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Article| Volume 118, ISSUE 1, P26-35, January 07, 2020
Identifying Heteroprotein Complexes in the Nuclear Envelope
Jared Hennen
School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota
Kwang-Ho Hur
John Kohler
Siddarth Reddy Karuka
Isaac Angert
G. W. Gant Luxton
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota
Joachim D. Mueller
Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota
Open ArchivePublished:November 21, 2019DOI:https://doi.org/10.1016/j.bpj.2019.11.020
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The nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ∼40-nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes, which are mediated by protein complexes within this subcellular compartment. Although fluorescence fluctuation spectroscopy is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional fluorescence fluctuation spectroscopy methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations. We previously addressed this challenge by developing time-shifted mean-segmented Q (tsMSQ) analysis and applied it to successfully characterize protein homo-oligomerization in the NE. However, many NE complexes, such as the linker of the nucleoskeleton and cytoskeleton complex, are formed by heterotypic interactions, which single-color tsMSQ is unable to characterize. Here, we describe the development of dual-color (DC) tsMSQ to analyze NE heteroprotein complexes built from proteins that carry two spectrally distinct fluorescent labels. Experiments performed on model systems demonstrate that DC tsMSQ properly identifies heteroprotein complexes and their stoichiometry in the NE by accounting for spectral cross talk and local volume fluctuations. Finally, we applied DC tsMSQ to study the assembly of the linker of the nucleoskeleton and cytoskeleton complex, a heteroprotein complex composed of Klarsicht/ANC-1/SYNE homology and Sad1/UNC-84 (SUN) proteins, in the NE of living cells. Using DC tsMSQ, we demonstrate the ability of the SUN protein SUN2 and the Klarsicht/ANC-1/SYNE homology protein nesprin-2 to form a heterocomplex in vivo. Our results are consistent with previously published in vitro studies and demonstrate the utility of the DC tsMSQ technique for characterizing NE heteroprotein complexes.
Protein complexes found within the nuclear envelope (NE) play a vital role in regulating cellular functions ranging from gene expression to cellular movement. However, the assembly state of these complexes within their native environment remains poorly understood, which is compounded by a general lack of fluorescence techniques suitable for quantifying the oligomeric state of NE protein complexes. This study aims at addressing this issue by introducing dual-color time-shifted mean-segmented Q analysis as a fluorescence fluctuation method specifically designed to identify the average oligomeric state of heteroprotein complexes within the NE of living cells.
Fluorescence fluctuation spectroscopy (FFS) refers to a collection of related biophysical techniques that exploit the stochastic intensity of fluorescently labeled biomolecules passing through a small observation volume created by confocal or two-photon microscopy (
Jameson D.M.
Ross J.A.
Albanesi J.P.
Fluorescence fluctuation spectroscopy: ushering in a new age of enlightenment for cellular dynamics.
Biophys. Rev. 2009; 1: 105-118
). The primary parameters accessible by FFS are the concentration, mobility, and oligomeric state of the labeled biomolecule (
). Although the original analysis of FFS results was based on the autocorrelation function (ACF), many other methods have been introduced over the years, each with its own strengths and weaknesses (
Petersen N.O.
Höddelius P.L.
Magnusson K.E.
Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application.
Biophys. J. 1993; 65: 1135-1146
Müller J.D.
Gratton E.
The photon counting histogram in fluorescence fluctuation spectroscopy.
Biophys. J. 1999; 77: 553-567
Cumulant analysis in fluorescence fluctuation spectroscopy.
Digman M.A.
Dalal R.
Mapping the number of molecules and brightness in the laser scanning microscope.
). An important advance in FFS was the introduction of dual-color (DC) FFS for identifying interactions between two species of biomolecules labeled with spectrally distinct fluorophores (
Schwille P.
Meyer-Almes F.J.
Rigler R.
Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution.
). In DC FFS, the emission of the fluorophores is separated by color into two detection channels. Heterotypic interactions between the two species lead to synchronized temporal fluctuations in both channels, which are recognized by the cross correlation function (CCF) of the two detected signals. In addition to CCF, other analysis techniques have been introduced for quantifying heterospecies interactions from FFS experiments (
Wu B.
Heterospecies partition analysis reveals binding curve and stoichiometry of protein interactions in living cells.
Tekmen M.
Dual-color photon-counting histogram.
Because FFS is an equilibrium technique that passively observes fluctuations, it is well suited for applications in live cells (
Wei L.N.
Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy.
). Cellular proteins can be conveniently labeled by genetic tagging with one of the many available fluorescent proteins (
Wiedenmann J.
Oswald F.
Nienhaus G.U.
Fluorescent proteins for live cell imaging: opportunities, limitations, and challenges.
IUBMB Life. 2009; 61: 1029-1042
). Brightness, which characterizes the intrinsic fluorescence intensity of a molecule, is an important FFS parameter because it contains information about the stoichiometry of fluorescently tagged protein complexes (
Unraveling protein-protein interactions in living cells with fluorescence fluctuation brightness analysis.
). For example, the brightness of monomeric proteins tagged with EGFP will increase upon their association into homo-oligomers as each protein complex contains several fluorescent labels. This concept has been generalized to include differently colored fluorophores to characterize heteroprotein complexes (
). Although FFS brightness analysis has been successfully used to quantify protein-protein interactions within the cytoplasm, nucleoplasm, and at the plasma membrane (
Macdonald P.J.
Mueller J.D.
Brightness analysis by Z-scan fluorescence fluctuation spectroscopy for the study of protein interactions within living cells.
