Non-Antigenic Modulation of Antigen Receptor (TCR) Cß-FG Loop Modulates Signalling: Implications of External Factors Influencing T-Cell Responses.

T-cell recognition of antigens is complex, leading to biochemical and cellular events that impart both specific and targeted immune responses. The end result is an array of cytokines that facilitate the direction and intensity of the immune reaction-such as T-cell proliferation, differentiation, macrophage activation, and B-cell isotype switching-all of which may be necessary and appropriate to eliminate the antigen and induce adaptive immunity. Using in silico docking to identify small molecules that putatively bind to the T-cell Cß-FG loop, we have shown in vitro using an antigen presentation assay that T-cell signalling is altered. The idea of modulating T-cell signalling independently of antigens by directly targeting the FG loop is novel and warrants further study.

Exploring the excited-state charge transfer fluorescence profile of 7-hydroxycoumarin and 2-methylimidazole - a combined X-ray diffraction and theoretical approach.

This study investigated the effect of 2-methylimidazole (2-MIM) addition on the fluorescence of ethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxylate using low-cost density functional theory (DFT) and Time-Dependent DFT calculations on single crystal X-ray geometries of ethyl-7-hydroxy-2-oxo-2H-chromene-3-carboxylate hydrate (1), 2-MIM (2), and the 1 : 1 co-crystal of (1) and (2), (3). At low concentrations (1 : 1-1 : 10) of 2-MIM, the fluorophore shows a decrease in the fluorescence intensity, but at higher concentrations (above 1 : 10) the fluorescence excitation maximum shifted from 354 nm to 405 nm, with a significant emission intensity increase. The changed excitation and emission profile at high concentrations is due to the deprotonation of the coumarin's phenolic group, which was confirmed by the increased shielding of the aromatic protons in the titration 1H NMR spectra. The experimental fluorescence data between the 1 : 1 and 1 : 10 ratios agreed with the theoretical fluorescence data, with a redshift and decreased intensity when comparing (1) and (3). The data indicated that combining the fluorophore with 2-MIM increased levels of vibronic coupling between 2-MIM and the fluorophore decreasing de-excitation efficiency. These increased vibronic changes were due to charge transfer between the fluorophore and 2-MIM in (3). The subtle movement of the proton, H(5) toward N(2') (0.07 Å) caused a significant decrease in fluorescence due to electron density distribution (EDD) changes. This was identified by comparison of the EDD in the excited (S1) and ground (S0) states plotted as an isosurface of EDD difference. For the higher concentrations, an alternative excitation pathway was explored by modifying the crystal geometry of (3) based on 1H NMR spectroscopy data to resemble excitoplexes. Theses excitoplex geometries reflected the fluorescence profile of the fluorophore with high concentrations of 2-MIM; there were dramatic changes in the theoretical fluorescence pathway, which was 100% vibronic coupling compared to 15.31% in the free fluorophore. At this concentration, the de-excitation pathway causes remodelling of the lactone ring via stretching/breaking the CO bond in the S1 causing increased fluorescence by movement of the transition dipole moment. These results reflect previous studies, but the methods used are less experimentally and computationally expensive. This study is among the first to explain charge transfer fluorescence using crystalline geometries. This study will be of interest to the fields of crystal engineering and fluorescence spectroscopy.

Discovery of 2',6-Bis(4-hydroxybenzyl)-2-acetylcyclohexanone, a Novel FtsZ Inhibitor.

Multi-drug resistance is increasing in the pathogenic bacterium S. pneumoniae, which is mainly responsible for meningitis and community-acquired pneumonia (CAP), highlighting the need for new anti-pneumococcal agents. We have identified a potential anti-pneumococcal agent, enol 3, which acts by hindering the cell division process by perturbing Z-ring dynamics inside the cell. Enol 3 was also shown to inhibit FtsZ polymerization and induce its aggregation in vitro but does not affect the activity of tubulin and alkaline phosphatase. Docking studies show that 3 binds near the T7 loop, which is the catalytic site of FtsZ. Similar effects on Z-ring and FtsZ assembly were observed in B. subtilis, indicating that 3 could be a broad-spectrum anti-bacterial agent useful in targeting Gram-positive bacteria. In conclusion, compound 3 shows strong anti-pneumococcal activity, prompting further pre-clinical studies to explore its potential.

