Phenol sensing in nature is modulated via a conformational switch governed by dynamic allostery.

The NtrC family of proteins senses external stimuli and accordingly stimulates stress and virulence pathways via activation of associated σ54-dependent RNA polymerases. However, the structural determinants that mediate this activation are not well understood. Here, we establish using computational, structural, biochemical, and biophysical studies that MopR, an NtrC protein, harbors a dynamic bidirectional electrostatic network that connects the phenol pocket to two distal regions, namely the "G-hinge" and the "allosteric linker." While the G-hinge influences the entry of phenol into the pocket, the allosteric linker passes the signal to the downstream ATPase domain. We show that phenol binding induces a rewiring of the electrostatic connections by eliciting dynamic allostery and demonstrates that perturbation of the core relay residues results in a complete loss of ATPase stimulation. Furthermore, we found a mutation of the G-hinge, ∼20 Å from the phenol pocket, promotes altered flexibility by shifting the pattern of conformational states accessed, leading to a protein with 7-fold enhanced phenol binding ability and enhanced transcriptional activation. Finally, we conducted a global analysis that illustrates that dynamic allostery-driven conserved community networks are universal and evolutionarily conserved across species. Taken together, these results provide insights into the mechanisms of dynamic allostery-mediated conformational changes in NtrC sensor proteins.

Nanoparticle and surfactant controlled switching between proton transfer and charge transfer reaction coordinates.

The reaction coordinates of a molecular photo-switch 2-(4'-diethylamino-2'-hydroxyphenyl)-1H-imidazo-[4,5-b]pyridine (DHP) was tuned with a nanoparticle and surfactant. DHP undergoes excited state intramolecular proton transfer (ESIPT) and emits normal and tautomer emissions in N,N-dimethylformamide. Silver nanoparticles suppress the ESIPT and induce twisted intramolecular charge transfer (TICT). Further addition of surfactants alters the process. Interestingly, different surfactants cause different effects. Accordingly, the luminescence characteristics are altered. The anionic surfactant sodium dodecyl sulfate (SDS) restores the ESIPT process by completely detaching the molecule from the nanoparticle. The nonionic surfactant Triton X-100 (TX-100), at lower concentration, enhances the TICT emission and the ESIPT process is also observed due to the release of some fluorophore from the nanoparticle complex. But at higher concentration the fluorophores are released completely and the ESIPT process is restored. The cationic surfactant cetyltrimethyl ammonium bromide (CTAB), at lower concentration, simply restores the ESIPT process by releasing the fluorophore. But at higher CTAB concentration, DHP enters the metalparticle-CTAB aggregate and shows enhanced ESIPT.

Racial disparities in COVID-19 hospitalizations do not lead to disparities in outcomes.

OBJECTIVES: The objective of the study is the identification of racial differences in characteristics and comorbidities in patients hospitalized for COVID-19 and the impact on outcomes. STUDY DESIGN: The study design is a retrospective observational study. METHODS: Data for all patients admitted to seven community hospitals in Michigan, United States, with polymerase chain reaction confirmed diagnosis of COVID-19 from March 10 to April 15, 2020 were analyzed. The primary outcomes of racial disparity in inpatient mortality and intubation were analyzed using descriptive statistics and multivariate regression models. RESULTS: The study included 336 Black and 408 White patients. Black patients were younger (62.9 ± 15.0 years vs 71.8 ± 16.4, P < .001), had a higher mean body mass index (32.4 ± 8.6 kg/m2 vs 28.8 ± 7.5, P < .001), had higher prevalence of diabetes (136/336 vs 130/408, P = .02), and presented later (6.6 ± 5.3 days after symptom onset vs. 5.4 ± 5.4, P = .006) compared with White patients. Younger Black patients had a higher prevalence of obesity (age <65 years, 69.9%) than older Black patients (age >65 years, 39.2%) and younger White patients (age < 65, 55.1%). Intubation did not reach statistical significance for racial difference (Black patients 61/335 vs. 54/406, P = .08). Mortality was not higher in Black patients (65/335 vs. 142/406 in White patients, odds ratio 0.61, 95% confidence interval: 0.37 to 0.99, 2-sided P = .05) in multivariate analysis, accounting for other risk factors associated with mortality. CONCLUSIONS: Higher prevalence of obesity and diabetes in young Black populations may be the critical factor driving disproportionate COVID-19 hospitalizations in Black populations. Hospitalized Black patients do not have worse outcomes compared with White patients.