Smith E.M.
Quantifying protein-protein interactions of peripheral membrane proteins by fluorescence brightness analysis.
Biophys. J. 2014; 107: 66-75
), its extension to the nuclear envelope (NE) has proven challenging (
Hennen J.
Hur K.H.
Quantitative brightness analysis of protein oligomerization in the nuclear envelope.
Biophys. J. 2017; 113: 138-147
The NE consists of an inner nuclear membrane (INM) and outer nuclear membrane (ONM) separated by a ∼40-nm-thick fluid layer, known as the lumen or perinuclear space. Although the NE has been identified as a critical signaling node of the cell (
Dauer W.T.
Worman H.J.
The nuclear envelope as a signaling node in development and disease.
Dev. Cell. 2009; 17: 626-638
), a mechanistic understanding of how these heteroprotein complexes assemble remains limited because of the lack of quantitative biophysical techniques suitable for use in the NE. To address this challenge, we explored the use of single-color (SC) FFS for characterizing homoprotein association in the NE of living cells (
Saunders C.A.
Luxton G.W.G.
Fluorescence fluctuation spectroscopy reveals differential SUN protein oligomerization in living cells.
Mol. Biol. Cell. 2018; 29: 1003-1011
). We found that slow undulations of the nuclear membranes give rise to local volume fluctuations that are not properly accounted for by conventional FFS methods (
). We overcame this obstacle by introducing mean-segmented Q (MSQ) analysis as well as time-shifted MSQ (tsMSQ), a significantly improved version of MSQ (
Quantitative brightness analysis of fluorescence intensity fluctuations in E. Coli.
PLoS One. 2015; 10: e0130063
Protein oligomerization and mobility within the nuclear envelope evaluated by the time-shifted mean-segmented Q factor.
Methods. 2019; 157: 28-41
). We demonstrated that these techniques successfully identify the mobility and homo-oligomeric state of NE proteins (
This study extends FFS analysis of heteroprotein complexes to the NE by introducing DC tsMSQ, a generalized form of regular tsMSQ. The DC tsMSQ framework includes heterospecies partitioning (HSP) (
) and effectively eliminates complications due to spectral cross talk in the characterization of heterotypic protein interactions as verified by control experiments. Our study demonstrates that local volume fluctuations of the NE are a significant challenge for conventional DC FFS analysis, which prompted the development of DC tsMSQ. We first verified the foundation of DC tsMSQ using pairs of interacting and noninteracting proteins measured in the cytoplasm and in the NE. In addition, control experiments using both luminal and nuclear membrane-associated proteins were conducted to illustrate the influence of the nuclear membrane undulations on DC tsMSQ analysis.
To demonstrate the power of DC tsMSQ, we applied it toward studying the assembly of the linker of nucleoskeleton and cytoskeleton (LINC) complex in the NE of living cells. This NE-spanning molecular bridge mediates mechanical force transmission into the nucleoplasm and is required for several fundamental cellular processes, including cell division, DNA damage repair, meiotic chromosome pairing, mechanoregulation of gene expression, and nuclear positioning (
Meinke P.
Schirmer E.C.
LINC'ing form and function at the nuclear envelope.
). The LINC complex is formed by a direct transluminal heterotypic interaction between the cytoskeletal-binding ONM Klarsicht/ANC-1/SYNE homology (KASH) proteins and the nuclear lamina-binding INM Sad1/UNC-84 (SUN) proteins (
Lombardi M.L.
Jaalouk D.E.
Lammerding J.
The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton.
). Previous in vitro biochemical and structural studies revealed that the luminal domain of SUN2 homotrimerizes and that the luminal domain of the KASH protein nesprin-2 binds in the grooves formed at the interface of two adjacent interacting SUN2 monomers (
Sosa B.A.
Rothballer A.
Schwartz T.U.
LINC complexes form by binding of three KASH peptides to domain interfaces of trimeric SUN proteins.
Cell. 2012; 149: 1035-1047
Wang W.
Shi Z.
Zhou Z.
Structural insights into SUN-KASH complexes across the nuclear envelope.
Cell Res. 2012; 22: 1440-1452
). Thus, the homotrimerization of the SUN2 luminal domain enables the recruitment of 3 nesprin-2 luminal domains, resulting in the assembly of a SUN2-nesprin-2 heterohexamer. We recently succeeded in directly measuring the homotrimerization of the EGFP-tagged luminal domain of SUN2 in the NE of living cells by SC FFS (
). Here, we utilize DC tsMSQ to directly observe the heterotypic interactions formed between the EGFP-tagged SUN2 luminal domain and an mCherry-tagged construct that encodes the last three spectrin-like repeats (SRs), the transmembrane domain, and the luminal KASH peptide of nesprin-2 in the NE. This work establishes the theoretical and practical framework necessary for future quantitative studies of LINC complex assembly within the NE of living cells as well as the characterization of additional heterotypic interactions between proteins within this relatively unexplored subcellular environment.