Patient beliefs associated with medication hesitancy in palliative care: A systematic review using the theory of planned behavior.

CONTEXT: The demand of palliative care is increasing due to the aging population and treatment hesitancy or intentional avoidance compromises symptom management. OBJECTIVES: To identify patient beliefs associated with medication hesitancy by using the theory of planned behavior (TPB) namely, attitudes, subjective norms, behavioral intention, and perceived behavioral control associated with medication hesitancy or intentional noncompliance by avoidance. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analysis guideline was followed to conduct a systematic literature search involving the CINAHL, Embase, MEDLINE, and PsycINFO databases from inception until March 2022. Hand-searched articles from reference lists and gray literature were included. Thematic analysis was conducted on qualitative data and triangulated with quantitative data. RESULTS: About 554 articles were retrieved from the literature search and 17 articles were included based on the eligibility criteria. Three subthemes that were identified under TPB constructs were attitude: negative attitude toward medications, passive attitude toward illness and inaccurate information about disease or medication; one subtheme was identified under subjective norms: perceived negative opinions from others; and one subtheme was identified under perceived behavioral control: perception of manageable symptoms. Quantitative data provided triangulation of qualitative findings related to fear of addiction and side effects, feelings of hopelessness, unclear direction and information, social stigma, endurable symptoms, and illness as determinants for medication avoidance. SIGNIFICANCE OF RESULTS: This systematic review highlighted some patient beliefs related to medication hesitancy or avoidance. Clinicians should take patient beliefs and concerns into consideration when creating treatment regimens for people receiving palliative care to optimize medication adherence and the quality of care.

The Antifungal and Synergistic Effect of Bisphosphonates in Cryptococcus.

New treatment strategies are required for cryptococcosis, a leading mycosis in HIV-AIDS patients. Following the identification of Cryptococcus proteins differentially expressed in response to fluconazole, we targeted farnesyl pryrophosphate synthetase (FPPS), an enzyme in the squalene biosynthesis pathway, using nitrogenous bisphosphonates. We hypothesized that these would disrupt squalene synthesis and thereby produce synergy with fluconazole, which acts on a downstream pathway that requires squalene. The susceptibilities of 39 clinical isolates from 6 different species of Cryptococcus were assessed for bisphosphonates and fluconazole, used both independently and in combination. Effective fluconazole-bisphosphonate combinations were then assessed for fungicidal activity, efficacy against biofilms, and ability to resolve cryptococcosis in an invertebrate model. The nitrogenous bisphosphonates risedronate, alendronate, and zoledronate were antifungal against all strains tested. Zoledronate was the most effective (geometric mean MIC = 113.03 mg/liter; risedronate = 378.49 mg/liter; alendronate = 158.4 mg/liter) and was broadly synergistic when combined with fluconazole, with a fractional inhibitory concentration index (FICI) of ≤0.5 in 92% of isolates. Fluconazole and zoledronate in combination were fungicidal in a time-kill assay, inhibited Cryptococcus biofilms, prevented the development of fluconazole resistance, and resolved infection in a nematode model. Supplementation with squalene eliminated bisphosphonate-mediated synergy, demonstrating that synergy was due to the inhibition of squalene biosynthesis. This study demonstrates the utility of targeting squalene synthesis for improving the efficacy of azole-based antifungal drugs and suggests bisphosphonates are promising lead compounds for further antifungal development.

Delivering anion transporters to lipid bilayers in water.

Cyclodextrins have been employed as delivery agents for lipophilic anion transporters, which allow their incorporation into lipid bilayers without using an organic solvent or pre-incorporation.

Understanding Hygroscopicity of Theophylline via a Novel Cocrystal Polymorph: A Charge Density Study.

