Zinc-Induced Polymerization of Killer-Cell Ig-like Receptor into Filaments Promotes Its Inhibitory Function at Cytotoxic Immunological Synapses.

The inhibitory function of killer cell immunoglobulin-like receptors (KIR) that bind HLA-C and block activation of human natural killer (NK) cells is dependent on zinc. We report that zinc induced the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy, which depolymerized after zinc chelation. Similar KIR filaments were isolated from lysates of cells treated with zinc, and membrane protrusions enriched in zinc were detected on whole cells by scanning electron microscopy and imaging mass spectrometry. Two independent mutations in the extracellular domain of KIR, away from the HLA-C binding site, impaired zinc-driven polymerization and inhibitory function. KIR filaments formed spontaneously, without the addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C(+) cells. Adding to the recent paradigm of signal transduction through higher order molecular assemblies, zinc-induced polymerization of inhibitory KIR represents an unusual mode of signaling by a receptor at the cell surface.

Deciphering CaMKII Multimerization Using Fluorescence Correlation Spectroscopy and Homo-FRET Analysis.

While kinases are typically composed of one or two subunits, calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is composed of 8-14 subunits arranged as pairs around a central core. It is not clear if the CaMKII holoenzyme functions as an assembly of independent subunits, as catalytic pairs, or as a single unit. One strategy to address this question is to genetically engineer monomeric and dimeric CaMKII and evaluate how their activity compares to the wild-type (WT) holoenzyme. Here a technique that combines fluorescence correlation spectroscopy and homo-FRET analysis was used to characterize assembly mutants of Venus-tagged CaMKIIα to identify a dimeric CaMKII. Spectroscopy was then used to compare how holoenzyme structure and function changes in response to activation with CaM in the dimeric mutant, WT-holoenzyme, and a monomeric CaMKII oligomerization-domain deletion mutant control. CaM triggered an increase in hydrodynamic volume in both WT and dimeric CaMKII without altering subunit stoichiometry or the net homo-FRET between Venus-tagged catalytic domains. Biochemical analysis revealed that the dimeric mutant also functioned like WT holoenzyme in terms of its kinase activity with an exogenous substrate, and for endogenous T286 autophosphorylation. We conclude that the fundamental functional units of CaMKII holoenzyme are paired catalytic-domains.

A single amino acid change in inhibitory killer cell Ig-like receptor results in constitutive receptor self-association and phosphorylation.

Signaling by immunoreceptors is often initiated by phosphorylation of cytosolic tyrosines, which then recruit effector molecules. In the case of MHC class I-specific inhibitory receptors, phosphorylation of cytosolic tyrosine residues within ITIMs results in recruitment of a protein tyrosine phosphatase that blocks activation signals. Recent work showed that signaling by an HLA-C-specific killer cell Ig-like receptor (KIR) is independent of signaling by activation receptors. It is not known how ITIM phosphorylation is initiated and regulated. In this article, we show that substitution of His-36 in the first Ig domain of KIR2DL1 with alanine (KIR2DL1-H36A) resulted in constitutive KIR2DL1 self-association and phosphorylation, as well as recruitment of tyrosine phosphatase SHP-1. Furthermore, substitution of His-36 with a similar bulky amino acid, phenylalanine, maintained the receptor in its unphosphorylated state, suggesting that steric hindrance by the His-36 side chain prevents constitutive KIR2DL1 self-association and ITIM phosphorylation. The equally strong phosphorylation of KIR2DL1 and KIR2DL1-H36A after inhibition of tyrosine phosphatase by pervanadate suggested that KIR2DL1-H36A is selectively protected from dephosphorylation. We propose that KIR phosphorylation is controlled by the accessibility of ITIM to tyrosine phosphatases and that KIR binding to HLA-C must override the hindrance that His-36 puts on KIR2DL1 self-association. Expression of KIR2DL1-H36A on NK cells led to stronger inhibition of lysis of HLA-C(+) target cells than did expression of wild-type KIR2DL1. These results revealed that ITIM phosphorylation is controlled by self-association of KIR and that His-36 serves as a gatekeeper to prevent unregulated signaling through KIR2DL1.

Covert Changes in CaMKII Holoenzyme Structure Identified for Activation and Subsequent Interactions.

