Wavelength-selective fluorescence as a novel tool to study organization and dynamics in complex biological systems

The dynamics exhibited by a given component of a large macromolecule such as a folded globular protein or an organized supramolecular assembly like the biological membrane is a function of its precise localization within the larger system. A set of approaches based on the red edge effect in fluorescence spectroscopy, which can be used to monitordirectly the environment and dynamics around a fluorophore in a complex biological system, is reviewed in this article. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases. This phenomenon arises from the slow rates of solvent relaxation around an excited-state fluorophore, which is a function of the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise optically silent water molecules. This makes REES and related techniques extremely useful in biology since hydration plays a crucial modulatory role in a large number of important cellular events

Perception Versus Actual Value of Quality of Drinking Water: A Case Study of Iron and Steel Industry in West Bengal, India

The study aims to understand employees’ knowledge, awareness, and overall perception of drinking water quality in the Iron and Steel Industry in Burnpur, India. Further, this study evaluated drinking water’s physicochemical and bacteriological properties collected from different company sites. This study uses a mixed-method approach with individual interviews of selected employees (n=342) and the laboratory test of eight selected drinking water sites. The results show that most employees considered drinking water acceptable to be excellent. However, only 30% of employees in Site 1 (Coke Oven By-Product department) have reported organoleptic properties of water under the excellent category. The result explained that other physicochemical and bacteriological properties are in good status in all sites except for a colony count, expressing their suitability for drinking purposes. In summary, employees’ perception of water quality aligns with their drinking water’s physicochemical and bacteriological properties

A small insulinomimetic molecule also improves insulin sensitivity in diabetic mice

Dramatic increase of diabetes over the globe is in tandem with the increase in insulin requirement. This is because destruction and dysfunction of pancreatic β-cells are of common occurrence in both Type1 diabetes and Type2 diabetes, and insulin injection becomes a compulsion. Because of several problems associated with insulin injection, orally active insulin mimetic compounds would be ideal substitute. Here we report a small molecule, a peroxyvanadate compound i.e. DmpzH[VO(O2)2(dmpz)], henceforth referred as dmp, which specifically binds to insulin receptor with considerable affinity (KD-1.17μM) thus activating insulin receptor tyrosine kinase and its downstream signaling molecules resulting increased uptake of [14C] 2 Deoxy-glucose. Oral administration of dmp to streptozotocin treated BALB/c mice lowers blood glucose level and markedly stimulates glucose and fatty acid uptake by skeletal muscle and adipose tissue respectively. In db/db mice, it greatly improves insulin sensitivity through excess expression of PPARγ and its target genes i.e. adiponectin, CD36 and aP2. Study on the underlying mechanism demonstrated that excess expression of Wnt3a decreased PPARγ whereas dmp suppression of Wnt3a gene increased PPARγ expression which subsequently augmented adiponectin. Increased production of adiponectin in db/db mice due to dmp effected lowering of circulatory TG and FFA levels, activates AMPK in skeletal muscle and this stimulates mitochondrial biogenesis and bioenergetics. Decrease of lipid load along with increased mitochondrial activity greatly improves energy homeostasis which has been found to be correlated with the increased insulin sensitivity. The results obtained with dmp, therefore, strongly indicate that dmp could be a potential candidate for insulin replacement therapy

Fluorophore environments in membrane-bound probes: a red edge excitation shift study