The charge density distribution in a novel cocrystal (1) complex of 1,3-dimethylxanthine (theophylline) and propanedioic acid (malonic acid) has been determined. The molecules crystallize in the triclinic, centrosymmetric space group P1̅, with four independent molecules (Z = 4) in the asymmetric unit (two molecules each of theophylline and malonic acid). Theophylline has a notably high hygroscopic nature, and numerous cocrystals have shown a significant improvement in stability to humidity. A charge density study of the novel polymorph has identified interesting theoretical results correlating the stability enhancement of theophylline via cocrystallization. Topological analysis of the electron density highlighted key differences (up to 17.8) in Laplacian (∇2ρ) between the experimental (EXP) and single-point (SP) models, mainly around intermolecular-bonded carbonyls. Further investigation via molecular electrostatic potential maps reaffirmed that the charge redistribution enhanced intramolecular hydrogen bonding, predominantly for N(2') and N(2) (61.2 and 61.8 kJ mol-1, respectively). An overall weaker lattice energy of the triclinic form (-126.1 kJ mol-1) compared to that of the monoclinic form (-133.8 kJ mol-1) suggests a lower energy threshold to overcome to initiate dissociation. Future work via physical testing of the novel cocrystal in both dissolution and solubility will further solidify the correlation between theoretical and experimental results.

Tubulin-Binding 3,5-Bis(styryl)pyrazoles as Lead Compounds for the Treatment of Castration-Resistant Prostate Cancer.

The microtubule-binding taxanes, docetaxel and cabazitaxel, are administered intravenously for the treatment of castration-resistant prostate cancer (CRPC) as the oral administration of these drugs is largely hampered by their low and highly variable bioavailabilities. Using a simple, rapid, and environmentally friendly microwave-assisted protocol, we have synthesized a number of 3,5-bis(styryl)pyrazoles 2a-l, thus allowing for their screening for antiproliferative activity in the androgen-independent PC3 prostate cancer cell line. Surprisingly, two of these structurally simple 3,5-bis(styryl)pyrazoles (2a and 2l) had concentrations which gave 50% of the maximal inhibition of cell proliferation (GI50) in the low micromolar range in the PC3 cell line and were thus selected for extensive further biologic evaluation (apoptosis and cell cycle analysis, and effects on tubulin and microtubules). Our findings from these studies show that 3,5-bis[(1E)-2(2,6-dichlorophenyl)ethenyl]-1H-pyrazole 2l 1) caused significant effects on the cell cycle in PC3 cells, with the vast majority of treated cells in the G2/M phase (89%); 2) induces cell death in PC3 cells even after the removal of the compound; 3) binds to tubulin [dissociation constant (Kd) 0.4 ± 0.1 µM] and inhibits tubulin polymerization in vitro; 4) had no effect upon the polymerization of the bacterial cell division protein FtsZ (a homolog of tubulin); 5) is competitive with paclitaxel for binding to tubulin but not with vinblastine, crocin, or colchicine; and 6) leads to microtubule depolymerization in PC3 cells. Taken together, these results suggest that 3,5-bis(styryl)pyrazoles warrant further investigation as lead compounds for the treatment of CRPC. SIGNIFICANCE STATEMENT: The taxanes are important components of prostate cancer chemotherapy regimens, but their oral administration is hampered by very low and highly variable oral bioavailabilities resulting from their poor absorption, poor solubility, high first-pass metabolism, and efficient efflux by P-glycoprotein. New chemical entities for the treatment of prostate cancer are thus required, and we report here the synthesis and investigation of the mechanism of action of some bis(styryl)pyrazoles, demonstrating their potential as lead compounds for the treatment of prostate cancer.

cyp51A Mutations, Extrolite Profiles, and Antifungal Susceptibility in Clinical and Environmental Isolates of the Aspergillus viridinutans Species Complex.

