Effect of UV Stress on the Structure and Function of Pro-apoptotic Bid and Anti-apoptotic Bcl-xl proteins.

Ultraviolet C (UV-C) radiation induces apoptosis in mammalian cells via the mitochondrion-mediated pathway. The Bcl-2 family of proteins are the regulators of the mitochondrial pathway of apoptosis and appears responsive to UV-C radiation. It is unknown how the structure and, effectively, the function of these proteins are directly impacted by UV-C exposure. Here, we present the effect of UV-C irradiation on the structure and function of pro-apoptotic Bid-FL and anti-apoptotic Bcl-xlΔC proteins. Using a variety of biophysical tools, we show that, following UV-C irradiation, the structures of Bcl-xlΔC and Bid-FL are irreversibly altered. Bcl-xLΔC is found to be more sensitive to UV stress than Bid-FL Interestingly, UV-C exposure shows dramatic chemical shift perturbations in consequence of dramatic structural perturbations (α-helix to ß-sheet) in the BH3- binding region, a crucial segment of Bcl-xlΔC. Furter it has been shown that UV-exposed Bcl-xlΔC has reduced efficacy of its interactions with pro-apoptotic tBid.

Curcumin, a potential initiator of apoptosis via direct interactions with Bcl-xL and Bid.

Apoptosis is a naturally occurring process during the growth and development of multicellular organisms and is increasingly active during times of cellular stress such as in response to intracellular DNA damage when removal of the host cell is paramount to prevent cancer. Unfortunately, once formed, cancer cells become impervious to apoptosis, creating a desperate need to identify an approach to induce apoptosis in these cells. An attractive option is to focus efforts on developing and locating compounds which activate apoptosis using natural compounds. Curcumin is a natural component in turmeric and is well-known for its pharmacological effects in preventing and combating many ailments and has been shown to decrease the rapid proliferation of a wide variety of tumor cells. However, to date, the apoptotic intermediates and interactions through which curcumin exerts its cytotoxic effects are unknown. Motivated by reports linking the intracellular modulation of the concentrations of Bid and Bcl-xL, following curcumin administration to cancer cells, we set out to probe for potential intermolecular interactions of these proteins with curcumin. Using several biophysical techniques, most notably, fluorescence, circular dichroism and nuclear magnetic resonance spectroscopy, we reveal binding interactions of curcumin with both Bcl-xLΔC and full-length Bid (Bid-FL) and prove that this binding is hydrophobically driven and localized to well-known functional regions of each protein. Specifically, our NMR studies show that while Bid-FL interacts with curcumin through its hydrophobic and pore forming helices (α6-α7), Bcl-xLΔC interacts with curcumin via its BH3 binding pocket (α2-α3-α4-α5), a critical region for mediating apoptosis.

Novel Low-Noise CMOS Bioamplifier for the Characterization of Neurodegenerative Diseases.

Conducting cells of the heart and nerve cells of the brain are having the ability to generate and transmit electrical signals. Recording of neural signals became an important research issue for better analysis and better control of neurological functions by using implantable devices. In neural recording systems, the most critical part is the power constraint neural amplifier. The major challenges of neural front ends are low power dissipation and low input-referred noise. This work describes a low-noise amplifier that uses Metal Oxide Semiconductor bipolar pseudo-resistor elements to amplify signals from 0.03 millihertz to 8.4 kilohertz. This design is suitable for neurodegenerative disorders like Parkinson's disease and Alzheimer's. This topology reduces major noise in low-frequency circuits. By choosing input devices as PMOS transistors and also by properly sizing the devices, flicker noise is reduced. Noise and power trade-off is quantified by calculating noise efficiency factor (NEF) which is improved by using the proposed design. The circuit is implemented in 180 nm technology and is operated with a dual power supply range of ±2.5 V.

High nitrous oxide fluxes from rice indicate the need to manage water for both long- and short-term climate impacts.