The origin of the longer wavelength emission in 2-(4-fluorophenylamino)-5-(2,4-dihydroxybenzeno)-1,3,4-thiadiazole and its analogue 2-phenylamino-5-(2-hydroxybenzono)-1,3,4-thiadiazole† ‡.

In aqueous solution, 2-(4-fluorophenylamino)-5-(2,4-dihydroxybenzeno)-1,3,4-thiadiazole (FABT) was found to emit dual emission and the longer wavelength emission was assigned to the combination of aggregation and conformational change. In a number of molecules that possess an intramolecular hydrogen bond between the proton donor and the acceptor, the longer wavelength emission is often observed due to the emission from the tautomer formed by excited state intramolecular proton transfer (ESIPT). Therefore, an analogue of FABT, 2-phenylamino-5-(2-hydroxybenzono)-1,3,4-thiadiazole (PHBT), was synthesized to determine the origin of the longer wavelength emission. The luminescence of PHBT and its methoxy derivatives was studied and compared with that of FABT. Theoretical calculations were also performed on both FABT and PHBT. Based on the experimental and theoretical investigations, the nonexistence of the keto tautomer in the ground state and the origin of the longer wavelength emission are divulged.

α-Synuclein Spontaneously Adopts Stable and Reversible α-Helical Structure in Water-Less Environment.

A highly stable, spontaneous, and reversible α-helical-structure formation in recombinant and chemically modified α-synuclein protein is demonstrated for the first time in a water-less (1.5 % w/w H2 O) polymer surfactant environment. Using a combination of circular dichroism and ATR-FTIR spectroscopy, we show that whilst native α-synuclein in aqueous solution shows a predominant unordered conformation (≈64 %), mixing with polyethylene glycol based anionic polymer surfactant (PS) and removing water reveals a 25 % unordered, 25 % α-helical, and 27 % ß-sheet structure. Interestingly, bioconjugation of native α-synuclein with a diamine molecule, to increase the positive charge on the protein chain, and subsequent electrostatic coupling with the PS forms a conjugate with a retained unordered structure. Removal of water from this system provides a highly stable α-helical (≈74 %) water-less liquid system. Surprisingly, the α-helical-to-unordered state transition is completely reversible and is achieved at ≈25-30 w/w% of water in the system. Moreover, the α-helix shows an extraordinary temporal stability (>6 months) in a waterless environment.

Tweaking the proton transfer triggered proton transfer of 3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazole.

The proton transfer of 3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazole (bis-HPTA), a fluorophore proficient in self-aided twin proton transfer, has been explored in the presence of dimethylformamide (DMF). The proton transfer is completely altered in the presence of DMF (compared to that in other solvents). Bis-HPTA forms a hydrogen bonded complex with DMF molecules, which shift the conformer equilibrium. Theoretical calculations predicted that the DMF complexes of bis-HPTA-II and bis-HPTA-III are more stable than the complexes of other conformers. Upon excitation, bis-HPTA-II transfers a proton to a DMF molecule to form an anion and bis-HPTA-III transfers a proton to a ring nitrogen to form a keto form. Silver particles reverse the effect of DMF. They regenerate bis-HPTA-I by removing the intermolecular hydrogen bond and thereby they reestablish the proton transfer triggered proton transfer (PTTPT).

Site-specific time-resolved FRET reveals local variations in the unfolding mechanism in an apparently two-state protein unfolding transition.

Protein folding/unfolding transitions between the native (N) and unfolded (U) states are usually describable as two-state, only because of the dominant presence of the N and/or U states, because of which high energy intermediates remain undetected. Delineation of the cooperativity underlying these transitions, and characterization of high energy intermediates that are populated sparsely, have been difficult challenges, especially under equilibrium conditions, and require the use of a sensitive probe that reports on both the structures and population distributions of the partially unfolded intermediates. In this study, the use of multisite time-resolved FRET to monitor structural change in five specific segments of the small protein monellin, has brought out local deviations from two-state behavior during unfolding. It is shown that in some segments of the protein structure, denaturant-induced unfolding proceeds first by gradual expansion of the N state, then by an all-or-none transition from the N state ensemble to the U state ensemble, followed finally by expansion of the U state. Segments encompassing the sole helix appear, however, to unfold completely through a gradual transition from the N to U states. Finally, it is shown that equilibrium unfolding of monellin is not only heterogeneous, but that the degree of non-cooperativity differs between the sole α-helix and different parts of the ß-sheet.

