Investigating the Photophysical Aspects of a Naphthalene-Based Excited-State Proton Transfer Dye 1-(1H-Benzo[d]imidazol-2-yl)naphthalen-2-ol: pH-Dependent Modulation of Photodynamics.

The steady state and time-resolved photophysical behavior of a proton transfer dye 1-(1H-benzo[d]imidazol-2-yl)naphthalen-2-ol (H-BINO) was investigated. The excited state intramolecular proton transfer (ESIPT) reaction in H-BINO is predominant in nonpolar solutions (toluene and DCM) with a lifetime of ∼1.0 ns. However, in polar media (DMF and MeOH), the excited state photodynamics is characterized by a complex equilibrium of emission from the locally excited state (0.1-2.3 ns), the phototautomer (0.5-1.2 ns), and the anionic emission (2.1-5.4 ns). In the solid state, emission from the various aggregated states dictates the photobehavior. Interestingly, the photodynamics in aqueous solution changes starkly as a function of pH with the anionic (2.1 ns) and phototautomeric (0.5-1.0 ns) emissions guiding the photodynamics as the pH of the medium increases. Optimized structural parameters at the proton donor and acceptor sites for the enol and keto forms and the calculated potential energy curve along the proton transfer coordinate at the density functional theory (DFT) level with the B3LYP/6-311G++(d,p) theory support a favorable and barrierless ESIPT process. The current results will surely boost the ongoing research on small molecule emissive materials.

Interrogating the nature of aggregates formed in a model azine based ESIPT coupled AIE active probe: stark differences in photodynamics in the solid state and aggregates in water.

A novel Schiff base 4-bromo-2-((E)-((E)-(1-(naphthalen-2-yl)ethylidene)hydrazono)methyl)phenol (BNHMP) was synthesized and characterized by NMR, ESI-MS, FTIR and single crystal X-ray diffraction studies. In the solution phase, BNHMP shows prominent emission from the keto-form, a consequence of excited state intramolecular proton transfer (ESIPT). The quantum yield and excited state lifetime decrease in polar solvent THF compared to relatively non-polar solvent DCM. Interestingly, in aqueous solution (pH 7.0), the quantum yield along with the excited state lifetime undergoes tremendous increment. Dynamic light scattering experiments and FESEM reveal the formation of aggregates in water as reflected by the increased hydrodynamic radius of BNHMP in water. Hence, aqueous phase studies revealed BNHMP to be an AIE active probe. On the other hand, BNHMP shows huge emission intensity in the solid state. Interestingly, the emission decay behavior of BNHMP changes upon excitation, as BNHMP shows very broad absorption in the solid state. Upon excitation at 360 nm, a triexponential decay pattern is found, which changes to a biexponential one upon excitation at 450 nm. Meticulous analysis of the fluorescence lifetimes led to the assignment of J and H aggregates coexisting in the solid state with the former dominating the photodynamics. A judicious comparison of the lifetime behavior in the solid state to that in water leads to the conclusion that BNHMP undergoes AIE by the formation of J and H aggregates to an equal extent, a phenomenon starkly different from the solid-state scenario. The current results hold significance as this is among a few reports where such comprehensive spectrodynamic dissection has been performed for an ESIPT-AIE active Schiff base in solution as well as in the solid phase, thereby giving a holistic vision of the nature and fate of aggregation occurring in such azine based systems and subsequently advancing the understanding of such systems in terms of their photo behavior.

"KRiShI": a manually curated knowledgebase on rice sheath blight disease.

Knowledgebase for rice sheath blight information (KRiShI) is a manually curated user-friendly knowledgebase for rice sheath blight (SB) disease that allows users to efficiently mine, visualize, search, benchmark, download, and update meaningful data and information related to SB using its easy and interactive interface. KRiShI collects and integrates widely scattered and unstructured information from various scientific literatures, stores it under a single window, and makes it available to the community in a user-friendly manner. From basic information, best management practices, host resistance, differentially expressed genes, proteins, metabolites, resistance genes, pathways, and OMICS scale experiments, KRiShI presents these in the form of easy and comprehensive tables, diagrams, and pictures. The "Search" tab allows users to verify if their input rice gene id(s) are Rhizoctonia solani (R. solani) responsive and/or resistant. KRiShI will serve as a valuable resource for easy and quick access to data and information related to rice SB disease for both the researchers and the farmers. To encourage community curation a submission facility is made available. KRiShI can be found at http://www.tezu.ernet.in/krishi .