Global rice cultivation is estimated to account for 2.5% of current anthropogenic warming because of emissions of methane (CH4), a short-lived greenhouse gas. This estimate assumes a widespread prevalence of continuous flooding of most rice fields and hence does not include emissions of nitrous oxide (N2O), a long-lived greenhouse gas. Based on the belief that minimizing CH4 from rice cultivation is always climate beneficial, current mitigation policies promote increased use of intermittent flooding. However, results from five intermittently flooded rice farms across three agroecological regions in India indicate that N2O emissions per hectare can be three times higher (33 kg-N2O⋅ha-1⋅season-1) than the maximum previously reported. Correlations between N2O emissions and management parameters suggest that N2O emissions from rice across the Indian subcontinent might be 30-45 times higher under intensified use of intermittent flooding than under continuous flooding. Our data further indicate that comanagement of water with inorganic nitrogen and/or organic matter inputs can decrease climate impacts caused by greenhouse gas emissions up to 90% and nitrogen management might not be central to N2O reduction. An understanding of climate benefits/drawbacks over time of different flooding regimes because of differences in N2O and CH4 emissions can help select the most climate-friendly water management regimes for a given area. Region-specific studies of rice farming practices that map flooding regimes and measure effects of multiple comanaged variables on N2O and CH4 emissions are necessary to determine and minimize the climate impacts of rice cultivation over both the short term and long term.

Aminoglycoside antibiotic resistance conferred by Hpa2 of MDR Acinetobacter baumannii: an unusual adaptation of a common histone acetyltransferase.

Antibiotic-resistant bacteria pose the greatest threat to human health. Among the list of such bacteria released by WHO, carbapenem-resistant Acinetobacter baumannii, for which almost no treatment exists, tops the list. A. baumannii is one of the most troublesome ESKAPE pathogens and mechanisms that have facilitated its rise as a successful pathogen are not well studied. Efforts in this direction have resulted in the identification of Hpa2-Ab, an uncharacterized histone acetyltransferase enzyme of GNAT superfamily. Here, we show that Hpa2-Ab confers resistance against aminoglycoside antibiotics using Escherichia coli DH5α strains in which Hpa2 gene is expressed. Resistivity for aminoglycoside antibiotics is demonstrated with the help of CLSI-2010 and KB tests. Isothermal titration calorimetry, MALDI and acetylation assays indicate that conferred resistance is an outcome of evolved antibiotic acetylation capacity in this. Hpa2 is known to acetylate nuclear molecules; however, here it is found to cross its boundary and participate in other functions. An array of biochemical and biophysical techniques were also used to study this protein, which demonstrates that Hpa2-Ab is intrinsically oligomeric in nature, exists primarily as a dimer and its interface is mainly stabilized by hydrophobic interactions. Our work demonstrates an evolved survival strategy by A. baumannii and provides insights into the mechanism that facilitates it to rise as a successful pathogen.

G-SERF Editing in Two-Dimensional Pure-Shift Total Correlation Spectroscopy: Scalar Coupling Measurements for a Group of Spins in Organic Molecules.

A novel G-SERF-PSYCHE-TOCSY (gradient encoded selective refocusing in pure shift yielded by chirp excitation version of total correlation spectroscopy) NMR pulse scheme has been proposed, which produces TOCSY chemical shift correlations, on one hand, and scalar coupling values for the spins scalarly coupled to irradiated resonances, by showing them as doublets along the indirect dimension, on the other. Therefore, recording such an experiment, for a group of spins with overlapping chemical shifts, in organic molecules can adequately provide scalar coupling information in a G-SERF manner along the indirect dimensions, and they can be assigned to particular spin pairs. Such COSY chemical shift correlations (which appear as doublets for the scalarly coupled spins) can be readily discriminated from the TOCSY peaks (which do not show such splitting) in the G-SERF-PSYCHE-TOCSY spectrum.

Multiple homonuclear band-selective decoupling NMR: Fast and unambiguous determination of diastereomeric excess.

Discrimination and quantification of chiral stereoisomers have been studied by different analytical methods, and NMR has emerged as a powerful one with the advancements in pure-shift NMR methods. In the present manuscript, an al-F1F2-MHOBS-DIAG NMR method for the quantification of diastereomeric excess ratio (dr) has been proposed and demonstrated, using hesperidin and naringin mixtures. This method enables simultaneous quantification of dr at multiple resonances, in a single experiment, and it takes only 10 min to record. The present method uses spectral aliasing and thus demands only very few indirect dwell increments. Further, the measured dr values are very reliable, because we consider several spins for the quantification.