Proton transfer triggered proton transfer: a self-assisted twin excited state intramolecular proton transfer.

The double excited state intramolecular proton transfer (ESIPT) of 3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazole (bis-HPTA), a molecule possessing two intramolecular hydrogen bonded donor-acceptor pairs, has been investigated. The molecule undergoes not only a single ESIPT, but also a rare twin ESIPT. The most interesting fact is that initially, only one acid-base pair is ESIPT active and the other pair is ESIPT inactive. The first proton transfer triggers the proton transfer in the second acid-base pair by creating appropriate conditions. This new type of sequential proton transfer is labeled as 'proton transfer triggered proton transfer' (PTTPT). The PTTPT has been demonstrated by steady state and time resolved fluorescence techniques. It was further substantiated by theoretical data and using a chemically modified system 3-(2-hydroxyphenyl)-5-(2-methoxyphenyl)-1H-1,2,4-triazole.

Bioactive compound from Aspergillus terreusDMTMGK004 synergistically contributes towards potential anti-pathogenicity.

This study focused on the evaluation of fungal compound for their anti-pathogenic potential against respiratory pathogens. Soil samples were collected from various geographical regions in Madurai, fungal strain was isolated and identified as Aspergillus terreusDMTMGK004 (MGK004). Secondary metabolites were extracted and evaluated for antioxidant potential. It exhibited significantly high anti-proliferative property against gastric adenocarcinoma (AGS) cell lines. Antimicrobial activity against Gram positive (Streptococcus pneumoniae) and Gram negative (Klebsiella pneumoniae and Haemophilus influenzae) respiratory pathogens were analysed and the minimum inhibitory concentration (MIC) values were determined. Furthermore, the time-killing assay illustrated that the metabolite eliminates 50% of the vegetative cells within few hours of the treatment. From the spectral data, the major functional groups present in the compound were determined as carbonyl group and phenolic hydroxyl group which contribute towards its bioactivity. The compound significantly depreciates the production of extracellular polysaccharides which results in the weakening of biofilm architecture and resistance towards serum killing and phagocytosis. It also induced cell membrane damage which leads to protein and nucleic acid leakage. Hence, the results of the present study could provide a better insinuation towards the formulation of new drug targeting respiratory pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The ubiquitous fungi Aspergillus terreus is well known for its secondary metabolite production. The fungus was evaluated for production of antagonistic molecule to reduce the growth of infectious agents causing respiratory infections. It exhibited the biological means of antioxidant, anti-proliferative and anti-pathogenic compound production. The compound exhibits killing effect against respiratory pathogens within two hours. It induced cell membrane damage leading to protein and nucleic acid leakage. It significantly reduced the production of extracellular polysaccharides. The results provide needed information to design innovative strategies for targeting pathogenic factors of the respiratory pathogens instead of killing it precisely.

Aggregation induced enhanced and exclusively highly Stokes shifted emission from an excited state intramolecular proton transfer exhibiting molecule.

The inner filter effect due to self-quenching dominates the normal emission of dyes at higher concentrations, which would limit their applications. Since normal emission was also observed with aggregation induced emission enhancement (AIEE) active excited state intramolecular proton transfer (ESIPT) exhibiting molecules, two new molecules are synthesized and studied to obtain normal emission free AIEE. The molecules are 4-(3-(benzo[d]thiazol-2-yl)-5-tert-butyl-4-hydroxybenzyl)-2-(benzo[d]thiazol-2-yl)-6-tert-butyl phenol (bis-HPBT) and its oxazole analogue (bis-HPBO). Of these molecules, bis-HPBT, which is weakly fluorescent in tetrahydrofuran solution, shows a sudden high enhancement in fluorescence upon addition of 70% water due to the formation of aggregates. Though the normal emission is also observed in tetrahydrofuran, it is completely eliminated in the aggregates, and the aggregates display exclusive tautomer emission. However, bis-HPBO does not emit such an exclusive tautomer emission in the water/tetrahydrofuran mixture. The enhancement in the fluorescence quantum yield of bis-HPBT in 70% water is ∼300 times higher than that in tetrahydrofuran. The modulated molecular structure of bis-HPBT is the cause of this outstanding AIEE. The observation of almost exclusive tautomer emission is a new additional advantage of AIEE from bis-HPBT over other ESIPT molecules. Since the tautomer emission is highly Stokes shifted, no overlap with the absorption spectrum occurs and therefore, the inner filter effect is averted. The aggregated structure acts as a good fluorescence chemosensor for metal ions as well as anions. The aggregated structure is cell permeable and can be used for cell imaging.