Brightness measurements were performed as previously described (
). Briefly, FFS data were acquired on a custom built two-photon microscope with a 63× C-Apochromat water-immersion objective with numerical aperture (NA) = 1.2 (ZEISS, Oberkochen, Germany) using an excitation wavelength of 1000 nm and an average power after the objective in the range of 0.3–0.4 mW. A dichroic mirror centered at 580 nm (FF580-FDi01; Semrock, Rochester, NY) was used to split the emission path into two channels. An additional 84-nm wide bandpass filter centered at 510 nm (FF01-510/84; Semrock) was placed before the green channel to remove any reflected fluorescence from mCherry. Photon counts were detected using avalanche photodiodes (SPCM-AQ-141 APD; PerkinElmer, Waltham, MA), recorded with a Flex04-12D card (Correlator.com, Bridgewater, NJ) sampled at 20 kHz and analyzed using programs written in IDL (Interactive Data Language) 8.7 (Research Systems, Boulder, CO). Z-scans were performed using an arbitrary waveform generator (model number 33522A; Agilent Technologies, Santa Clara, CA) to move a PZ2000 piezo stage (Applied Scientific Instrumentation, Eugene, OR) axially. The waveform created by the generator was a linear ramp function with a peak-to-peak amplitude of 1.6 V, corresponding to 24.1 μm of axial travel and a period of 10 s for a speed of 4.82 μm/s.
Measurement procedure
EGFP (G) calibration measurements were performed in the cytoplasm of EGFP-expressing U2OS cells, as previously described (
), to obtain its brightness in both the green and red channels (λg,G and λr,G, respectively). Additional calibration measurements were performed in the cytoplasm of U2OS cells expressing EGFP-RARLBD-mCherry (
) to obtain the brightness λr,Ch of mCherry (Ch) in the red channel, which accounts for the previously described two-state brightness of this fluorescent protein (
). These calibration values were then converted to Q values for the NE using Q = γ2λTs, where γ2 is the shape factor for a two-dimensional Gaussian point spread function, and Ts is the sampling time (
Sanchez-Andres A.
Molecular brightness determined from a generalized form of Mandel's Q-parameter.
). Measurements in the NE were performed as previously described (
Angert I.
Investigating LINC complex protein homo-oligomerization in the nuclear envelopes of living cells using fluorescence fluctuation spectroscopy.
Methods Mol. Biol. 2018; 1840: 121-135
) by first using epifluorescence to identify cells expressing the relevant constructs. FFS data were then acquired by taking z-scans through the nucleus, which were analyzed as previously described (
Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations.
Anal. Biochem. 2015; 480: 11-20
). Next, the two-photon beam was focused on the ventral NE followed by the dorsal NE and ∼60 s of intensity fluctuation data were obtained at each location. These data were analyzed as described in Theory to obtain the normalized HSP brightness vector b = (bg, br) (
Characterization of ternary protein systems in vivo with tricolor heterospecies partition analysis.
Biophys. J. 2016; 110: 1158-1167
). The normalized brightness values were corrected for two-state brightness and fluorescence resonance energy transfer as previously described (
Fluorescence fluctuation spectroscopy of mCherry in living cells.
). The average number of molecules in the observation volume was determined by Ng=〈Fg〉/λg,G and Nr=(〈Fr〉−fct×〈Fg〉)/λr,Ch, where 〈Fi〉=〈ki〉/TS and fct is the spectral cross talk of EGFP given by Qr,G/Qg,G (
). We limited our measurements to expression levels of N ≥ 10, which, for our experiments, corresponds to a minimum signal to background ratio of 10:1. These conditions ensure that the influence of background on the recovered FFS parameters is negligible.
Experiments were conducted using transiently transfected U2OS cells (ATCC, Manassas, VA) maintained in DMEM with 10% fetal bovine serum (HyClone Laboratories, Logan, UT). U2OS cells were subcultured into 24-well glass-bottom plates (Cellvis, Sunnyvale, CA) before transfection. GenJet (SignaGen Laboratories, Rockville, MD) was used to transiently transfect cells 12–24 h before measurement, according to the instructions of the manufacturer. The growth medium was replaced with Dulbecco's phosphate-buffered saline containing calcium and magnesium (BioWhittaker, Walkerville, MD) immediately before measuring.
Restriction enzymes were either purchased from New England BioLabs (Ipswich, MA) or Promega (Madison, WI). Calf Intestinal Phosphatase, Phusion DNA polymerase, T4 DNA ligase, and T4 polynucleotide kinase were also purchased from New England BioLabs. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. The Wizard SV Gel and PCR Clean-Up System were purchased from Promega, whereas the GeneJet Plasmid Midiprep Kit was purchased from Thermo Fisher Scientific (Waltham, MA).
DNA constructs
The generation of SS-EGFP, SS-EGFP-torsinANTD-2xLeu, SS-EGFP-SUN2595–731, and SS-EGFP-SUN2261–731 constructs were described previously (
). The generation of SS-mCherry-KDEL, the SUN domain-linked SS-EGFP-SUN2595–731-mCherry (SS-EGFP-SL-mCherry), and mCherry-SR-KASH2 constructs are described in Supporting Materials and Methods, Section 6.
Dual-channel tsMSQσρ
The single channel tsMSQ was generalized to dual-channel tsMSQσρ (Supporting Materials and Methods, Section 1) with the subscripts σ and ρ specifying the detection channel. A single diffusing species is described by the following:
tsMSQσρ,D(T;Qρ,τD)=QρtsFD(T;τD),
whereas the exponential correlation process caused by the local volume fluctuations of the NE is given by the following:
tsMSQσρ,E(T;Aρ,τ0)=AρtsFE(T;τ0),
as derived in Supporting Materials and Methods, Section 2. A sample consisting of a single diffusing species in the lumen of the NE experiences local volume fluctuations and is described by the addition of Eqs. 1 and 2 (
), as follows:
tsMSQσρ,DE(T)=QρtsFD(T;τD)+AρtsFE(T;τ0).