Single-Cell Chromatin Accessibility Data Combined with GWAS Improves Detection of Relevant Cell Types in 59 Complex Phenotypes.

Several disease risk variants reside on non-coding regions of DNA, particularly in open chromatin regions of specific cell types. Identifying the cell types relevant to complex traits through the integration of chromatin accessibility data and genome-wide association studies (GWAS) data can help to elucidate the mechanisms of these traits. In this study, we created a collection of associations between the combinations of chromatin accessibility data (bulk and single-cell) with an array of 201 complex phenotypes. We integrated the GWAS data of these 201 phenotypes with bulk chromatin accessibility data from 137 cell types measured by DNase-I hypersensitive sequencing and found significant results (FDR adjusted p-value ≤ 0.05) for at least one cell type in 21 complex phenotypes, such as atopic dermatitis, Graves' disease, and body mass index. With the integration of single-cell chromatin accessibility data measured by an assay for transposase-accessible chromatin with high-throughput sequencing (scATAC-seq), taken from 111 adult and 111 fetal cell types, the resolution of association was magnified, enabling the identification of further cell types. This resulted in the identification of significant correlations (FDR adjusted p-value ≤ 0.05) between 15 categories of single-cell subtypes and 59 phenotypes ranging from autoimmune diseases like Graves' disease to cardiovascular traits like diastolic/systolic blood pressure.

Observation of Imbert-Fedorov shift in monolayer MoS2 via quantum weak measurement.

We report the experimental evidence of the Imbert-Fedorov (IF) shift in monolayer MoS2 for a fundamental Gaussian beam. Using Jones vector formalism, we have shown a novel, to the best of our knowledge, pathway to apply the quantum weak measurement technique for easy and accurate determination of the IF shift. We have revealed the dependence of IF shift over a large range of angles of incidence along with the mode of polarization of the incident light. Our experimental findings via the weak value amplification scheme are in good agreement with the theoretical analysis. The present method is a general one and can also be implemented for other materials to observe such tiny transverse shifts.

Temperature differentially modulates the transcriptome response in Oryza sativa to Xanthomonas oryzae pv. oryzae infection.

Bacterial blight is caused by the pathogen Xanthomonas oryzae pv. oryzae (Xoo). Genome scale integrative analysis on the interaction of high and low temperatures on the molecular response signature in rice during the Xoo infection has not been conducted yet. We have analysed a unique RNA-Seq dataset generated on the susceptible rice variety IR24 under combined exposure of Xoo with low 29/21 °C (day/night) and high 35/31 °C (day/night) temperatures. Differentially regulated key genes and pathways in rice plants during both the stress conditions were identified. Differential dynamics of the regulatory network topology showed that WRKY and ERF families of transcription factors play a crucial role during signal crosstalk events in rice plants while responding to combined exposure of Xoo with low temperature vs. Xoo with high temperatures. Our study suggests that upon onset of high temperature, rice plants tend to switch its focus from defence response towards growth and reproduction.

Triazoles inhibit cholesterol export from lysosomes by binding to NPC1.

Niemann-Pick C1 (NPC1), a membrane protein of lysosomes, is required for the export of cholesterol derived from receptor-mediated endocytosis of LDL. Lysosomal cholesterol export is reportedly inhibited by itraconazole, a triazole that is used as an antifungal drug [Xu et al. (2010) Proc Natl Acad Sci USA 107:4764-4769]. Here we show that posaconazole, another triazole, also blocks cholesterol export from lysosomes. We prepared P-X, a photoactivatable cross-linking derivative of posaconazole. P-X cross-linked to NPC1 when added to intact cells. Cross-linking was inhibited by itraconazole but not by ketoconazole, an imidazole that does not block cholesterol export. Cross-linking of P-X was also blocked by U18666A, a compound that has been shown to bind to NPC1 and inhibit cholesterol export. P-X also cross-linked to purified NPC1 that was incorporated into lipid bilayer nanodiscs. In this in vitro system, cross-linking of P-X was inhibited by itraconazole, but not by U18666A. P-X cross-linking was not prevented by deletion of the N-terminal domain of NPC1, which contains the initial binding site for cholesterol. In contrast, P-X cross-linking was reduced when NPC1 contained a point mutation (P691S) in its putative sterol-sensing domain. We hypothesize that the sterol-sensing domain has a binding site that can accommodate structurally different ligands.