The past decade has seen an increase in aspergillosis in humans and animals due to Aspergillus viridinutans species complex members. Azole resistance is common to these infections, carrying a poor prognosis. cyp51A gene mutations are the main cause of acquired azole resistance in Aspergillus fumigatus This study aimed to determine if the azole-resistant phenotype in A. viridinutans complex members is associated with cyp51A mutations or extrolite profiles. The cyp51A gene of clinical and environmental isolates was amplified using novel primers, antifungal susceptibility was tested using the Clinical and Laboratory Standards Institute methodology, and extrolite profiling was performed using agar plug extraction. Very high azole MICs were detected in 84% of the isolates (31/37). The MICs of the newer antifungals luliconazole and olorofim (F901318) were low for all isolates. cyp51A sequences revealed 113 nonsynonymous mutations compared to the sequence of wild-type A. fumigatus M172A/V and D255G, previously associated with A. fumigatus azole resistance, were common among all isolates but were not correlated with azole MICs. Two environmental isolates with nonsusceptibility to itraconazole and high MICs of voriconazole and isavuconazole harbored G138C, previously associated with azole-resistant A. fumigatus Some novel mutations were identified only among isolates with high azole MICs. However, cyp51A homology modeling did not cause a significant protein structure change for these mutations. There was no correlation between extrolite patterns and susceptibility. For A. viridinutans complex isolates, cyp51A mutations and the extrolites that they produced were not major causes of antifungal resistance. Luliconazole and olorofim show promise for treating azole-resistant infections caused by these cryptic species.

A Selective, Dual Emission ß-Alanine Aminopeptidase Activated Fluorescent Probe for the Detection of Pseudomonas aeruginosa, Burkholderia cepacia, and Serratia marcescens.

Selective detection of ß-alanyl aminopeptidase (BAP)-producing Pseudomonas aeruginosa, Serratia marcescens, and Burkholderia cepacia was achieved by employing the blue-to-yellow fluorescent transition of a BAP-specific enzyme substrate, 3-hydroxy-2-(p-dimethylaminophenyl)flavone derivative, incorporating a self-immolative linker to ß-alanine. Upon cellular uptake and accumulation of the substrate by viable bacterial colonies, blue fluorescence was generated, while hydrolysis of the N-terminal peptide bond by BAP resulted in the elimination of the self-immolative linker and the restoration of the original fluorescence of the flavone derivative.

Surveillance for azole resistance in clinical and environmental isolates of Aspergillus fumigatus in Australia and cyp51A homology modelling of azole-resistant isolates.

Background: The prevalence of azole resistance in Aspergillus fumigatus is uncertain in Australia. Azole exposure may select for resistance. We investigated the frequency of azole resistance in a large number of clinical and environmental isolates. Methods: A. fumigatus isolates [148 human, 21 animal and 185 environmental strains from air (n = 6) and azole-exposed (n = 64) or azole-naive (n = 115) environments] were screened for azole resistance using the VIPcheck™ system. MICs were determined using the Sensititre™ YeastOne YO10 assay. Sequencing of the Aspergillus cyp51A gene and promoter region was performed for azole-resistant isolates, and cyp51A homology protein modelling undertaken. Results: Non-WT MICs/MICs at the epidemiological cut-off value of one or more azoles were observed for 3/148 (2%) human isolates but not amongst animal, or environmental, isolates. All three isolates grew on at least one azole-supplemented well based on VIPcheck™ screening. For isolates 9 and 32, the itraconazole and posaconazole MICs were 1 mg/L (voriconazole MICs 0.12 mg/L); isolate 129 had itraconazole, posaconazole and voriconazole MICs of >16, 1 and 8 mg/L, respectively. Soil isolates from azole-exposed and azole-naive environments had similar geometric mean MICs of itraconazole, posaconazole and voriconazole (P > 0.05). A G54R mutation was identified in the isolates exhibiting itraconazole and posaconazole resistance, and the TR34/L98H mutation in the pan-azole-resistant isolate. cyp51A modelling predicted that the G54R mutation would prevent binding of itraconazole and posaconazole to the haem complex. Conclusions: Azole resistance is uncommon in Australian clinical and environmental A. fumigatus isolates; further surveillance is indicated.

A latent green fluorescent styrylcoumarin probe for the selective growth and detection of Gram negative bacteria.

A novel, green fluorescent ß-alanylstyrylcoumarin derivative was synthesized and evaluated for its performance as a fluorogenic enzyme substrate on a range of clinically relevant microorganisms. The substrate was selectively hydrolysed by ß-alanyl aminopeptidase producing P. aeruginosa resulting in an on-to-off fluorescent signal. Growth inhibitory effect of the substrate was observed on Gram positive bacteria and yeasts. Meanwhile, Gram negative species, despite their extremely protective cell envelope, showed ready uptake and accumulation of the substrate within their healthy growing colonies displaying intense green fluorescence.