The time-shifted correlation functions tsFD and tsFE are defined in terms of second-order binning functions B2,D(T) and B2,E(T) (
Time-integrated fluorescence cumulant analysis in fluorescence fluctuation spectroscopy.
). Explicit formulas are found in Supporting Materials and Methods, Section 3. Because Eqs. 1 and 2 must reproduce the single channel case for σ = ρ, the amplitudes Qρ and Aρ are given by Qρ = γ2λρTs and Aρ = c2λρTsN, respectively (
). The brightness of the fluorescent molecule in the ρ channel is λρ, the number of molecules in the observation volume is N, and the shape factor of the observation volume is γ2. The factor c is determined by the fluctuations in the gap distance h separating the INM and ONM, c=〈δh2〉/〈h〉 (
). The diffusion time and the characteristic time of the volume fluctuations are given by τD and τ0, respectively.
The two detection channels used for DC FFS are labeled as green (g) and red (r). We define the DC tsMSQ function as follows:
tsMSQ(T)=[tsMSQgg(T)tsMSQgr(T)].
We refer to tsMSQgg as the autocorrelation tsMSQ of the green detection channel and tsMSQgr as the cross correlation tsMSQ of the green and red channel. Thus, DC tsMSQ of a diffusing species is described by the following:
tsMSQD(T)=QtsFD(T;τD),
and in the presence of volume fluctuations, the exponential correlation process,
tsMSQE(T)=AtsFE(T,τ0),
is added, resulting in the following:
tsMSQDE(T)=tsMSQD(T)+tsMSQE(T).
DC tsMSQ reduces to the same functional form as SC tsMSQ but with vector amplitudes Q = (Qg, Qr) and A = (Ag, Ar), which are scaled by tsFD and tsFE, respectively. Finally, for a fit to a model with S diffusing species, the tsMSQD term is replaced by the following:
tsMSQD(T)=∑i=1SfiQitsFD(T;τD,i),
where Qi and τD,i are the Q-vector and diffusion time of the ith species, respectively. The fractional intensity fi of the ith species is defined by the ratio of the green-channel intensity of the ith species to the total intensity 〈Fg〉=∑j=1S〈Fg,j〉 of the green channel, fi=〈Fg,i〉/〈Fg〉, as described in the Supporting Materials and Methods. The total Q-vector, Q¯, of the sample is defined by the following:
Q¯=∑i=1SfiQi,
and represents the ensemble average over all species.
DC tsMSQ and HSP
We previously demonstrated that the complications in interpreting the results of FFS experiments in the presence of spectral cross talk are avoided by HSP analysis (
). HSP requires that there is no spectral leakage of the red-emitting mCherry into the green emission channel, which is accomplished by choosing the appropriate filters (
). Thus, the two fluorescent proteins EGFP (G) and mCherry (Ch) are characterized by their respective Q-vectors QG = (Qg,G, Qr,G) and Qch = (0, Qr,Ch). The Q-vector (Eq. 9) determined by DC tsMSQ analysis may be expressed as a linear combination of the Q-vectors of EGFP and mCherry, as follows:
Q¯=bgQG+brQCh,
where the coefficients bg and br represent the normalized brightness associated with the corresponding fluorescent proteins (
Determining the stoichiometry of protein heterocomplexes in living cells with fluorescence fluctuation spectroscopy.
). The tuple (bg, br) characterizes the brightness vector of the heterospecies present in the sample. The heterospecies comprises all EGFP-labeled proteins and heteroprotein complexes carrying both EGFP and mCherry. Monomeric or oligomeric complexes that only contain the mCherry label are partitioned out by HSP. For example, a noninteracting monomeric EGFP-labeled species is described by an HSP brightness vector of (1, 0), whereas a heterodimer containing one EGFP and one mCherry label are described by an HSP brightness vector of (1, 1). An HSP brightness vector of (1, y) describes an EGFP-labeled protein that on average is associated with y mCherry-labeled proteins (
). A graphical representation of these examples is provided in Fig. S1. The HSP brightness vector (bg, br) is affected by fluorescent labels with dark states and multiple brightness states as well as by fluorescence resonance energy transfer, which bias the interpretation (
). These effects were accounted and corrected for as previously described (
). A derivation of HSP for DC tsMSQ is found in Supporting Materials and Methods, Section 5.
Initial experiments were performed on a noninteracting pair of proteins located in the lumen of the NE (Fig. 1 A). Specifically, we used SS-EGFP and SS-mCherry-KDEL, which have been found to be monomeric proteins within the NE by SC FFS (Fig. S2; (
)). EGFP and mCherry were chosen as labels because this pair has been characterized extensively by DC FFS and have a wide separation in their emission color (
). The signal sequence (SS) of the luminal ATPase torsinA, which is cleaved after protein expression, ensures the presence of EGFP in the lumen of the NE (
Liu Z.
Zolkiewska A.
Zolkiewski M.
Characterization of human torsinA and its dystonia-associated mutant form.
Biochem. J. 2003; 374: 117-122
). The C-terminus of SS-mCherry was additionally fused to the endoplasmic reticulum retention signal KDEL to ensure its efficient targeting to the lumen. DC FFS data were collected at the NE of U2OS cells co-expressing SS-EGFP and SS-mCherry-KDEL. The ACFs of the fluorescence collected by the green and red detection channels were calculated (Ggg and Grr, respectively) as well as the CCF Ggr of both channels. We observed a positive CCF amplitude (Fig. 2), which was not unexpected for a noninteracting pair of fluorescent proteins because spectral cross talk leads to a positive CCF component (
Bacia K.
Practical guidelines for dual-color fluorescence cross-correlation spectroscopy.