Use of mutant 125I-perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells.

Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study, we generate an (125)I-labeled mutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to (125)I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The (125)I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines, we show that this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.

Fabrication of thiosemicarbazone-based Pd(II) complexes: structural elucidations, catalytic activity towards Suzuki-Miyaura coupling reaction and antitumor activity against TNBC cells.

Currently, there are many uses of metal complexes, especially in the fields of medicinal chemistry and catalysis. Thus, fabrication of new complexes which perform as a catalyst and chemotherapeutic drug is always a beneficial addition to the literature. Herein, we report three heterocyclic thiosemicarbazone-based Pd(II) complexes [Pd(HL1)Cl] (C1), [Pd(L2)(PPh3)] (C2) and [Pd(L3)(PPh3)]Cl (C3) having coligands Cl and PPh3. Thiosemicarbazone ligands (H2L1, H2L2 and HL3) and the complexes (C1-C3) were characterized methodically using several spectroscopic techniques. Single-crystal X-ray diffraction methods reveal that the structural environment around the metal center of C2 is square planar, while for C1 and C3 it is a slighty distorted square plane. The supramolecular network of compounds was built via hydrogen bonds, C-H⋯π and π⋯π interactions. Density functional theory (DFT) study of the structure of the complexes supports experimental findings. The application of these complexes as catalysts toward Suzuki-Miyaura coupling reactions has been examined with various aryl halides and phenyl boronic acid in PEG 400 solvent. The complexes displayed good biomolecular interactions with DNA/protein, with a binding constant value of the order of 105 M-1. C3 showed greater binding efficacy toward these biomolecules than the other complexes, which might be due to the cationic nature of C3. Furthermore, antitumor activity of the complexes was studied against the human triple-negative breast cancer (TNBC) cell line MDA-MB-231. It was found that C3 was more toxic (IC50 = 10 ± 2.90 µM) toward MDA-MB-231 cells than the other complexes. A known chemotherapeutic drug, 5-fluorouracil, was included as positive control. The programmed cell death mechanism of C3 was confirmed. Additionally, complex-induced apoptosis was confirmed and occurred via a mitochondria-dependent (intrinsic) pathway.

Gold-Catalyzed Aryl-Alkenylation of Alkenes.

Herein, we report the gold-catalyzed aryl-alkenylation of unactivated alkenes with alkenyl iodides and bromides employing ligand-enabled gold redox catalysis. The present methodology followed the π-activation pathway rather than the migratory insertion pathway, which is predominant in other transition metal catalysis such as Pd, Ni, Cu, etc. Detailed mechanistic investigations such as 31P NMR, deuterium labeling, and HRMS studies have been carried out to underpin mechanistic insights.

A biphenyl thiosemicarbazide based fluorogenic chemosensor for selective recognition of Cd2+: application in cell bioimaging.

A diversified biphenyl thiosemicarbazide based chemosensor (HBMC) has been fabricated and reported for the specific detection of Cd2+ in a MeOH : H2O (4 : 1) solution. We observed a chromogenic change from colorless to light yellow colour, and it showed a "turn-on" fluorogenic change from non fluorescent to blooming cyan colour. In fluorometric titration a sharp "turn-on" emission for Cd2+ was observed with a ∼16 fold increase in fluorescence intensity value at 496 nm by incremental addition of Cd2+ ions in the MeOH : H2O (4 : 1) solution. The reversibility of the chemosensor (HBMC) was confirmed by a sequential addition of the EDTA solution. Again the binding stoichiometry of HBMC with Cd2+ was found to be 2 : 1, as confirmed by Job's plot analysis and HRMS spectra of the HBMC-Cd2+ complex. The mechanism for Cd2+ sensing in MeOH : H2O (4 : 1) is based upon the inhibition of CN isomerization and ESIPT process and simultaneously turning on the CHEF (chelation enhanced fluorescence) process. The limit of detection for Cd2+ was found to be in the order of 10-8 (M), which implies that HBMC is an efficient probe to detect Cd2+ at the microscopic level. A reusability study was performed and on-sight detection of cadmium ions by the chemosensor (HBMC) was established by dip-stick experiment. In vitro detection of Cd2+ in human breast cancer cells (MDA-MB-231) by HBMC discloses its cell permeability and biocompatible nature. Computational studies (DFT and TDDFT) with the probe HBMC and HBMC-Cd2+ complex were also performed.

