatic activity to be retained while increasing the enzyme thermostability. It is demonstrated that the use of a zwitterionic amino acid tyrosine as a reducing and capping agent to synthesise gold nanoparticles allows efficient immobilization of phytase enzyme through charge-switchable electrostatic interactions. The detailed kinetic and thermodynamic studies reveal that the proposed enzyme immobilization strategy improves the overall quality of phytase by reducing the activation energy required for substrate hydrolysis and broadening the temperature window in which immobilized enzyme is able to operate. The outcomes of this study indicate that the underlying zwitterionic nature of 20 natural amino acids along with significant variability in their isoelectric points and hydropathy indices as well the ability of some of the amino acids to reduce metal ions is likely to offer significant opportunities for tailoring nano-bio interfaces in a rational manner for a range of biological applications.

quEHRy: a question answering system to query electronic health records.

OBJECTIVE: We propose a system, quEHRy, to retrieve precise, interpretable answers to natural language questions from structured data in electronic health records (EHRs). MATERIALS AND METHODS: We develop/synthesize the main components of quEHRy: concept normalization (MetaMap), time frame classification (new), semantic parsing (existing), visualization with question understanding (new), and query module for FHIR mapping/processing (new). We evaluate quEHRy on 2 clinical question answering (QA) datasets. We evaluate each component separately as well as holistically to gain deeper insights. We also conduct a thorough error analysis for a crucial subcomponent, medical concept normalization. RESULTS: Using gold concepts, the precision of quEHRy is 98.33% and 90.91% for the 2 datasets, while the overall accuracy was 97.41% and 87.75%. Precision was 94.03% and 87.79% even after employing an automated medical concept extraction system (MetaMap). Most incorrectly predicted medical concepts were broader in nature than gold-annotated concepts (representative of the ones present in EHRs), eg, Diabetes versus Diabetes Mellitus, Non-Insulin-Dependent. DISCUSSION: The primary performance barrier to deployment of the system is due to errors in medical concept extraction (a component not studied in this article), which affects the downstream generation of correct logical structures. This indicates the need to build QA-specific clinical concept normalizers that understand EHR context to extract the "relevant" medical concepts from questions. CONCLUSION: We present an end-to-end QA system that allows information access from EHRs using natural language and returns an exact, verifiable answer. Our proposed system is high-precision and interpretable, checking off the requirements for clinical use.

Development of A Rapid, Low-Cost Portable Detection Assay for Enterococci in Wastewater and Environmental Waters.

Waterborne diseases are known as a leading cause of illness and death in both developing and developed countries. Several pathogens can be present in contaminated water, particularly waters containing faecal material; however, routine monitoring of all pathogens is not currently possible. Enterococcus faecalis, which is present in the microflora of human and animals has been used as a faecal indicator in water due to its abundance in surface water and soil. Accurate and fast detection methods are critical for the effective monitoring of E. faecalis in the environment. Although conventional and current molecular detection techniques provide sufficient sensitivity, specificity and throughput, their use is hampered by the long waiting period (1-6 days) to obtain results, the need for expensive laboratory equipment, skilled personnel, and cold-chain storage. Therefore, this study aimed to develop a detection system for E. faecalis that would be simple, rapid, and low-cost, using an isothermal DNA amplification assay called recombinase polymerase amplification (RPA), integrated with a lateral flow assay (LFA). The assay was found to be 100% selective for E. faecalis and capable of detecting rates as low as 2.8 × 103 cells per 100 mL from water and wastewater, and 2.8 × 104 cells per 100 mL from saline water. The assay was completed in approximately 30 min using one constant temperature (38 °C). In addition, this study demonstrated the quantitation of E. faecalis using a lateral flow strip reader for the first time, enhancing the potential use of RPA assay for the enumeration of E. faecalis in wastewater and heavily contaminated environmental waters, surface water, and wastewater. However, the sensitivity of the RPA-LFA assay for the detection of E. faecalis in tap water, saline water and in wastewater was 10-1000 times lower than that of the Enterolert-E test, depending on the water quality. Nevertheless, with further improvements, this low-cost RPA-LFA may be suitable to be used at the point-of-need (PON) if conjugated with a rapid field-deployable DNA extraction method.

Cubosome Lipid Nanocarriers As a Drug Delivery Vehicle for Intracellular Mycobacterium tuberculosis Infections.