Nat. Protoc. 2007; 2: 2842-2856
). This cross talk-induced CCF function is predicted by (
) the following:
CCFct(τ)=Gg(τ)fct〈Fg〉〈Fr〉,
with 〈Fg〉 and 〈Fr〉 representing the mean fluorescence intensities of the green and red channel, respectively. The cross talk in our setup is caused by the detection of the long wavelength emission of EGFP in the red detection channel (
Patterson G.
Day R.N.
Piston D.
Fluorescent protein spectra.
J. Cell Sci. 2001; 114: 837-838
), which is characterized by the cross talk intensity fraction fct. The observed CCF has to significantly exceed this baseline for a positive identification of heterotypic protein interactions (
). The computed CCFct (τ) (solid blue lines, Fig. 2) is not significantly different from the observed CCF for the data taken at low expression levels (Fig. 2 A) but is significantly below the experimental CCF at medium (Fig. 2 B) and high expression levels (Fig. 2 C). This observation is counterintuitive as it suggests the onset of interactions between EGFP and mCherry-KDEL at higher concentrations. We further noticed a significant change in the shape of the ACF of both channels as well as the CCF with increasing concentration (Fig. 2), which was unexpected because the diffusion process is independent of concentration. These changes in the ACF are specific to the NE environment (
) and are not observed in the cytoplasm as confirmed by a control experiment performed in the cytoplasm of cells expressing EGFP and mCherry. Specifically, measurements performed in these cells showed a concentration-independent shape of the ACF as well as a strong overlap between the computed CCFct (τ) and the CCF over a wide range of expression levels (Fig. S3), indicating the absence of significant interactions between EGFP and mCherry within the cytoplasm.
Figure 1FFS at the NE. (A) Shown is an illustration of a cell expressing potentially interacting green and red fluorescently labeled NE proteins with the two-photon excitation volume (blue oval) focused at its NE (orange circle), which consists of the INM and ONM separated by a ∼40-nm-thick lumen. (B) Shown is an illustration of the time-dependent local volume fluctuations caused by nuclear membrane undulations, which give rise to coupled intensity variations of the noninteracting green and red fluorescently labeled proteins. To see this figure in color, go online.
Figure 2ACF and CCF analysis of DC FFS data collected in the NE of cells expressing SS-EGFP and SS-mCherry-KDEL at low (A), medium (B), and high (C) levels of protein expression. The number of proteins in the observation volume is given by NEGFP and NmCherry. The red and green channel ACFs are shown with red triangles and green squares, respectively. The CCF is graphed as black circles, and the calculated CCF from spectral cross talk (CCFct) is shown by the blue line. All correlation curves are divided by the amplitude of the ACF of the green channel, Ggg(Ts), with a lag time equal to the sampling time Ts. To see this figure in color, go online.
The unusual behavior of the experimental ACF of FFS data collected within the NE was traced to undulations of the nuclear membranes (
). These membrane undulations introduce local volume fluctuations that modulate the fluorescence intensity signals received from SS-EGFP and SS-mCherry-KDEL (Fig. 1 B). This additional fluctuation process not only affects the ACF but also the CCF as the volume fluctuations lead to concomitant intensity variations in both the green and red channels. We previously demonstrated that the slow membrane undulations lead to biases in ACF analysis (
). To overcome this challenge, we replaced ACF with MSQ analysis and, more recently, with the improved tsMSQ algorithm (
). Both methods have proven to be robust for measuring homotypic protein interactions within the NE of living cells (
). To extend cross correlation analysis to DC FFS data obtained in the NE, we generalize SC to DC tsMSQ. Two subscripts are added to distinguish the different tsMSQ functions. The SC tsMSQ from the green channel is identified by tsMSQgg, whereas the cross correlation tsMSQ between the green and the red channel is described by tsMSQgr (Fig. 3). The autocorrelation tsMSQgg is calculated from the fluorescence intensity of a single channel using the standard tsMSQ algorithm (Fig. 3 A). The cross correlation tsMSQgr follows the same procedure but utilizes the intensity traces of both channels (Fig. 3 B).
Figure 3Conceptual illustration of the DC tsMSQ algorithm. (A) The fluorescence intensity trace of the green channel, kg, is divided into n segments of period T. The time-shifted Q value of segment i, tsQgg(i)(T), is calculated from kg and a copy of kg, according to Eq. S3. Averaging over the time-shifted Q values determines the tsMSQgg for segment time T. (B) The algorithm for calculating the cross correlation tsMSQgr follows the same procedure as described in (A) but replaces the copy of kg with the intensity of the red channel, kr. (C) The autocorrelation tsMSQgg (green squares) and cross correlation tsMSQgr (orange circles) are constructed by repeating the procedures shown in (A and B) for a range of segment times T. To see this figure in color, go online.