Receptor model-based source apportionment and ecological risk assessment of metals in sediment of river Ganga, India.

Ganga river surface sediment was sampled from 11 locations, which revealed average concentrations (mg/kg) of metals in the order Mn (296.93) > Zn (61.94) > Cr (54.82) > Cu (30.19) > Pb (24.42) > Cd (0.36). Sediment quality guidelines showed metals rarely to occasionally exhibit adverse biological effects. Indices like potential ecological risk, contamination security index, hazard quotients, multiple probable effect concentrations quality, mean probable effects level quotients, mean effects range median quotient suggest nil to a very low level of pollution with low ecological risk. Contamination factor, geo accumulation index, enrichment factor, quantification of contamination revealed that Pb and Cd originated from anthropogenic activities. APCS-MLR model revealed that metals contributed from natural sources (Zn, Mn, Cr; 20.18 %), industrial-agricultural (Cd; 21.35 %); and discharge of paints, Pb batteries, fossil fuel (Pb; 8.49 %). Present findings will serve as an effective guideline for managing and ameliorating pollution in the river system.

Time-course transcriptome analysis identifies rewiring patterns of transcriptional regulatory networks in rice under Rhizoctonia solani infection.

Sheath Blight (SB) disease in rice is caused by the infection from the fungal pathogen Rhizoctonia solani (R. solani). SB is one of the most severe rice diseases that can cause up to 50% yield losses in rice. Naturally occurring rice varieties resistant to SB have not been reported yet. We have performed a Time-Series RNA-Seq analysis on a widely cultivated rice variety BPT-5204 for identifying transcriptome level response signatures during R. solani infection at 1st, 2nd and 5th day post infection (dpi). In total, 428, 3225 and 1225 genes were differentially expressed in the treated rice plants on 1, 2 and 5 dpi, respectively. GO and KEGG enrichment analysis identified significant processes and pathways differentially altered in the rice plants during the fungal infection. Machine learning and network based integrative approach was used to construct rice Transcriptional Regulatory Networks (TRNs) for the three time points. TRN analysis identified SUB1B, MYB30 and CCA1 as important regulatory hub transcription factors in rice during R. solani infection. Jasmonic acid, salicylic acid, ethylene biogenesis and signaling were induced on infection. SAR was up regulated, while photosynthesis and carbon fixation processes were significantly down regulated. Involvement of MAPK, CYPs, peroxidase, PAL, chitinase genes were also observed in response to the fungal infection. The integrative analysis identified seven putative SB resistance genes differentially regulated in rice during R. solani infection.

Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection.

Niemann-Pick C1 (NPC1) is a lysosomal membrane protein that exports cholesterol derived from receptor-mediated uptake of LDL, and it also mediates cellular entry of Ebola virus. Cholesterol export is inhibited by nanomolar concentrations of U18666A, a cationic sterol. To identify the target of U18666A, we synthesized U-X, a U18666A derivative with a benzophenone that permits ultraviolet-induced crosslinking. When added to CHO cells, U-X crosslinked to NPC1. Crosslinking was blocked by U18666A derivatives that block cholesterol export, but not derivatives lacking blocking activity. Crosslinking was prevented by point mutation in the sterol-sensing domain (SSD) of NPC1, but not by point mutation in the N-terminal domain (NTD). These data suggest that the SSD contains a U18666A-inhibitable site required for cholesterol export distinct from the cholesterol-binding site in the NTD. Inasmuch as inhibition of Ebola requires 100-fold higher concentrations of U18666A, the high affinity U16888A-binding site is likely not required for virus entry.

Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis.

