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

DNA Damage Response to Lesions Involving Both Strands of the Double-helix

DNA damage response is vital to genome maintenance, cell survival and successful transmission of genetic information to daughter cells. This response is extremely important since DNA is subject to damage daily either by endogenous metabolic errors and byproducts or by exposure to genotoxic agents. Different types of lesions are formed as a result of such insults to the DNA; the most toxic of such lesions are those that affect both strands of the double-helix. During my dissertation work, I studied cellular response to DNA lesions such as double-strand breaks and interstrand crosslinks using the model system Drosophila melanogaster. Double-strand breaks are repaired primarily by two mechanisms: homology mediated repair (HR) and nonhomologous end joining (NHEJ). Here I discuss the importance of homology mediated repair by studying repair defects in mutants defective for either of the two genes: 1) nbs gene encodes for the protein Nibrin, which is part of a well characterized protein complex MRN, comprising two other proteins Mre11 and Rad50 2) okra encodes the Drosophila homolog of the Rad54 protein. While the MRN complex is hypothesized to be required during early steps of HR such as break resection, Rad54 is believed to be involved in chromatin remodeling and facilitating the role of the strand invasion protein, Rad51. I have addressed several questions here about the function of MRN in responding to double-strand breaks, using mutations in the nbs gene. Since the NBS protein is known to target the MRN complex to the nucleus, study of NBS in isolation should be reflective of the nuclear function of the MRN complex. The requirement of MRN for NHEJ and /or HR appears to differ in different organism. I found that Drosophila NBS is required for HR and not NHEJ. In addition, I found that in contrast to other studies, MRN may function in late steps of HR, post break resection in Drosophila. Study of defects in responding to DNA damage, specifically double-strand breaks (DSBs), in haploinsufficient nbs mutant backgrounds provided valuable clues into underlying molecular mechanisms that lead to carcinogenesis in human carriers of nbs mutation. I tested to verify if DmRad54 is functionally conserved. This study showed that not only does DmRad54 facilitate DmRad51 function during first round of strand invasion, but it is also required multiple times while repairing the break, during the several rounds of strand invasion and synthesis that is characteristic of HR in pre-meiotic germline cells in Drosophila. The second type of toxic lesion discussed here are the interstrand crosslinks (ICLs). Multiple repair mechanisms integrate to repair interstrand crosslinks in the bacteria Escherichia coli and the budding yeast Saccharomyces cerevisiae. Nucleotide excision repair (NER) and HR proteins are required for ICL repair, among others. Also, since DSB intermediates are formed while resolving ICLs, HR proteins seem to be integral in responding to crosslinks. I tested mutants defective in two genes, mus301 and mus302, both of which are hypersensitive to crosslinking agents, for defects in DSB repair. I found that while mus302 mutants, which have previously been implicated in NER, can repair double-strand breaks normally; mus301 mutants are severely defective in HR, when the only available homologous template for repair is the sister chromatid

Knot Ready: Lessons From India on Delaying Marriage for Girls

Examines trends in and predictors of age at marriage. Assesses the effectiveness of ten program and policy interventions, underlying factors, and lessons for expanding viable strategies: empower girls, mobilize communities, and influence decision makers

DNA damage responses in Drosophila nbs mutants with reduced or altered NBS function

The MRN complex, composed of MRE11, RAD50 and NBS, plays important roles in responding to DNA double-strand breaks (DSBs). In metazoans, functional studies of genes encoding these proteins have been challenging because complete loss-of-function mutations are lethal at the organismal level and because NBS has multiple functions in DNA damage responses. To study functions of Drosophila NBS in DNA damage responses, we used a separation-of-function mutation that causes loss of the forkhead-associated (FHA) domain. Loss of the FHA domain resulted in hypersensitivity to ionizing radiation and defects in gap repair by homologous recombination, but had only a small effect on the DNA damage checkpoint response and did not impair DSB repair by end joining. We also found that heterozygosity for an nbs null mutation caused reduced gap repair and loss of the checkpoint response to low-dose irradiation. These findings shed light on possible sources of the cancer predisposition found in human carriers of NBN mutations

ELECTRICAL RESISTIVITY BASED SHALLOW CRUSTAL DEFORMATION ANALYSIS IN AN ACTIVE SEISMIC ZONE

This paper aims to assess the shallow crustal deformation in a highly seismic-prone zone ofnortheast India. For that purpose, Tripura University campus has been selected which isnearly 8 km away from Agartala city. The field work was conducted by using electricalresistivity meter. Vertical Electrical Sounding or VES method of resistivity survey wasadopted for measuring the electrical resistivity and structural analysis of the study area.Total four VES points were recorded namely near the Department of Physics, Department ofGeography and Disaster Management, Teachers’ Residential area and Girls’ Hostel. Therecorded data was measured and two different electrical profiles were drawn i.e. (a) Physicsbuilding to Geography buildings and (b) teachers’ quarters to Girls’ Hostel. From theelectrical resistivity profiles, a clear break in resistivity characters has been observed in thewestern part which is located near the Tripura University Girls’ Hostel. This clearly provesthat a deformation occurred in this area below 4 m depth and it can be very dangerous if ahigh magnitude seismic slip takes place. From this observation, this hostel is detected as themost risk prone building. Apart from that, the teachers’ quarters near the Girls’ hostel havealso been detected as equally risk prone. From this point of view it can be recommended thatconstruction of heavy buildings should be restricted in Tripura University campus as thesubsurface deformation zone can create faulting during high magnitude earthquake

ELECTRICAL RESISTIVITY BASED SHALLOW CRUSTAL DEFORMATION ANALYSIS IN AN ACTIVE SEISMIC ZONE

This paper aims to assess the shallow crustal deformation in a highly seismic-prone zone of northeast India. For that purpose, Tripura University campus has been selected which is nearly 8 km away from Agartala city. Thefield work was conducted by using electrical resistivity meter. Vertical Electrical Sounding or VES method of resistivity survey was adoptedfor measuring the electrical resistivity and structural analysis of the study area. Total four VES points were recorded namely near the Department of Physics, Department of Geography and Disaster Management, Teachers\u27 Residential area and Girls\u27 Hostel. The recorded data was measured and two different electrical profiles were drawn i.e. (a) Physics building to Geography buildings and (b) teachers\u27 quarters to Girls\u27 Hostel. From the electrical resistivity profiles, a clear break in resistivity characters has been observed in the western part which is located near the Tripura University Girls\u27 Hostel. This clearly proves that a deformation occurred in this area below 4 m depth and it can be very dangerous if a high magnitude seismic slip takes place. From this observation, this hostel is detected as the most risk prone building. Apart from that, the teachers\u27 quarters near the Girls\u27 hostel have also been detected as equally risk prone. From this point of view it can be recommended that construction of heavy buildings should be restricted in Tripura University campus as the subsurface deformation zone can createfaulting dlling high magnitude earthquake

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

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