We reanalyzed the FFS data of SS-EGFP and SS-mCherry-KDEL taken in the NE (Fig. 2) using the experimental tsMSQgg (green squares) and tsMSQgr (red circles) curves (Figs. 4 A and S4, A and B). For two noninteracting proteins undergoing simple diffusion in the lumen, the cross correlation tsMSQgr should be entirely determined by the cross talk fct from EGFP into the red channel and the membrane undulations of the NE, which contribute a correlation amplitude proportional to the average intensity (
). Thus, the tsMSQgr curve for two noninteracting luminal proteins is predicted to be the following
tsMSQgr(−)(T)=fcttsMSQgg,D(T;τD,Qg)+〈Fr〉〈Fg〉tsMSQgg,E(T;τ0,Ag),
where tsMSQgg,D(T, τD, Qg) and tsMSQgg,E(T, τ0, Ag) are model functions (Eqs. 1 and 2) that describe the diffusion and exponential components of tsMSQgg, respectively, whereas 〈Fi〉 represents the mean intensity in the ith channel. The green-channel tsMSQgg was fit to a single species diffusion model with an exponential correlation term (Eq. 3) to account for nuclear membrane undulations, enabling the calculation of the predicted cross correlation tsMSQgr(−) in the absence of interactions from Eq. 12 (blue line, Figs. 4 A and S4, A and B). The predicted tsMSQgr(−) and the experimental tsMSQgr closely overlap, demonstrating the absence of protein interactions between luminal EGFP and mCherry-KDEL. These results demonstrate that tsMSQgg accurately predicts the cross correlation tsMSQgr(−) curve of a noninteracting pair of proteins within the NE.
Figure 4DC tsMSQ analysis of measurements performed in the NE of cells (n = 16) co-expressing SS-EGFP and SS-mCherry-KDEL. (A) Shown are tsMSQ curves (symbols) with predicted tsMSQgr(−) curve (blue line) derived from a fit to tsMSQgg (black line) and standard residuals (sr). The tsMSQgg and tsMSQgr curves are shown with green squares and red circles, respectively. (B) Shown are tsMSQ curves (symbols) with fit to Eq. 7 (black). (C) Shown is br versus bg with standard deviations (error bars). (D) Shown are bg (green circles) and br (red squares), with respective standard deviations (error bars), versus Ng with means (dashed lines) and SDs of bg = 1.0 ± 0.2 and br = 0.16 ± 0.13. To see this figure in color, go online.
This initial validation of the theory prompted us to analyze FFS data with DC tsMSQ, which simultaneously describes the green-channel autocorrelation and the cross correlation tsMSQ (Eq. 4), tsMSQ = [tsMSQgg, tsMSQgr]. We fit both tsMSQ curves to Eq. 7, which describes a single diffusion species in the presence of volume fluctuations. The fit and data are in very good agreement (Figs. 4 B and S4, C and D) with reduced chi-squared values close to one. From these fits, we obtained the mean fit parameters and their uncertainties, which were derived from the covariance matrix. The analysis was performed on n = 16 cells to collect fit parameters and uncertainties over a range of expression values. The amplitude A = (Ag, Ar) is expected to increase linearly with the fluorescence intensity, which agrees with the data (Fig. S5 A). The characteristic time of the volume fluctuations is independent of the concentration with a mean time of 0.3 s, closely mirroring results obtained in previous work (Fig. S6 A; (
)).
The fitted Q-vector Q¯=(Q¯g,Q¯r) was converted into the normalized brightness b = (bg, br) (Fig. 4 C), as described in Theory. The values of the brightness plot scatter around b = (1, 0), which is consistent for the SS-EGFP species containing one EGFP label and zero mCherry labels. These b values showed no dependence on the concentration of SS-EGFP (Fig. 4 D). As discussed in Theory, DC tsMSQ was designed to identify HSP parameters. Thus, any purely red-emitting species is filtered out by HSP. As a consequence, we expected the noninteracting SS-mCherry-KDEL species to be invisible to the analysis, which is confirmed by the brightness plot. Consequently, the recovered diffusion times apply to the SS-EGFP species (Fig. S7 A). Their values are concentration independent with a mean value of 1.7 ms, which is consistent with previous results (
As an additional control, we reanalyzed the cytoplasmic EGFP and mCherry data (Fig. S3) using DC tsMSQ with a fit to a single diffusing species (Eq. 5). The data and fit closely match (Fig. S8, A–C), with the recovered HSP brightness values b = (bg, br) centered near (1, 0) in a brightness plot, which is consistent with the EGFP species (Fig. S8, D and E). The fitted diffusion time is concentration independent with a mean value of 0.8 ms (Fig. S7 B), which agrees with previously published experiments (
Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy.
After establishing that DC tsMSQ properly identifies noninteracting proteins within the NE and the cytoplasm, we next examined the tandem heterodimer SS-EGFP-SL-mCherry. This construct carries both EGFP and mCherry separated by a linker (SL) and mimics a strongly interacting pair of proteins forming a heterodimeric complex. As expected, DC FFS measurements of SS-EGFP-SL-mCherry in the NE produced a cross correlation tsMSQgr curve that significantly exceeded the predicted baseline tsMSQgr(−) for noninteracting proteins (Fig. 5 A). The DC tsMSQ curves were readily modeled by a fit to Eq. 7 (Fig. 5 A) with reduced chi-squared values of 1.06. The HSP brightness values were centered around (1, 1), reflecting the presence of complexes with an average composition of one EGFP and one mCherry label as expected for the heterodimer (Fig. 5 B). The diffusion time of the heterodimer identified by fitting is concentration independent (Fig. S7 C). The A and τ0 values obtained for the volume fluctuations agree with the expected behavior (Figs. S5 B and S6 B). These results demonstrate that DC tsMSQ can be used to accurately identify the presence of heterodimeric protein complexes in the NE of living cells.
Figure 5DC tsMSQ analysis of measurements obtained in the NE of cells (n = 24) expressing SS-EGFP-SL-mCherry. (A) Shown are tsMSQ curves (symbols) with fits (black lines), standard residuals (sr), and predicted tsMSQgr(−) curve (blue line). The tsMSQgg and tsMSQgr curves are shown with green squares and red circles, respectively. (B) Shown is br versus bg with standard deviations (error bars). (C) Shown are bg (green circles) and br (red squares), with respective standard deviations (error bars), versus Ng with means (dashed lines) and SDs of bg = 0.96 ± 0.06 and br = 1.1 ± 0.3. To see this figure in color, go online.