Between 8 to 14 calcium-calmodulin (Ca(2+)/CaM) dependent protein kinase-II (CaMKII) subunits form a complex that modulates synaptic activity. In living cells, the autoinhibited holoenzyme is organized as catalytic-domain pairs distributed around a central oligomerization-domain core. The functional significance of catalytic-domain pairing is not known. In a provocative model, catalytic-domain pairing was hypothesized to prevent ATP access to catalytic sites. If correct, kinase-activity would require catalytic-domain pair separation. Simultaneous homo-FRET and fluorescence correlation spectroscopy was used to detect structural changes correlated with kinase activation under physiological conditions. Saturating Ca(2+)/CaM triggered Threonine-286 autophosphorylation and a large increase in CaMKII holoenzyme hydrodynamic volume without any appreciable change in catalytic-domain pair proximity or subunit stoichiometry. An alternative hypothesis is that two appropriately positioned Threonine-286 interaction-sites (T-sites), each located on the catalytic-domain of a pair, are required for holoenzyme interactions with target proteins. Addition of a T-site ligand, in the presence of Ca(2+)/CaM, elicited a large decrease in catalytic-domain homo-FRET, which was blocked by mutating the T-site (I205K). Apparently catalytic-domain pairing is altered to allow T-site interactions.

Stepwise evaluation for the risk of metabolic unhealthiness and significant non-alcoholic fatty liver disease in India.

Background: Non-communicable diseases including metabolic health disorders are becoming area of concern for low/middle income countries with poor health-care resources. Present study was planned to assess the prevalence of metabolically unhealthy (MU) subjects in the community and proportion of the MU subjects having the risk of significant Non-alcoholic Fatty Liver Disease (NAFLD) using a step-wise evaluation strategy in a resource-poor setting. Methods: Study was performed in 19 community development blocks of Birbhum district, West Bengal, India. Every fifth member in the electoral list was included for the first step evaluation (n = 79,957/1,019,365, 7.8%) to detect any metabolic risk. Subjects with any metabolic risk in the first step (n = 9819/41,095, 24%) were taken for second step evaluation with Fasting blood glucose (FBG) and ALT. Subjects with elevated FBG and/or ALT in the second step (n = 1403/5283, 27%) were taken into third step evaluation. Finding: At least one risk factor was found in 51.4% (n = 41,095/79,957). 63% (n = 885/1403) of the subjects with metabolic abnormality (third step) had MU state making its overall prevalence of 1.1% (n = 885/79,957). 53% of MU subjects (n = 470/885) had 'persistently elevated ALT' suggesting the risk of having significant NAFLD. Interpretation: Step-wise evaluation strategy could detect the subjects at risk, actually having MU state and proportion of MU subjects at risk of having 'persistently elevated ALT' (surrogate of significant NAFLD) in the community with minimum utilization of scarce resources. Funding: This study was funded by Bristol Myers Squibb Foundation, USA, under the program 'Together on Diabetes Asia' (Project Number: 1205 - LFWB).

Engineering resonance energy transfer for advanced immunoassays: the case of celiac disease.

Celiac disease (CD) is an immune-mediated disorder affecting genetically predisposed subjects. It is caused by the ingestion of wheat gluten and related prolamins. A final diagnosis for this disease can be obtained by examination of jejunal biopsies. Nevertheless, different analytical approaches have been established to detect the presence of anti-tissue transglutaminase antibodies that represent a serological hallmark of the disease. In this work, we explored a new method for the diagnosis of CD based on the detection of serum anti-transglutaminase antibodies by resonance energy transfer (RET) between donor molecules and acceptor molecules. In particular, we labeled the liver transglutaminase (tTG) enzyme from guinea pig and the rabbit anti-tTG antibodies with a couple of fluorescence probes that are able to make RET if they are located within with Förster distance. We labeled tTG with the fluorescence probe DyLight 594 as donor and the anti-tTG antibodies with the fluorescence probe DyLight 649 as acceptor. However, due to the large size of the formed complex (tTG/anti-tTG), and consequently to the low efficiency energy transfer process between the donor-acceptor molecules, we explored a new experimental approach that allows us to extend the utilizable range of RET between donor:acceptor pairs by using one single molecule as donor and multiple molecules as energy acceptors, instead of using a single acceptor molecule as usually occurs in RET experiments. The obtained results clearly show that the use of one donor and multiacceptor strategy enables for a simple and rapid detection of serum anti-transglutaminase antibodies. In addition, our results point out that it is possible to consider this approach as a new method for a wide variety of analytical assays.