Perturbation of cationic equilibrium by cucurbit-7-uril.

The effect of cucurbit-7-uril (CB-7) on a cationic mixture with same charge has been investigated by studying monocationic mixtures of 2-(4'-N,N-dimethylaminophenyl)imidazo[4,5-b]pyridine (DMAPIP-b) and 2-(4'-N,N-dimethylaminophenyl)imidazo[4,5-c]pyridine (DMAPIP-c). The pKa of both the guests increases in CB-7. DMAPIP-b forms all three monocations in the ground and the excited states in aqueous as well as in CB-7 solution. However, CB-7 shifts the equilibrium more towards the less polar MC2 and MC3. On the other hand, DMAPIP-c exists only as MC1 and MC3 in aqueous solution, however, in CB-7 it exists not only as MC1 and MC3 but also as MC2 in CB-7 in the ground state. In the excited state, as in aqueous solution MC1 forms MC2 by biprotonic phototautomerism in CB-7. The association constants of monocations suggest a pyridyl nitrogen position dependence. All the MC-CB-7 complexes are optimized by density functional theory (DFT).

Mode of DNA binding with γ-butyrolactone receptor protein CprB from Streptomyces coelicolor revealed by site-specific fluorescence dynamics.

BACKGROUND: The γ-butyrolactone (GBL) binding transcription factors in Streptomyces species are known for their involvement in quorum sensing where they control the expression of various genes initiating secondary metabolic pathways. The structurally characterized member of this family CprB from Streptomyces coelicolor had earlier been demonstrated to bind a multitude of sequences containing a specific binding signature. Though structural breakthrough has been obtained for its complex with a consensus DNA sequence there is, however a dearth of information regarding the overall and site specific dynamics of protein-DNA interaction. METHODS: To delineate the effect of CprB on the bound DNA, changes in motional dynamics of the fluorescent probe 2-aminopurine were monitored at three conserved base positions (5th, 12th and 23rd) for two DNA sequences: the consensus and the biologically relevant cognate element, on complex formation. RESULTS: The changes in lifetime and generalized order parameter revealed a similarity in the binding pattern of the protein to both sequences with greater dynamic restriction at the end positions, 5th and 23rd, as compared to the middle 12th position. Also differences within this pattern demonstrated the influence of even small changes in sequence on protein interactions. CONCLUSIONS: Here the study of motional dynamics was instrumental in establishing a structural footprint for the cognate DNA sequence and explaining the dynamics for the consensus DNA from structural correspondence. GENERAL SIGNIFICANCE: Motional dynamics can be a powerful tool to efficiently study the mode of DNA binding to proteins that interact differentially with a plethora of DNA sequences, even in the absence of structural breakthrough.

Aggregation induced enhanced emission of 2-(2'-hydroxyphenyl)benzimidazole.

In this study, the aggregation induced emission enhancement (AIEE) of 2-(2'-hydroxyphenyl)benzimidazole (HPBI) is reported. To investigate the AIEE process of HPBI, absorption/fluorescence spectroscopy, fluorescence imaging and field emission scanning electron microscopy were employed. A comparative study with 2-phenylbenzimidazole (PBI) divulges the significance of the hydroxyl group in the AIEE process. Further, molecular dynamics simulations have been carried out with explicit solvent molecules to follow the aggregation process of HPBI with time. The obtained molecular dynamics simulation results not only predicted the formation of aggregates but also provided detailed insight and information on the molecular interactions. The cellular studies showed aggregates yield higher fluorescence in the visible region inside HeLa cells in comparison to monomeric compounds which failed to exhibit any visible fluorescence inside the cell. The obtained aggregates were further found to be biocompatible and therefore can be used for bio-imaging applications.