When human fibroblasts take up plasma low density lipoprotein (LDL), its cholesterol is liberated in lysosomes and eventually reaches the endoplasmic reticulum (ER) where it inhibits cholesterol synthesis by blocking activation of SREBPs. This feedback protects against cholesterol overaccumulation in the plasma membrane (PM). But how does ER know whether PM is saturated with cholesterol? In this study, we define three pools of PM cholesterol: (1) a pool accessible to bind 125I-PFO*, a mutant form of bacterial Perfringolysin O, which binds cholesterol in membranes; (2) a sphingomyelin(SM)-sequestered pool that binds 125I-PFO* only after SM is destroyed by sphingomyelinase; and (3) a residual pool that does not bind 125I-PFO* even after sphingomyelinase treatment. When LDL-derived cholesterol leaves lysosomes, it expands PM's PFO-accessible pool and, after a short lag, it also increases the ER's PFO-accessible regulatory pool. This regulatory mechanism allows cells to ensure optimal cholesterol levels in PM while avoiding cholesterol overaccumulation.

Identification of putative active site residues of ACAT enzymes.

In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

Identification of the interaction site within acyl-CoA:cholesterol acyltransferase 2 for the isoform-specific inhibitor pyripyropene A.

Targeted deletion of acyl-CoA:cholesterol acyltransferase 2 (ACAT2) (A2), especially in the liver, protects hyperlipidemic mice from diet-induced hypercholesterolemia and atherosclerosis, whereas the deletion of ACAT1 (A1) is not as effective, suggesting ACAT2 may be the more appropriate target for treatment of atherosclerosis. Among the numerous ACAT inhibitors known, pyripyropene A (PPPA) is the only compound that has high selectivity (>2000-fold) for inhibition of ACAT2 compared with ACAT1. In the present study we sought to determine the PPPA interaction site of ACAT2. To achieve this goal we made several chimeric proteins where parts of ACAT2 were replaced by the analogous region of ACAT1. Differences in the amino acid sequence and the membrane topology were utilized to design the chimeras. Among chimeras, A2:1-428/A1:444-550 had 50% reduced PPPA selectivity, whereas C-terminal-truncated ACAT2 mutant A2:1-504 (C-terminal last 22 amino acids were deleted) remained selectively inhibited, indicating the PPPA-sensitive site is located within a region between amino acids 440 and 504. Three additional chimeras within this region helped narrow down the PPPA-sensitive site to a region containing amino acids 480-504, representing the fifth putative transmembrane domain of ACAT2. Subsequently, for this region we made single amino acid mutants where each amino acid in ACAT2 was individually changed to its ACAT1 counterpart. Mutation of Q492L, V493L, S494A resulted in only 30, 50, and 70% inhibition of the activity by PPPA, respectively (as opposed to greater than 95% with the wild type enzyme), suggesting these three residues are responsible for the selective inhibition by PPPA of ACAT2. Additionally, we found that PPPA non-covalently interacts with ACAT2 apparently without altering the oligomeric structure of the protein. The present study provides the first evidence for a unique motif in ACAT2 that can be utilized for making an ACAT2-specific drug.

CGI-58 facilitates the mobilization of cytoplasmic triglyceride for lipoprotein secretion in hepatoma cells.

Comparative Gene Identification-58 (CGI-58) is a member of the alpha/beta-hydrolase family of proteins. Mutations in the human CGI-58 gene are associated with Chanarin-Dorfman syndrome, a rare autosomal recessive genetic disease in which excessive triglyceride (TG) accumulation occurs in multiple tissues. In this study, we investigated the role of CGI-58 in cellular lipid metabolism in several cell models and discovered a role for CGI-58 in promoting the packaging of cytoplasmic TG into secreted lipoprotein particles in hepatoma cells. Using both gain-of-function and loss-of-function approaches, we demonstrate that CGI-58 facilitates the depletion of cellular TG stores without altering cellular cholesterol or phospholipid accumulation. This depletion of cellular TG is attributable solely to augmented hydrolysis, whereas TG synthesis was not affected by CGI-58. Furthermore, CGI-58-mediated TG hydrolysis can be completely inhibited by the known lipase inhibitors diethylumbelliferyl phosphate and diethyl-p-nitrophenyl phosphate, but not by p-chloro-mercuribenzoate. Intriguingly, CGI-58-driven TG hydrolysis was coupled to increases in both fatty acid oxidation and secretion of TG. Collectively, this study reveals a role for CGI-58 in coupling lipolytic degradation of cytoplasmic TG to oxidation and packaging into TG-rich lipoproteins for secretion in hepatoma cells.