Fast and simultaneous determination of 1 H-1 H and 1 H-19 F scalar couplings in complex spin systems: Application of PSYCHE homonuclear broadband decoupling.

The present manuscript focuses on fast and simultaneous determination of 1 H-1 H and 1 H-19 F scalar couplings in fluorinated complex steroid molecules. Incorporation of broadband PSYCHE homonuclear decoupling in the indirect dimension of zero-quantum filtered diagonal experiments (F1-PSYCHE-DIAG) suppresses 1 H-1 H scalar couplings; however, it retains 1 H-19 F scalar couplings (along F1 dimension) for the 19 F coupled protons while preserving the pure-shift nature for 1 H resonances uncoupled to 19 F. In such cases, along the direct dimensions, 1 H-1 H scalar coupling multiplets deconvolute and they appear as duplicated multiplets for the 19 F coupled protons, which facilitates unambiguous discrimination of 19 F coupled 1 H chemical sites from the others. Further, as an added advantage, data acquisition has been accelerated by invoking the known ideas of spectral aliasing in the F1-PSYCHE-DIAG scheme and experiments demand only ~10 min of spectrometer times.

Probing the Potential Role of Non-B DNA Structures at Yeast Meiosis-Specific DNA Double-Strand Breaks.

A plethora of evidence suggests that different types of DNA quadruplexes are widely present in the genome of all organisms. The existence of a growing number of proteins that selectively bind and/or process these structures underscores their biological relevance. Moreover, G-quadruplex DNA has been implicated in the alignment of four sister chromatids by forming parallel guanine quadruplexes during meiosis; however, the underlying mechanism is not well defined. Here we show that a G/C-rich motif associated with a meiosis-specific DNA double-strand break (DSB) in Saccharomyces cerevisiae folds into G-quadruplex, and the C-rich sequence complementary to the G-rich sequence forms an i-motif. The presence of G-quadruplex or i-motif structures upstream of the green fluorescent protein-coding sequence markedly reduces the levels of gfp mRNA expression in S. cerevisiae cells, with a concomitant decrease in green fluorescent protein abundance, and blocks primer extension by DNA polymerase, thereby demonstrating the functional significance of these structures. Surprisingly, although S. cerevisiae Hop1, a component of synaptonemal complex axial/lateral elements, exhibits strong affinity to G-quadruplex DNA, it displays a much weaker affinity for the i-motif structure. However, the Hop1 C-terminal but not the N-terminal domain possesses strong i-motif binding activity, implying that the C-terminal domain has a distinct substrate specificity. Additionally, we found that Hop1 promotes intermolecular pairing between G/C-rich DNA segments associated with a meiosis-specific DSB site. Our results support the idea that the G/C-rich motifs associated with meiosis-specific DSBs fold into intramolecular G-quadruplex and i-motif structures, both in vitro and in vivo, thus revealing an important link between non-B form DNA structures and Hop1 in meiotic chromosome synapsis and recombination.

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates.

Cyclophilin catalyzes the ubiquitous process "peptidyl-prolyl cis-trans isomerization," which plays a key role in protein folding, regulation, and function. Here, we present a detailed characterization of the unfolding of yeast mitochondrial cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and I2. The I1 state has native-like secondary structure and shows strong anilino-8-naphthalenesulphonate binding due to increased exposure of the solvent-accessible cluster of non-polar groups. Thus, it has some features of a molten globule. The I2 state is more unfolded, but it retains some residual secondary structure, and shows weak anilino-8-naphthalenesulphonate binding. Chemical shift perturbation analysis by 1H-15N heteronuclear single quantum coherence spectra reveals disruption of the tertiary contacts among the regions close to the active site in the first step of unfolding, i.e., the N-I1 transition. Both of the intermediates, I1 and I2, showed a propensity to self-associate under stirring conditions, but their kinetic profiles are different; the native protein did not show any such tendency under the same conditions. All these observations could have significant implications for the function of the protein.

Aetiology of acute encephalitis syndrome in Uttar Pradesh, India from 2014 to 2016.