A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the Red Edge Excitation Shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases. In this paper, we report the red edge excitation shift of a membrane-bound phospholipid molecule whose headgroup is covalently labeled with a 7-nitrobenz- 2-oxa- 1,3-diazol-4-y1 (NBD) moiety. When incorporated into model membranes of dioleoyl-sn-glycero- 3-phosphocholine (DOPC), the NBD-labeled phospholipid (NBD-PE), exhibits a red edge excitation shift of 10 nm. In addition, fluorescence polarization of NBD-PE in membranes shows both excitation and emission wavelength dependence. The nonpolar membrane probe 1,6-diphenyl- 1,3,5-hexatriene (DPH) does not show red edge excitation shift in model membranes. The lifetime of NBD-PE in DOPC vesicles was found to be dependent on both excitation and emission wavelengths. These wavelength-dependent lifetimes are correlated to the reorientation of solvent dipoles around the excited-state dipole of the NBD moiety in the membrane. The magnitude of the red shift in the emission maximum for NBD-PE was found to be independent of temperature, between 12 and 54 ° C , and of the physical state (gel or fluid) of the membrane. Taken together, these observations are indicative of the motional restriction experienced by this fluorophore in the membrane. Red edge excitation shift promises to be a powerful tool in probing membrane organization and dynamics

Motionally restricted tryptophan environments at the peptide-lipid interface of gramicidin channels

The tryptophans in the gramicidin channel play a crucial role in the organization and function of the channel. The localization and dynamics of these tryptophans have been studied using fluorescence spectroscopy, especially utilizing environment-induced effects on the rates of solvent relaxation around these residues in membranes. When incorporated into model membranes of dioleoyl-sn-glycero-3-phosphocholine (DOPC), the tryptophans in the gramicidin channel exhibit a red edge excitation shift (REES) of 6 nm. In addition, fluorescence polarization shows both excitation and emission wavelength dependence. Fluorescence lifetime analysis shows a biexponential decay, corresponding to a short- and a long-lifetime component. The mean lifetime was found to be dependent on both excitation and emission wavelengths. Analysis of time-resolved emission spectra (TRES) shows a heterogeneous environment for the tryptophans consistent with the lifetime information. Taken together, these observations point out the motional restriction experienced by the tryptophans in the gramicidin channel. This is consistent with other studies in which such restrictions are thought to be imposed due to hydrogen bonding between the indole rings of the tryptophans and the neighboring lipid carbonyls. The significance of such organization in terms of functioning of the channel is brought out by the fact that substitution, photodamage, or chemical modification of these tryptophans is known to give rise to channels with conformation and reduced conductivity

Depth-dependent solvent relaxation in membranes: wavelength-selective fluorescence as a membrane dipstick

Membrane penetration depth represents an important parameter which can be used to define the conformation and topology of membrane proteins and probes. We have previously characterized a set of fluorescence spectroscopic approaches, collectively referred to as wavelength-selective fluorescence, as a powerful tool to monitor microenvironments in the vicinity of reporter fluorophores embedded in the membrane. Since several membrane parameters that characterize local environments such as polarity, fluidity, segmental motion, degree of water penetration, and the ability to form hydrogen bonds are known to vary as a function of depth of penetration into the membrane, we propose that wavelength-selective fluorescence could provide a novel approach to investigate the depth of membrane penetration of a reporter fluorophore. We test this hypothesis by demonstrating that chemically identical fluorophores, varying solely in terms of their localization at different depths in the membrane, experience very different local environments, as judged by wavelength-selective fluorescence parameters. We used two anthryoloxy stearic acid derivatives where the anthroyloxy group has previously been found to be either shallow (2-AS) or deep (12-AS). Our results show that the anthroyloxy moiety of 2- and 12-AS experiences different local membrane microenvironments, as reflected by varying extents of red-edge excitation shift (REES) as well as varying degrees of wavelength dependence of fluorescence polarization and lifetime and rotational correlation times. We attribute these results to differential rates of solvent reorientation in the immediate vicinity of the anthroyloxy group as a function of its membrane penetration depth. We thus provide evidence, for the first time, of depth-dependent solvent relaxation which can be used as a membrane dipstick

Red edge excitation shift of a deeply embedded membrane probe: implications in water penetration in the bilayer