Exploring the Binding of Barbital to a Synthetic Macrocyclic Receptor. A Charge Density Study.

Experimental charge density distribution studies, complemented by quantum mechanical theoretical calculations, of a host-guest system composed of a macrocycle (1) and barbital (2) in a 1:1 ratio (3) have been carried out via high-resolution single-crystal X-ray diffraction. The data were modeled using the conventional multipole model of electron density according to the Hansen-Coppens formalism. The asymmetric unit of macrocycle 1 contained an intraannular ethanol molecule and an extraannular acetonitrile molecule, and the asymmetric unit of 3 also contained an intraannular ethanol molecule. Visual comparison of the conformations of the macrocyclic ring shows the rotation by 180° of an amide bond attributed to competitive hydrogen bonding. It was found that the intraannular and extraannular molecules inside were orientated to maximize the number of hydrogen bonds present, with the presence of barbital in 3 resulting in the greatest stabilization. Hydrogen bonds ranging in strength from 4 to 70 kJ mol-1 were the main stabilizing force. Further analysis of the electrostatic potential among 1, 2, and 3 showed significant charge redistribution when cocrystallization occurred, which was further confirmed by a comparison of atomic charges. The findings presented herein introduce the possibility of high-resolution X-ray crystallography playing a more prominent role in the drug design process.

Methods for the detection and identification of pathogenic bacteria: past, present, and future.

In order to retard the rate of development of antibacterial resistance, the causative agent must be identified as rapidly as possible, so that directed patient treatment and/or contact precautions can be initiated. This review highlights the challenges associated with the detection and identification of pathogenic bacteria, by providing an introduction to the techniques currently used, as well as newer techniques that are in development. Focusing on the chemical basis for these techniques, the review also provides a comparison of their advantages and disadvantages.

A Carbocyclic Curcumin Inhibits Proliferation of Gram-Positive Bacteria by Targeting FtsZ.

Inhibition of FtsZ assembly has been found to stall bacterial cell division. Here, we report the identification of a potent carbocyclic curcumin analogue (2d) that inhibits Bacillus subtilis 168 cell proliferation by targeting the assembly of FtsZ. 2d also showed potent inhibitory activity (minimum inhibitory concentrations of 2-4 mg/L) against several clinically important species of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In addition, 2d displayed a significantly reduced inhibitory effect on human cervical cancer cells in comparison to its effect on bacterial cells. Using live cell imaging of GFP-FtsZ by confocal microscopy, 2d was found to rapidly perturb the cytokinetic FtsZ rings in Bacillus subtilis cells. The immunofluorescence imaging of FtsZ also showed that 2d destroyed the Z-ring in bacteria within 5 min. Prolonged treatment with 2d produced filamentous bacteria, but 2d had no detectable effect either on the nucleoids or on the membrane potential of bacteria. 2d inhibited FtsZ assembly in vitro, whereas it had minimal effects on tubulin assembly. Interestingly, 2d strongly enhanced the GTPase activity of FtsZ and reduced the GTPase activity of tubulin. Furthermore, 2d bound to purified FtsZ with a dissociation constant of 4.0 ± 1.1 µM, and the binding of 2d altered the secondary structures of FtsZ. The results together suggested that the non-natural curcumin analogue 2d possesses powerful antibacterial activity against important pathogenic bacteria, and the evidence indicates that 2d inhibits bacterial proliferation by targeting FtsZ.

Rapid access to N-(indol-2-yl)amides and N-(indol-3-yl)amides as unexplored pharmacophores.

Preparation of N-(indol-2-yl)amides and N-(indol-3-yl)amides are scarce in the scientific literature due to unstable intermediates impeding current reported syntheses. We have employed cheap and readily available substrates in the Curtius rearrangement of indole-3-carboxazide to afford N-(indol-3-yl)amides. The reaction is observed for alkyl and aryl carboxylic acids and both N-substituted or 1H-indole derivatives are tolerated. This approach was extended to the preparation of N-(indol-2-yl)amides from the corresponding indole-2-carboxazides.

A comparison of the experimental and theoretical charge density distributions in two polymorphic modifications of piroxicam.