Mycobacterium tuberculosis (MTB) causes the infectious disease tuberculosis (TB), responsible for more deaths than any other single infectious disease in history. Intracellular MTB are slow growing and difficult to target with traditional antitubercular drugs, leading to the emergence of multidrug resistance in TB infection, which is a major global public health issue. Recent advances in innovative lipid nanotechnologies for drug delivery have demonstrated promising outcomes for chronic infectious diseases but have not yet been tested as potential delivery systems for intracellular infections such as TB. The current study evaluates the potential of monoolein (MO)-based cationic cubosomes for the encapsulation and delivery of the first line antitubercular drug rifampicin (RIF) against an MTB-H37Ra in vitro culture model. In particular, we show that the use of cationic cubosomes as delivery vehicles reduced the minimum inhibitory concentration (MIC) of RIF by 2-fold against actively replicating MTB-H37Ra (compared to that of the free drug) and also shortened the lifecycle duration of axenic MTB-H37Ra from 5 to 3 days. The cubosome-mediated delivery was also found to be effective against intracellular MTB-H37Ra within THP-1 human macrophages, with a 2.8 log reduction in viability of the bacilli after 6 days incubation at the MIC. The killing time was also reduced from 8 to 6 days without distressing the host macrophages. Mechanistic studies on the uptake of RIF-loaded cationic cubosomes using total internal reflection fluorescence microscopy (TIRFM) demonstrated the capacity of these lipid particles to effectively target intracellular bacteria. Overall, these results demonstrate that cationic cubosomes are a potent delivery system for the antitubercular drug RIF for therapeutic management of TB.

Prospective Subunit Nanovaccine against Mycobacterium tuberculosis Infection─Cubosome Lipid Nanocarriers of Cord Factor, Trehalose 6,6' Dimycolate.

An improved vaccine is urgently needed to replace the now more than 100-year-old Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis (TB) disease, which represents a significant burden on global public health. Mycolic acid, or cord factor trehalose 6,6' dimycolate (TDM), a lipid component abundant in the cell wall of the pathogen Mycobacterium tuberculosis (MTB), has been shown to have strong immunostimulatory activity but remains underexplored due to its high toxicity and poor solubility. Herein, we employed a novel strategy to encapsulate TDM within a cubosome lipid nanocarrier as a potential subunit nanovaccine candidate against TB. This strategy not only increased the solubility and reduced the toxicity of TDM but also elicited a protective immune response to control MTB growth in macrophages. Both pre-treatment and concurrent treatment of the TDM encapsulated in lipid monoolein (MO) cubosomes (MO-TDM) (1 mol %) induced a strong proinflammatory cytokine response in MTB-infected macrophages, due to epigenetic changes at the promoters of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in comparison to the untreated control. Furthermore, treatment with MO-TDM (1 mol %) cubosomes significantly improved antigen processing and presentation capabilities of MTB-infected macrophages to CD4 T cells. The ability of MO-TDM (1 mol %) cubosomes to induce a robust innate and adaptive response in vitro was further supported by a mathematical modeling study predicting the vaccine efficacy in vivo. Overall, these results indicate a strong immunostimulatory effect of TDM when delivered through the lipid nanocarrier, suggesting its potential as a novel TB vaccine.

Self-assembled histidine acid phosphatase nanocapsules in ionic liquid [BMIM][BF4] as functional templates for hollow metal nanoparticles.

We report the biomacromolecular self-assembly of histidine acid phosphatase (HAP), an enzyme of significant biomedical and industrial importance, in the ionic liquid (IL) 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF(4)]). The spontaneous self-assembly of HAP enzyme in [BMIM][BF(4)] results in the formation of HAP nanocapsules. The HAP enzyme molecules were found to retain their enzymatic activity after the self-assembly process, which enabled us to utilize self-assembled HAP capsules as self-catalyzing templates for the synthesis of a range of hollow metal nanoparticles (Au, Ag, Pd, and Ni) without employing any additional reducing agent. The hollow metal nanospheres with HAP encapsulated within their cavity were found to retain enzymatic activity for at least up to four cycles, as demonstrated in the case of Au-coated HAP capsules as the model system.

Facile approach for the dispersion of regenerated cellulose in aqueous system in the form of nanoparticles.

This study reports a facile method to disperse cellulose in deionized water, wherein a critical condition of regenerated cellulose is discovered, where it completely disperses up to a maximum of 5 g L(-1) concentration in deionized water with the help of ultrasonication. The dispersed cellulose is characterized by TEM and DLS, the latter among which shows 200 nm hydrodynamic radii of cellulose nanoparticles dispersed in deionized water. FTIR analysis of dispersed cellulose reveals that dispersed cellulose losses its crystallinity during regeneration and dispersion step employed in this study. The dispersed cellulose reported in this study is able to form free-standing, transparent films, which were characterized by SEM, XRD, TGA, EDX, and FTIR spectroscopy and show resistance against dissolution in water. Additionally, the dispersed cellulose is able to undergo at least three times faster enzymatic hydrolysis in comparison to pristine microcrystalline cellulose under similar reaction conditions. The dispersed cellulose reported here could be a better material for reinforcement, preparation of hydrogels, and drug delivery applications under physiological environment.