Switching between cis and trans anions of 2-(2'-hydroxyphenyl)benzimidazole: a molecular rotation perturbed by chemical stabilization.

Despite the fact that 2-(2'-hydroxyphenyl)benzimidazole (HPBI) exists as the cis and trans enol in neutral form, it was reported to exist only in the trans form upon deprotonation in an aqueous medium. It was also shown that the dianion formed in the ground state can be re-protonated to form the trans-anion in the excited state. In the present study, the cis-anion and dianion could be obtained upon proper stabilization in suitable environments. Switching between the cis and trans-anion was demonstrated to be possible by changing the environment. Theoretical calculations were performed to substantiate the existence of the cis-anion and dianion. In contrast to a literature report, it was shown that the 'OH' group deprotonates before the 'NH' group to form a monoanion not only in protic solvents but also in aprotic solvents.

An unusual deprotonation trend in 2-(2'-hydroxyphenyl)pyridoimidazoles.

The anion sensitivity and the deprotonation nature of the nitrogenous analogues of 2-(2'-hydroxyphenyl)benzimidazole (HPBI) are investigated in a polar aprotic medium. It is observed that the substitution of pyridyl nitrogen enhances the anion sensitivity. However, despite the enhanced sensitivity of the nitrogenous analogues the deprotonation of these molecules in the presence of strong anions is less favored as compared to HPBI. This anomalous trend observed for the nitrogenous analogs is discussed and explained using theoretical calculations and experimental findings. It is also found that the sensitivity towards anions and the formation of anions also depend on the position of the pyridyl nitrogen.

Interactions of a few azole derivatives with a transport protein: role of heteroatoms.

The interaction of a few azole derivatives, 2-(4'-N,N-dimethylaminophenyl)benzimidazole, 2-(4'-N,N-dimethylaminophenyl)benzoxazole, 2-(4'-N,N-dimethylaminophenyl)oxazolo[4,5-b]pyridine with bovine serum albumin (BSA) were examined by absorption and fluorescence spectroscopy. The results were compared with the previously studied imidazopyridine derivative 2-(4'-N,N-dimethylaminophenyl)imidazo[4,5-b]pyridine. Displacement studies were carried out with site selective probes to locate the binding site of these ligands. The spectral shifts and the binding constant vary depending on the nature of the ligand. The fluorescence intensity of both oxazole derivatives 2-(4'-N,N-dimethylaminophenyl)benzoxazole and 2-(4'-N,N-dimethylaminophenyl) oxazolo[4,5-b]pyridine increases substantially in the presence of BSA, whereas the intensity of 2-(4'-N,N-dimethylaminophenyl)benzimidazole decreases. However, hypsochromic shift is observed in presence of BSA. The results obtained from the docking studies are also in good agreement with the experimental results. The location and orientation of binding depend upon the nature of the ligand. The studies revealed that apart from hydrophobic interaction, hydrogen bonding also plays a vital role in the molecular binding. Oxazoles have higher binding affinity than imidazoles and substitution of extra nitrogen further increases the binding affinity.

Effect of Melatonin on Glutamate: BDNF Signaling in the Cerebral Cortex of Polychlorinated Biphenyls (PCBs)-Exposed Adult Male Rats.