Experimental charge density distribution studies of two polymorphic forms of piroxicam, ß-piroxicam (1) and piroxicam monohydrate (2), were carried out via high-resolution single crystal X-ray diffraction experiments and multipole refinement. The asymmetric unit of (2) consists of two discrete piroxicam molecules, (2a) and (2b), and two water molecules. Geometry differs between (1) and (2) due to the zwitterionic nature of (2) which results in the rotation of the pyridine ring around the C(10)-N(2) bond by approximately 180°. Consequently, the pyridine and amide are no longer co-planar and (2) forms two exclusive, strong hydrogen bonds, H(3)O(4) and H(2)O(3), with bond energies of 66.14 kJ mol-1 and 112.82 kJ mol-1 for (2a), and 58.35 kJ mol-1 and 159.51 kJ mol-1 for (2b), respectively. Proton transfer between O(3) and N(3) in (2) results in significant differences in surface electrostatic potentials. This is clarified by the calculation of atomic charges in the zwitterion that shows the formally positive charge of the pyridyl nitrogen which is redistributed over the whole of the pyridine ring instead of concentrating at N-H. Similarly, the negative charge of the oxygen is distributed across the benzothiazine carboxamide moiety. The multipole derived lattice energy for (1) is -304 kJ mol-1 and that for (2) is -571 kJ mol-1, which is in agreement with the experimentally determined observations of higher solubility and dissolution rates of (1) compared to (2).

Identification of dual PPARα/γ agonists and their effects on lipid metabolism.

The three peroxisome proliferator-activated receptor (PPAR) isoforms; PPARα, PPARγ and PPARδ, play central roles in lipid metabolism and glucose homeostasis. Dual PPARα/γ agonists, which stimulate both PPARα and PPARγ isoforms to similar extents, are gaining popularity as it is believed that they are able to ameliorate the unwanted side effects of selective PPARα and PPARγ agonists; and may also be used to treat dyslipidemia and type 2 diabetes mellitus simultaneously. In this study, virtual screening of natural product libraries, using both structure-based and ligand-based drug discovery approaches, identified ten potential dual PPARα/γ agonist lead compounds (9-13 and 16-20). In vitro assays confirmed these compounds to show no statistically significant toxicity to cells, with the exception of compound 12 which inhibited cell growth to 74.5%±3.5 and 54.1%±3.7 at 50µM and 100µM, respectively. In support of their potential as dual PPARα/γ agonists, all ten compounds upregulated the expression of cholesterol transporters ABCA1 and ABCG1 in THP-1 macrophages, with indoline derivative 16 producing the greatest elevation (2.3-fold; 3.3-fold, respectively). Furthermore, comparable to the activity of established PPARα and PPARγ agonists, compound 16 stimulated triacylglycerol accumulation during 3T3-L1 adipocyte differentiation as well as fatty acid ß-oxidation in HuH7 hepatocytes.

Experimental and theoretical charge density distribution in Pigment Yellow 101.

The charge density distribution in 2,2'-dihydroxy-1,1'-naphthalazine (Pigment Yellow 101; P.Y.101) has been determined using high-resolution X-ray diffraction and multipole refinement, along with density functional theory calculations. Topological analysis of the resulting densities highlights the localisation of single/double bonds in the central C=N-N=C moiety of the molecule in its ground state. The density in the N-N is examined in detail, where we show that very small differences between experiment and theory are amplified by use of the Laplacian of the density. Quantification of hydrogen bonds highlights the importance of the intramolecular N-H···O interaction, known to be vital for retention of fluorescence in the solid state, relative to the many but weak intermolecular contacts located. However, a popular method for deriving H-bond strengths from density data appears to struggle with the intramolecular N-H···O interaction. We also show that theoretical estimation of anisotropic displacements for hydrogen atoms brings little benefit overall, and degrades agreement with experiment for one intra-molecular contact.

Correction: Experimental and theoretical charge density distribution in Pigment Yellow 101.

Correction for 'Experimental and theoretical charge density distribution in Pigment Yellow 101' by Jonathan J. Du et al., Phys. Chem. Chem. Phys., 2015, DOI: 10.1039/c4cp04302b.