Distinct mechanism of Tb3+ and Eu3+ binding to NCS1.

Lanthanides have been frequently used as biomimetic compounds for NMR and fluorescence studies of Ca2+ binding proteins due to having similar physical properties and coordination geometry to Ca2+ ions. Here we report that a member of the neuronal calcium sensor family, neuronal calcium sensor 1, complexes with two lanthanide ions Tb3+ and Eu3+. The affinity for Tb3+ is nearly 50 times higher than that for Ca2+ (Kd,Tb3+ = 0.002 ± 0.0001 µM and Kd, Ca2+ = 91 nM) whereas Eu3+ binding is notably weaker, Kd,Eu3+ = 26 ± 1 µM. Interestingly, despite having identical charge and similar ionic radii, Tb3+ and Eu3+ ions exhibit a distinct binding stoichiometry for NCS1 with one Eu3+ and two Tb3+ ions bound per NCS1 monomer, as demonstrated in fluorescence titration and mass spectrometry studies. These results suggest that the lanthanides' affinity for the individual EF hands is fine-tuned by a small variation in the ion charge density as well as EF hand binding loop amino acid sequence. As observed previously for other lanthanide:protein complexes, the emission intensity of Ln3+ is enhanced upon complexation with the protein, likely due to the displacement of water molecules by oxygen atoms from the coordinating amino acid residues. The overall shape of the Tb3+NCS1 and Eu3+NCS1 monomer shows high levels of similarity compared to the Ca2+ bound protein based on their collision cross section. However, the distinct occupation of EF hands impacts NCS1 oligomerization and affinity for the D2R peptide that mimics the NCS1 binding site on the D2R receptor. Specifically, the Tb3+NCS1 complex populates the dimer and has comparable affinity for the D2R peptide, whereas Eu3+ bound NCS1 remains in the monomeric form with a negligible affinity for the D2R peptide.

Nanomolar affinity of EF-hands in neuronal calcium sensor 1 for bivalent cations Pb2+, Mn2+, and Hg2.

Abiogenic metals Pb and Hg are highly toxic since chronic and/or acute exposure often leads to severe neuropathologies. Mn2+ is an essential metal ion but in excess can impair neuronal function. In this study, we address in vitro the interactions between neuronal calcium sensor 1 (NCS1) and divalent cations. Results showed that non-physiological ions (Pb2+ and Mn2+) bind to EF-hands in NCS1 with nanomolar affinity and lower equilibrium dissociation constant than the physiological Ca2+ ion. (Kd, Pb2+ = 7.0 ± 1.0 nM; Kd, Mn2+ = 34.0 ± 6.0 nM; K). Native ultra-high resolution mass spectrometry (FT-ICR MS) and trapped ion mobility spectrometry-mass spectrometry (nESI-TIMS-MS) studies provided the NCS1-metal complex compositions-up to four Ca2+ or Mn2+ ions and three Pb2+ ions (M⋅Pb1-3Ca1-3, M⋅Mn1-4Ca1-2, and M⋅Ca1-4) were observed in complex-and similarity across the mobility profiles suggests that the overall native structure is preserved regardless of the number and type of cations. However, the non-physiological metal ions (Pb2+, Mn2+, and Hg2+) binding to NCS1 leads to more efficient quenching of Trp emission and a decrease in W30 and W103 solvent exposure compared to the apo and Ca2+ bound form, although the secondary structural rearrangement and exposure of hydrophobic sites are analogous to those for Ca2+ bound protein. Only Pb2+ and Hg2+ binding to EF-hands leads to the NCS1 dimerization whereas Mn2+ bound NCS1 remains in the monomeric form, suggesting that other factors in addition to metal ion coordination, are required for protein dimerization.

Tobacco Industry Branding Strategies and Its Influence on Young Adults.

Every year, tobacco industries are spending millions of dollars targeting youths and non-smokers with tactful advertisements and promotion, which has impact on early initiation, increasing overall smoking rate and further upsurge the disease and early death. The study aimed to explore the tobacco industry's branding strategies that influences youth to early initiation of tobacco in Bangladesh. This study was a cross-sectional design with mixed method approach and implemented during March - December 2019. Survey was conducted using semi-structured questionnaire through face-to-face interview among 208 students in different educational institutions of the selected areas of Dhaka city as well as twenty-five (25) KIIs were conducted with different experts personnel. Descriptive and inferential analysis were performed for quantitative data. Thematic analysis was done for qualitative data. Among student respondents, 71.0% were smokers and their average age of initiation was 13.8 years. Study revealed that 21% students attended or exposed to different types of tobacco industry's (TI) promotional programs including one-to-one/group campaign (61%), corporate programs (23%), seminar (21%), career counseling (21%) and sports event (2%). Among them, 70.0% students received free promotional items, such as T-shirt, lighter, cap, wrist belt, free cigarette, etc. at these programs and 65% students were interested in the motivational speech given by the TI representatives. Three-fourths (75%) were familiar with misleading branding terms such as light, tar, full flavor, filter and menthol. The initiation age of the smoking was associated with observing the arrangement of the smoking product in the retail shop (p <0.05), attracted to cigarette stick and packet color (p<0.05) and attracted to smell/flavor (p<0.05) and attracted to role model/celebrity's smoking (p<0.05). Among retailers, 59.3% received TI assistance including showcase decoration with brand color, receiving model box and mounting board. Besides, 59.3% retailers mentioned promoting new brands to the non-user including students through promotional campaign, convince/persuasion, giving free samples, etc. TI is undertaking aggressive marketing and promoting brands targeting the youth around educational institutions that are ultimately contributing to early smoking initiation. A Strong monitoring system should be in place to stop direct and indirect branding and promotional activities.