The biological membrane is a highly organized anisotropic molecular assembly. While the center of the bilayer is nearly isotropic, the upper portion, only a few angstroms away toward the membrane surface, is highly ordered. How this organization correlates with the degree of water penetration into the bilayer interior is not clear. In general, it is believed that there is not much water in the deeper hydrocarbon regions of the bilayer. In this study, we have utilized the phenomenon of wavelength-selective fluorescence to address this question. We show here that when the same fluorescent group (i.e., 7-nitrobenz-2-oxa-1,3-diazol-4-yl or NBD) is localized at different depths within the bilayer (viz., near the membrane interface in case of the headgroup-labeled NBD-phosphatidylethanolamine (NBD-PE) and near the center of the bilayer in NBD-cholesterol), the degrees to which their fluorescence properties exhibit solvent-induced effects are markedly different. For example, the headgroup-labeled NBD-PE exhibits a much stronger red edge excitation shift (REES) relative to that of NBD-cholesterol. This indicates lesser restriction to mobility in this region as compared to the polar/hydrocarbon interface. In the gel phase, however, REES of NBD-PE did not show any significant change while NBD-cholesterol exhibited no REES. In addition, NBD-cholesterol exhibits a stronger dependence of fluorescence polarization on excitation wavelength in fluid membranes. We attribute these results to the more compact arrangement of the lipid acyl chains in the gel phase which results in lesser water penetration. Since the hydrophobic core of the lipid bilayer is made up of methyl and methylene groups, the only solvent dipoles capable of any interaction with the dipole of the fluorophore giving rise to the REES effect in the fluid phase have to be water molecules that have penetrated deep into the bilayer close to the NBD moiety of NBD-cholesterol. Our results indicate that at least in the fluid phase of the membrane, penetration of water in the deep hydrocarbon region of the bilayer does indeed occur

Effectiveness and feasibility of methanol extracted latex of Calotropis procera as larvicide against dengue vectors of western Rajasthan, India

Background & objectives:Identification of novel effective larvicide from natural resources is essential to combat developing resistances, environmental concerns, residue problems and high cost of synthetic insecticides. Results of earlier laboratory findings have shown that Calotropis procera extracts showed larvicidal, ovicidal and refractory properties towards ovipositioning of dengue vectors; further, latex extracted with methanol was found to be more effective compared to crude latex. For testing efficacy and feasibility of extracted latex in field, the present study was undertaken in different settings of Jodhpur City, India against dengue vectors. Methods:Study areas were selected based on surveillance design for the control of dengue vectors. During the study period domestic and peri-domestic breeding containers were treated with methanol extracted latex and mortality was observed after 24 h as per WHO guidelines. Latex was manually collected from internodes of Calotropis procera and extracted using methanol (AR) grade. Results:Methanol extracted latex of C. procera was found effective and feasible larvicide against dengue vectors in the field conditions. Cement tanks, clay pots and coolers (breeding sites) were observed as key containers for the control of dengue transmission. Interpretation & conclusion:Today environmental safety is considered to be very important. Herbal composition prepared by the extraction of latex of C. procera can be used as an alternative approach for the control of dengue vectors. This will reduce the dependence on expensive products and stimulate local efforts to enhance the public involvement

Micellar organization and dynamics: a wavelength-selective fluorescence approach

Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in fluorescence spectroscopy, which can be used to monitor directly the environment and dynamics around a fluorophore in a complex biological system. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band, is termed the red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases. We have previously shown that REES and related techniques (wavelength-selective fluorescence approach) offer a novel way to monitor organization and dynamics of membrane-bound probes and peptides. In this paper, we report REES of NBDPE, a phospholipid whose headgroup is covalently labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) moiety, when incorporated into micelles formed by a variety of detergents (SDS, Triton X-100, CTAB and CHAPS) which differ in their charge and organization. In addition, fluorescence polarization of NBD-PE in these micelles shows both excitation and emission wavelength dependence. The lifetime of NBD-PE was found to be dependent on both excitation and emission wavelengths. These wavelength-dependent lifetimes are correlated to the reorientation of solvent dipoles around the excited-state dipole of the NBD group in micellar environments. Taken together, these observations are indicative of the motional restriction experienced by the fluorophore when bound to micelles. Wavelength-selective fluorescence promises to be a powerful tool for studying micellar organization and dynamics