Predictors of COVID-19 Vaccine Confidence: Findings from Slums of Four Major Metro Cities of India.

There are limited studies on COVID vaccine confidence at the household level in urban slums, which are at high risk of COVID-19 transmission due to overcrowding and poor living conditions. The objective was to understand the reasons influencing COVID-19 vaccine confidence, in terms of barriers and enablers faced by communities in urban slums and informal settlements in four major metro cities in India. A mixed method approach was adopted, where in field studies were conducted during April-May 2021. First, a survey of at least 50 subjects was conducted among residents of informal urban settlements who had not taken any dose of the COVID-19 vaccine in Mumbai, Bengaluru, Kolkata and Delhi; second, a short interview with five subjects who had taken at least one dose of the vaccine in each of the four cities to understand the factors that contributed to positive behaviour and, finally, an in-depth interview of at least 3 key informants in each city to ascertain the vaccination pattern in the communities. The reasons were grouped under contextual, individual/group and vaccine/vaccination specific issues. The most frequent reason (27.7%) was the uncertainty of getting the vaccine. The findings show the need for increasing effectiveness of awareness campaigns, accessibility and the convenience of vaccination, especially among vulnerable groups, to increase the uptake.

Polarized fluorescent nanospheres.

Fluorescent beads (nanoparticles, nanospheres) are commonly used in fluorescence spectroscopy and microscopy. Due to the random distribution of dye and high dye to nanoparticle ratio, the fluorescence polarization observed from the beads is low. Therefore beads are not used for polarization study. We demonstrate that photoselective bleaching creates beads with highly polarized fluorescence. First, the beads were immobilized in a PVA polymer. Second, the beads-doped PVA film was exposed to the illumination within the dye absorption band. A progressive decrease of absorption was observed. Next, photophysical properties of photobleached and not bleached films dissolved in water were compared.

Self-quenching of uranin: Instrument response function for color sensitive photo-detectors.

Concentration is a key determining factor in the fluorescence properties of organic fluorophores. We studied self-quenching of disodium fluorescein (uranin) fluorescence in polyvinyl alcohol (PVA) thin films. The concentration dependent changes in brightness and anisotropy were followed by a lifetime decrease. We found that at a concentration of 0.54 M, the lifetime decreases to 7 ps. At a concentration of 0.18 M the lifetime was 10 ps with the relatively high quantum yield of 0.002. In these conditions the fluorescence intensity decay was homogeneous (well approximated by a single lifetime). We realized that such a sample was an ideal fluorescence lifetime standard for spectroscopy and microscopy, and therefore characterized instrument response functions for a time-domain technique. We show that self-quenched uranin enables measurements free of the color effect, making it a superior choice for a lifetime reference over scattered light.

Photophysical properties of Cerulean and Venus fluorescent proteins.

Cerulean and Venus are recently developed fluorescent proteins, often used as a donor-acceptor pair by researchers in Forster resonance energy transfer-based colocalization studies. We characterized the fluorescent properties of these two proteins in a broad spectral range (form ultraviolet to visible region). Excitation spectra, lifetimes, and polarization spectra show significant energy transfer from aromatic amino acids to the fluorescent protein chromophore. High steady-state anisotropy values and the lack of a fast component in anisotropy decays show that the fluorescent protein chromophore is rigidly fixed within the protein structure. Furthermore, we show that the chromophores are not accessible to external quenchers, such as acrylamide or potassium iodide (KI), allowing the removal of "unwanted" background in the environment with external quencher, while leaving the Cerulean/Venus fluorescence unchanged.

Collisional quenching of erythrosine B as a potential reference dye for impulse response function evaluation.

We studied the collisional quenching of the erythrosine B fluorophore by potassium iodide. The quenching follows a Stern-Volmer dependence up to the highest quencher concentration. The lifetime of erythrosine B decreases to 24 ps in 5.02 M of potassium iodide. The quantum yield of erythrosine B in the presence of 5.02 M KI is 0.0035. The relatively high brightness makes this compound attractive as an ultrashort reference in time-resolved measurements. In both frequency- and time-domain fluorescence techniques, there is a need for lifetime standards with extremely short decay times. Mimicking the instantaneous scattering at longer wavelengths allows color-effect-free measurements in the emission region. Another motivation is the problem of obtaining the impulse response function in the case of two-photon excitation. Time-resolved microscopy also benefits from fast-decaying dyes because the impulse response function can be evaluated at the emission wavelength of the investigated specimen without changing filters. We demonstrated that impulse response functions for commonly used detectors are practically the same for scattering as for quenched erythrosine B emission. We also analyzed a complex fluorescence decay using both elastic scattering and quenched erythrosine B emission as a response function.