BACKGROUND & OBJECTIVES: It is imperative to know the aetiology of acute encephalitis syndrome (AES) for patient management and policy making. The present study was carried out to determine the prevalence of common aetiological agents of AES in Uttar Pradesh (UP) state of India. METHODS: Serum and/or CSF samples were collected from AES patients admitted at Gandhi Memorial and Associated Hospital, King George's Medical University, Lucknow, a tertiary care centre, UP during 2014-16. Cerebrospinal fluid (CSF) and serum samples from cases were tested for IgM antibodies against Japanese encephalitis virus (anti-JEV), and dengue virus (anti-DENV) by ELISA; and for enterovirus, herpes simplex virus (HSV) and varicella zoster virus (VZV) by real-time PCR. Serum samples of cases having sufficient CSF volume, were also tested for anti-scrub typhus IgM antibodies and for Neisseria meningitides, Streptococcus pneumoniae and Haemophilus influenzae. RESULTS: JEV and DENV (8% each) were the most common identified aetiology from the 4092 enrolled patients. Enterovirus, HSV and VZV, each were detected in <1% AES cases. Co-positivity occurred in 48 cases. Scrub typhus (31.8%) was the most common aetiology detected. Haemophilus influenzae and S. pneumoniae were detected in 0.97 and 0.94% cases, respectively, however, N. meningitides was not detected in any of the cases. About 40% of the JEV/DENV positive AES cases were adults. The gap between the total number of AES cases and those with JEV/ DENV infection increased during monsoon and post-monsoon seasons. INTERPRETATION & CONCLUSION: Scrub typhus, JEV and DENV are the main aetiological agents of AES in UP. DENV and JEV can no longer be considered paediatric diseases. The prevalence of non-JEV/DENV aetiology of AES increases in the monsoon and post-monsoon seasons.

Non-Uniform-Sampling Ultrahigh Resolution TOCSY NMR: Analysis of Complex Mixtures at Microgram Levels.

Non-uniform sampling in combination with homonuclear broadband decoupling along an indirect dimension, and indirect covariance processing are used to record ultrahigh resolution two-dimensional TOCSY spectra in less than half an hour, for typical sample concentrations in the mm range. TOCSY correlations belonging to protons separated by as little as ≈2 Hz can be distinctly discerned. The utility of the technique for low concentrations has been demonstrated.

Hadamard Homonuclear Broadband Decoupled TOCSY NMR: Improved Efficacy in Detecting Long-range Chemical Shift Correlations.

The importance of Hadamard encoding pulses in one-dimensional pure shift yielded by the chirp excitation version of selective total correlation spectroscopy (1D PSYCHE-TOCSY) experiments is discussed for chemical-shift analysis of complex natural products at ultrahigh resolution. Herein, we adapted Hn Hadamard matrices to 1D PSYCHE-TOCSY and observed an overall circa square root of n-fold enhancement in the signal-to-noise (S/N) ratio when compared to conventional 1D PSYCHE-TOCSY recorded by refocusing only one spin at a time. This enhancement in S/N facilitates the observation of very weak long-range chemical-shift correlations from Hadamard-encoded PSYCHE-TOCSY (HE-PSYCHE-TOCSY). The proposed method will have a significant impact on structure determination of complex isolated/ synthetic natural products.

Synthesis and biological evaluation of novel N1-phenylsulphonyl indole derivatives as potent and selective 5-HT6R ligands for the treatment of cognitive disorders.

A series of 1-[3-(4-methyl piperazin-1-ylmethyl) phenylsulfonyl]-1H-indole and 1-[3-(4-ethyl piperazin-1-ylmethyl) phenylsulfonyl]-1H-indole derivatives were designed and synthesized as 5-HT6 receptor (5-HT6R) ligands. The lead compound 1-[4-methyl-3-(4-methyl piperazin-1-yl methyl) phenylsulfonyl]-1H-indole dihydrochloride (6b), in this series, has shown potent in vitro binding affinity, selectivity, good pharmacokinetics (PK) profile and activity in the animal models of cognition.

Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters.

Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules.

Design, synthesis and pharmacological evaluation of indolylsulfonamide amines as potent and selective 5-HT6 receptor antagonists.

A series of N'-[3-(indole-1-sulfonyl) aryl]-N,N-dimethyl ethane-1,2-diamines and N'-[3-(indole-1-sulfonyl) aryl]-N,N-dimethyl propane-1,3-diamines was designed and synthesized as 5-HT6 receptor ligands. These compounds, when screened in a functional reporter gene-based assay, displayed potent antagonistic activity with Kb values in the range of 1.8-60 nM. The lead compound 9y has shown good ADME surrogate properties, acceptable pharmacokinetic profile and is active in animal models of cognition like novel object recognition test and Morris water maze. It was selected for detailed profiling.