Various epidemiological survey suggests that the central nervous system is the target for many environmental contaminants. One among them is Aroclor 1254, a mixture of polychlorinated biphenyls (PCBs) which explore a spectrum of biochemical and neurotoxic responses in humans and laboratory animals. Learning and motor coordination deficits are the profound effects of PCBs which may be related to cerebral dysfunction. The aim of the study is to elicit the protective effect of melatonin (Mel), a potent, blood brain permeable antioxidant against the effect of Aroclor 1254 on the signaling of glutamate-principal excitatory neurotransmitter and brain derived neurotrophic factor (BDNF) in the cerebral cortex of adult rats which plays a key role in brain functions. Adult male Wistar rats were grouped into four and treated intraperitonealy (i.p) Group I with corn oil (Control), Group II with PCBs (2 mg/kg/bwt), Group III with PCBs + Mel (2 mg/kg/bwt + 5 mg/kg/bwt) and Group IV with Mel (5 mg/kg/bwt). The protein expression of glutamate signaling molecules and mRNA expressions of GLAST, BDNF signaling molecules were analyzed. The results suggest that simultaneous melatonin treatment significantly attenuated the NMDA receptor mediated glutamate excitotoxicity and protects the inhibition of BDNF signaling caused by PCBs exposure in cerebral cortex of adult male rats. Schematic pathway illustrating the proposed mechanism by which melatonin protects against A1254 mediated glutamate induced neurodegeneration in the cerebral cortex of adult male rats. PCBs induced neurodegeneration is caused by the overactivation of NMDAR, followed by the activation of voltage dependent calcium channels leading to the increase in intracellular Ca(2+) that stimulates calpain. Calpain inturn inhibits the PKA α and neurtrophin BDNF, its receptor and downstream signaling MAPK pathway leading to neurodegeneration. Melatonin had scavenged the ROS produced by PCBS and decreased the NMDAR expression which inturn protected the cells from neurodegeneration.

Depth-Dependent Organization and Dynamics of Archaeal and Eukaryotic Membranes: Development of Membrane Anisotropy Gradient with Natural Evolution.

The lipid composition of archaea is unique and has been correlated with increased stability under extreme environmental conditions. In this article, we have focused on the evolution of membrane organization and dynamics with natural evolution. Dynamic anisotropy along the membrane normal (i.e., gradients of mobility, polarity, and heterogeneity) is a hallmark of fluid phase diester or diether phospholipid membranes. We monitored gradients of mobility, polarity, and heterogeneity along the membrane normal in membranes made of a representative archaeal lipid using a series of membrane depth-dependent fluorescent probes, and compared them to membranes prepared from a typical diether lipid from higher organisms (eukaryotes). Our results show that the representative dynamic anisotropy gradient along the membrane normal is absent in membranes made from archaeal lipids. We hypothesize that the dynamic gradient observed in membranes of diester and diether phospholipids is a consequence of natural evolution of membrane lipids in response to the requirement of carrying out complex cellular functions by membrane proteins.

Relay proton transfer triggered twisted intramolecular charge transfer.

The mechanism for the dual emission of 2-(4'-N,N-dimethylaminophenyl)imidazo[4,5-c]pyridine (DMAPIP-c) in protic solvents was investigated by synthesizing and studying its analogues. Theoretical calculations were carried out to corroborate the experimental findings. The deprotonation studies suggest that the enhancement in the TICT emission of anionic forms of DMAPIP-c is limited to a protic environment. The spectral characteristics of DMAPIP-c were also studied in a methanol-acetonitrile binary solvent mixture. Unlike DMAPIP-c, the methyl derivatives do not emit dual fluorescence in protic solvents. The relative intensity of the TICT emission (with respect to that of normal emission) rises with the methanol amount in the acetonitrile-methanol binary solvent mixture. The studies also show that a 1:3 hydrogen bonded complex is formed between DMAPIP-c and methanol and it is responsible for the TICT emission. Based on the results a relay proton transfer tiggered TICT emission is proposed. TDDFT calculations were performed to predict the emission energies.

A single fluorophore to address multiple logic gates.

Logic gates with different radixes have been constructed using a biologically active molecule, 2-(4'-N,N-dimethylaminophenyl)imidazo[4,5-b]pyridine (DMAPIP-b). Taking advantage of the multiple binding sites of the fluorophore, a series of different molecular logic gates are developed using fluorescence intensities at different wavelengths. The high emission of the molecule is drastically quenched in the presence of Fe(3+). It is regained by the addition of an equivalent amount of F(-). The fluorescence On-Off nature has been used to construct molecular full subtractor and molecular keypad lock system with Boolean logic. A ternary system is generated by considering three defined fluorescence intensities at particular wavelengths. The smooth dependency of emission intensities with analyte concentration is utilized to construct an infinite-valued fuzzy logic system. The fuzzy logic system is further coupled with a neuro-adaptation method to predict more accurately the dependency of molecular intensity on external inputs.