Fluorescence intensity decays of 2-aminopurine solutions: lifetime distribution approach.

The fluorescent adenine analog 2-aminopurine (2AP) has been used extensively to monitor conformational changes and macromolecular binding events involving nucleic acids because its fluorescence properties are highly sensitive to changes in chemical environment. Furthermore, site-specific incorporation of 2AP permits local DNA and RNA conformational events to be discriminated from the global structural changes monitored by UV-Vis spectroscopy and circular dichroism. However, although the steady-state fluorescence properties of 2AP have been well defined in diverse settings, interpretation of 2AP fluorescence lifetime parameters has been hampered by the heterogeneous nature of multiexponential 2AP intensity decays observed across populations of microenvironments. To resolve this problem, we tested the utility of multiexponential versus continuous Lorentzian lifetime distribution models to describe fluorescence intensity decays from 2AP in diverse chemical backgrounds and within the context of RNA. Heterogeneity was introduced into 2AP intensity decays by mixing solvents of differing polarities or by adding quenchers under high viscosity to evaluate the transient effect. Heterogeneity of 2AP fluorescence within the context of a synthetic RNA hairpin was introduced by structural remodeling using a magnesium salt. In each case except folded RNA (which required a bimodal distribution), 2AP lifetime properties were well described by single Lorentzian distribution functions, abrogating the need to introduce additional discrete lifetime subpopulations. Rather, heterogeneity in fluorescence decay processes was accommodated by the breadth of each distribution. This approach also permitted solvent relaxation effects on 2AP emission to be assessed by comparing lifetime distributions at multiple wavelengths. Together, these studies provide a new perspective for the interpretation of 2AP fluorescence lifetime properties that will further the utility of this fluorophore in analyses of the complex and heterogeneous structural microenvironments associated with nucleic acids.

Binding of 8-anilino-1-naphthalenesulfonate to lecithin:cholesterol acyltransferase studied by fluorescence techniques.

The solvatochromic fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) has been used to study the hydrophobicity and conformational dynamics of lecithin:cholesterol acyltransferase (LCAT). The ANS to LCAT binding constant was estimated from titrations with ANS, keeping a constant concentration of LCAT (2 microM). Apparent binding constant was found to be dependent on the excitation. For the direct excitation of ANS at 375 nm the binding constant was 4.7 microM(-1) and for UV excitation at 295 nm was 3.2 microM(-1). In the later case, not only ANS but also tryptophan (Trp) residues of LCAT is being excited. Fluorescence spectra and intensity decays show an efficient energy transfer from tryptophan residues to ANS. The apparent distance from Trp donor to ANS acceptor, estimated from the changes in donor lifetime was about 3 nm and depends on the ANS concentration. Steady-state and time-resolved fluorescence emission and anisotropies have been characterized. The lifetime of ANS bound to LCAT was above 16 ns which is characteristic for it being in a hydrophobic environment. The ANS labeled LCAT fluorescence anisotropy decay revealed the correlation time of 42 ns with a weak residual motion of 2.8 ns. These characteristics of ANS labeled LCAT fluorescence show that ANS is an excellent probe to study conformational changes of LCAT protein and its interactions with other macromolecules.

Fluorescence polarization standard for near infrared spectroscopy and microscopy.

We present studies of polarized absorption [linear dichroism (LD)] and fluorescence polarization of the styryl derivative (LDS 798) embedded in oriented poly(vinyl alcohol) (PVA) films. These films were oriented by progressive stretching up to eight folds. Both vertical and horizontal components of absorptions and fluorescence were measured and dichroic ratios were determined for different film stretching ratios. The dichroic ratio and fluorescence anisotropy values were analyzed as a function of PVA film stretching ratio by fitting according to the previously developed theory. For maximum stretching ratios, exceptionally high anisotropy (approximately 0.8) and polarization (approximately 0.9) values have been measured. The stretched films have high polarization values also for isotropic excitation in a wide spectral range (500-700 nm). Such films can be conveniently used as high polarization standards and we envision they will also have applications in near infrared (NIR) imaging microscopy, where they can be used for correcting an instrumental factor in polarization measurements.