Reduced dimensionality (3,2)D NMR experiments and their automated analysis: implications to high-throughput structural studies on proteins.

Protein NMR spectroscopy has expanded dramatically over the last decade into a powerful tool for the study of their structure, dynamics, and interactions. The primary requirement for all such investigations is sequence-specific resonance assignment. The demand now is to obtain this information as rapidly as possible and in all types of protein systems, stable/unstable, soluble/insoluble, small/big, structured/unstructured, and so on. In this context, we introduce here two reduced dimensionality experiments ­ (3,2)D-hNCOcanH and (3,2)D-hNcoCAnH ­ which enhance the previously described 2D NMR-based assignment methods quite significantly. Both the experiments can be recorded in just about 2-3 h each and hence would be of immense value for high-throughput structural proteomics and drug discovery research. The applicability of the method has been demonstrated using alpha-helical bovine apo calbindin-D9k P43M mutant (75 aa) protein. Automated assignment of this data using AUTOBA has been presented, which enhances the utility of these experiments. The backbone resonance assignments so derived are utilized to estimate secondary structures and the backbone fold using Web-based algorithms. Taken together, we believe that the method and the protocol proposed here can be used for routine high-throughput structural studies of proteins.

Complete backbone and DENQ side chain NMR assignments in proteins from a single experiment: implications to structure-function studies.

Resonance assignment is the first and the most crucial step in all nuclear magnetic resonance (NMR) investigations on structure-function relationships in biological macromolecules. Often, the assignment exercise has to be repeated several times when specific interactions with ligands, substrates etc., have to be elucidated for understanding the functional mechanisms. While the protein backbone serves to provide a scaffold, the side chains interact directly with the ligands. Such investigations will be greatly facilitated, if there are rapid methods for obtaining exhaustive information with minimum of NMR experimentation. In this context, we present here a pulse sequence which exploits the recently introduced technique of parallel detection of multiple nuclei, e.g. (1)H and (13)C, and results in two 3D-data sets simultaneously. These yield complete backbone resonance assignment ((1)H(N), (15)N, (13)CO, (1)Hα/(13)Cα, and (1)Hß/(13)Cß chemical shifts) and side chain assignment of D, E, N and Q residues. Such an exhaustive assignment has the potential of yielding accurate 3D structures using one or more of several algorithms which calculate structures of the molecules very reliably on the basis of NMR chemical shifts alone. The side chain assignments of D, E, N, and Q will be extremely valuable for interaction studies with different ligands; D and E side chains are known to be involved in majority of catalytic activities. Utility of this experiment has been demonstrated with Ca(2+) bound M-crystallin, which contains largely D, E, N and Q residues at the metal binding sites.

A reduced dimensionality NMR pulse sequence and an efficient protocol for unambiguous assignment in intrinsically disordered proteins.

Resonance assignment in intrinsically disordered proteins poses a great challenge because of poor chemical shift dispersion in most of the nuclei that are commonly monitored. Reduced dimensionality (RD) experiments where more than one nuclei are co-evolved simultaneously along one of the time axes of a multi-dimensional NMR experiment help to resolve this problem partially, and one can conceive of different combinations of nuclei for co-evolution depending upon the magnetization transfer pathways and the desired information content in the spectrum. Here, we present a RD experiment, (4,3)D-hNCOCAnH, which uses a combination of CO and CA chemical shifts along one of the axes of the 3-dimensional spectrum, to improve spectral dispersion on one hand, and provide information on four backbone atoms of every residue-HN, N, CA and CO chemical shifts-from a single experiment, on the other. The experiment provides multiple unidirectional sequential (i → i - 1) amide (1)H correlations along different planes of the spectrum enabling easy assignment of most nuclei along the protein backbone. Occasional ambiguities that may arise due to degeneracy of amide proton chemical shifts are proposed to be resolved using the HNN experiment described previously (Panchal et al. in J Biomol NMR 20:135-147, 2001). Applications of the experiment and the assignment protocol have been demonstrated using intrinsically disordered α-synuclein (140 aa) protein.