Although the fluorescence intensity of luminal proteins is affected by volume fluctuations in the NE (Fig. 1 B), the fluorescence intensity of membrane-associated proteins is not affected by these volume fluctuations as demonstrated in previous work using SC FFS (
). To test this difference between luminal and membrane-bound proteins in the context of DC tsMSQ, we performed DC FFS experiments in the NE of cells expressing the membrane-bound SS-EGFP-torsinANTD-2xLeu and the luminal SS-mCherry-KDEL. Because torsinANTD-2xLeu is a transmembrane domain (
Vander Heyden A.B.
Naismith T.V.
Hanson P.I.
Static retention of the lumenal monotopic membrane protein torsinA in the endoplasmic reticulum.
), its presence ensures that EGFP is anchored to the nuclear membrane.
Although the fluorescence signal generated by the luminal SS-mCherry-KDEL includes volume fluctuations, the fluorescence signal generated from the membrane-bound SS-EGFP-torsinANTD-2xLeu does not. Because DC tsMSQ filters out any purely red fluorescing species, the data are expected to only contain the membrane-bound green fluorescing species. Consequently, we fit the DC tsMSQ curves to a model containing only a diffusing species and no exponential process (Eq. 5), which agreed well with the data (χν2 = 1.07). The fit recovered HSP brightness values b that are clustered around (1, 0) (Fig. 6 B) and show no concentration dependence (Fig. 6 C), which is consistent with SS-EGFP-torsinANTD-2xLeu being monomeric. The diffusion times for SS-EGFP-torsinANTD-2xLeu (Fig. S7 D) have a mean and SD of 16 ± 4 ms, which is in agreement with previously published results for this construct (
Figure 6DC tsMSQ analysis of measurements performed in the NE of cells (n = 20) co-expressing SS-EGFP-torsinANTD-2xLeu and SS-mCherry-KDEL. (A) Shown are tsMSQ curves (symbols) with fits (black lines) and standard residuals (sr). The tsMSQgg and tsMSQgr curves are shown with green squares and red circles, respectively. (B) Shown is br versus bg with standard deviations (error bars). (C) Shown are bg (green circles) and br (red squares), with respective standard deviations (error bars), versus Ng with means (dashed lines) and SDs of bg = 0.97 ± 0.18 and br = 0.08 ± 0.12. To see this figure in color, go online.
We next used DC tsMSQ to study the assembly of LINC complexes composed of SUN2 and nesprin-2 in vivo by investigating the ability of the SUN2 luminal domain to form a heterotypic interaction with the luminal domain of nesprin-2. Based on previously published in vitro biochemical and structural studies, it is expected that a homotrimer of SUN2 interact with three nesprin-2 KASH peptides to form a heterohexamer (
). Moreover, SUN2 homotrimerization was shown to be critical for KASH binding (
Ke H.
Feng W.
Coiled-coil domains of SUN proteins as intrinsic dynamic regulators.
Structure. 2016; 24: 80-91
). To begin to test this model of LINC complex assembly in the NE of living cells, we first performed measurements on cells co-expressing mCherry-SR-KASH2 with SS-EGFP-SUN2595–731. The SUN domain (SUN2595–731) contains the KASH-binding sites and has previously been shown to remain monomeric using SC tsMSQ (
). Thus, the SUN domain permits us to directly test for the presence of monomer-monomer interactions with nesprin-2.
Fitting the DC tsMSQ curves to Eq. 7 agreed well with the data (χν2 = 0.88) (Fig. 7 A). A plot of br versus bg revealed that the data were clustered around (1, 0), indicating a noninteracting monomeric EGFP-labeled protein species (Fig. 7 B). Neither bg nor br showed any concentration dependence, and both have means consistent with SS-EGFP-SUN2595–731 being unable to interact with mCherry-SR-KASH2 (Fig. 7 C). In addition, the values of τD, A, and τ0 all agreed with the expectation of a noninteracting SS-EGFP-SUN2595–731 monomer (Figs. S5 C, S6 C, and S7 E). Taken together, these results support the model in which the luminal domain of nesprin-2 is unable to interact with monomers of the SUN2 luminal domain.
Figure 7DC tsMSQ analysis of measurements performed in the NE of cells (n = 20) co-expressing SS-EGFP-SUN2595–731 and mCherry-SR-KASH2. (A) Shown are tsMSQ curves (symbols) with fits (black lines) and standard residuals (sr). The tsMSQgg and tsMSQgr curves are shown with green squares and red circles, respectively. (B) Shown is br versus bg with standard deviations (error bars). (C) Shown are bg (green circles) and br (red squares), with respective standard deviations (error bars), versus Ng with means (dashed lines) and SDs of bg = 0.96 ± 0.13 and br = 0.0 ± 0.2. To see this figure in color, go online.