Fluorescence polarization and fluctuation analysis monitors subunit proximity, stoichiometry, and protein complex hydrodynamics.

Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1-10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9 ± 1.2 subunit, but values ranged from 10-14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2 ± 1.3 subunits in the holoenzyme with values ranging from 9-12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved.

Metal-enhanced immunoassays.

The surface-confined assay format is one of the most convenient detection formats used in many immunoassays. Fluorescence emission from monolayers of dyes requires a strong excitation and good detection system. Such samples are susceptible to artifacts due to background fluorescence from substrates. We demonstrate that using silver nanostructures deposited on the slide substrate can significantly enhance measured fluorescence, reduce unwanted background and increase photostability of the used probes. Using thin layers of polymer doped with fluorescein, we tested two nanostructures--silver island films (SIFs) deposited on glass slides and self-assembled colloidal structures (SACS) deposited on thin silver film. The SACS surfaces show extraordinary fluorescence enhancements: over 100-folds in hot spots. We applied these surfaces for enhanced Alexa488 model immunoassay.

Fluorescent properties of antioxidant cysteine ABZ analogue.

The antioxidant properties of aminobenzamide cysteine (ABZ Cys) makes it a molecule that can potentially be used as a drug in oxidative stress related diseases and delivered in the form of a nanoparticles. Here we have studied the photo-physical properties of ABZ Cys, a fluorescent analogue of a popular antioxidant N-acetyl cysteine (NAC). We have compared ABZ Cys steady state and time-resolved fluorescence properties with its parent compounds anthranilic acid and anthranilamide in solution as well as in poly-vinyl alcohol (PVA) polymer films. ABZ Cys did not show any significant shift in absorption after entrapment in PVA film, but there was a shift towards shorter wavelengths in the emission peak compared to the phosphate buffer solution. Fluorescence lifetimes and quantum yields indicated a slight quenching of ABZ Cys fluorescence in comparison to the cysteine-less parent compounds. We also demonstrated that very low concentrations of ABZ Cys, such as 100 nM, are readily detected by a commercial spectrofluorometer. Hence we have established the possible use of ABZ Cys in biomedical applications.

Fluorescence detection of MMP-9. I. MMP-9 selectively cleaves Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys peptide.

MMP-9 enzyme recognizes a peptide sequence Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys and cleaves the peptide into two parts. We synthesized a dual fluorophore beacon consisting of 5-FAM and Cy5 dyes. The fluorescence emission of the fluorescein moiety is dramatically quenched by Cy5 molecule due to Förster Resonance Energy Transfer (FRET) and the fluorescence of Cy5 is strongly enhanced. Upon addition of MMP-9 enzyme, the fluorescence of 5-FAM intensifies and Cy5 decreases. The control MMP-2 enzyme does not cause any changes in either 5-FAM or Cy5 fluorescence. We believe that our observation will help in early detection of elevated MMP-9 levels under disease conditions.

Fluorescence instrument response standards in two-photon time-resolved spectroscopy.

We studied the fluorescence properties of several potential picosecond lifetime standards suitable for two-photon excitation from a Ti:sapphire femtosecond laser. The fluorescence emission of the selected fluorophores (rose bengal, pyridine 1, and LDS 798) covered the visible to near-infrared wavelength range from 550 to 850 nm. We suggest that these compounds can be used to measure the appropriate instrument response functions needed for accurate deconvolution of fluorescence lifetime data. Lifetime measurements with multiphoton excitation that use scatterers as a reference may fail to properly resolve fluorescence intensity decays. This is because of the different sensitivities of photodetectors in different spectral regions. Also, detectors often lose sensitivity in the near-infrared region. We demonstrate that the proposed references allow a proper reconvolution of measured lifetimes. We believe that picosecond lifetime standards for two-photon excitation will find broad applications in multiphoton spectroscopy and in fluorescence lifetime imaging microscopy (FLIM).

Photophysical properties of a new DyLight 594 dye.

We describe spectral properties of novel fluorescence probe DyLight 594. Absorption and fluorescence spectra of this dye are in the region of Alexa 594 fluor spectra. The quantum yield of DyLight 594 in conjugated form to IgG is higher than corresponding quantum yield of Alexa 594 by about 50%. The new DyLight dye also shows slightly longer lifetime and photostability. These favorable properties and high anisotropy value, as well as a high cross-section for two-photon excitation, make this fluorophore attractive as a fluorescence probe in biochemical/biological studies involving fluorescence methods.