Unlike SS-EGFP-SUN2595–731, the SUN2 luminal domain-encoding SS-EGFP-SUN2261–731 homotrimerizes in the NE of living cells as determined by SC FFS (
). Thus, we expected that measurements performed in the NE of cells co-expressing SS-EGFP-SUN2261–731 and mCherry-SR-KASH2 would identify heterotypic interactions between these constructs. Proper analysis by DC tsMSQ of data obtained in cells expressing both proteins required the inclusion of two diffusing species (Eq. 8) to describe the data (Fig. 8 A). The two diffusion times have means of 1.7 and 180 ms (Fig. S7 F), which are consistent with a previous SC FFS study of SS-EGFP-SUN2261–731 (
).The tsMSQgr amplitude is significantly higher than the noninteracting prediction (Fig. 8 A), indicating the presence of heteroprotein association. Plotting br versus bg from n = 53 cells revealed that both bg and br increase together (Fig. 8 B), implying that an increase in the average oligomeric state of SS-EGFP-SUN2261–731 is associated with an increase in the number of bound mCherry-SR-KASH2. This result supports the model that nesprin-2 interacts with homotrimers of SUN2 within the NE. We further notice that br ≤ bg, indicating that at most, one mCherry-SR-KASH2 can associate with each SS-EGFP-SUN2261–731 protein within the homotrimer.
Figure 8DC tsMSQ analysis of measurements performed in the NE of cells (n = 53) co-expressing SS-EGFP-SUN2261–731 and mCherry-SR-KASH2. (A) Shown are tsMSQ curves (symbols) with fits (black lines) and associated standard residuals (sr) with χν2 = 0.88 and predicted tsMSQgr(−) curve (blue line). The tsMSQgg and tsMSQgr curves are shown with green squares and red circles, respectively. (B) br versus bg with standard deviations (error bars) are correlated and increase with concentration as indicated. (C) Shown is bg versus Ng with standard deviations (error bars) and a line provided to guide the eye. (D) Shown is br versus Ng with standard deviations (error bars) separated by Nr/Ng ratios greater (blue squares) or less (magenta circles) than 4:1, with lines provided to guide the eye. To see this figure in color, go online.
These data show that bg ranges between 1 and 3, signifying a limiting homotrimeric state for the SS-EGFP-SUN2261–731 in the NE. The concentration dependence of the oligomerization of SS-EGFP-SUN2261–731 is visualized by a plot of bg versus Ng (Fig. 8 C). The brightness increases with Ng, approaching a homotrimeric brightness state, which is identical to the behavior observed for SS-EGFP-SUN2261–731 in the absence of mCherry-SR-KASH2 (
). We expect to observe an increase in br with Ng because the luminal domain of nesprin-2 is predicted to bind to homotrimers of the SUN2 luminal domain, which are populated at a higher Ng (Fig. 8 C). However, the amount of bound mCherry-SR-KASH2 also depends on its concentration, which varies significantly from cell to cell. Thus, the variability in the expression ratio of mCherry-SR-KASH2 to SS-EGFP-SUN2261–731 is responsible for the large scatter in the observed br values (Fig. 8 D). To visualize the dependence of br on the expression ratio of these two constructs, the data were separated into sets with an Nr/Ng above and below 4:1 (Fig. 8 D). Data with a Nr/Ng ratio above 4:1 exhibit a strong increase in br up to values approaching 3, whereas the lower ratio data only show a modest increase in br with Ng. This reflects the relative reduction in mCherry-SR-KASH2 concentration between both data sets. Notably, the highest brightness values measured for bg and br approach 3 (Fig. 8, B–D), which suggests the formation of a SUN2-nesprin-2 heterohexamer in vivo as predicted by in vitro models (
Although CCF analysis has been widely used to identify heterotypic protein interactions from FFS measurements performed in cells (
Majoul I.V.
Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis.
), the presence of nuclear membrane undulations is a significant barrier for its application to FFS measurements performed in the NE. The local volume changes caused by nuclear membrane undulations lead to coupled changes in the emission intensity of both the red and green fluorescent labels (Fig. 1 B), which result in a spurious cross correlation in the CCF, even when accounting for spectral cross talk. The DC tsMSQ theory developed in this article provides a comprehensive method that incorporates both spectral cross talk and volume fluctuations, thus overcoming the issues present in CCF analysis of FFS data measured in the NE. Although this article has focused on the use of tsMSQ to account for the presence of volume fluctuations, the advantages of tsMSQ extend to any slow fluctuation process. Bias introduced by slow fluctuations is a general challenge for FFS analysis, and tsMSQ provides the ability to identify and account for the effect of these slow processes (
). Once a slow fluctuation process has been detected, new models incorporating the process can be developed to account for its effect on the tsMSQ curve. For example, the original formulation of MSQ analysis was used to account for the presence of photodepletion (
We experimentally verified DC tsMSQ using model systems representing noninteracting protein pairs as well as heterodimeric protein complexes and recovered diffusion times and HSP brightness values. The label stoichiometry determined by HSP brightness matched the expected values of the control samples and reliably distinguished interacting from noninteracting proteins. Furthermore, the parameters of the volume fluctuation process agreed with our previously published SC tsMSQ results (
), thus providing additional support for the DC tsMSQ model.
Next, we used DC tsMSQ to investigate the assembly mechanism of LINC complexes, which are formed by the heterotypic interaction of the luminal domains of SUN and KASH proteins (
Crisp M.
Liu Q.
Hodzic D.
Coupling of the nucleus and cytoplasm: role of the LINC complex.
J. Cell Biol. 2006; 172: 41-53
). Previously published in vitro biochemical and structural studies revealed that a homotrimer of the SUN2 luminal domain interacts with three nesprin-2 luminal domains (
). Deep KASH peptide-binding grooves are formed at the interface of two adjacent SUN domains upon SUN2 luminal domain homotrimerization; therefore, SUN2 homotrimerization is believed to be a necessary precursor to the assembly of a SUN2-nesprin-2 heterohexamer (
To test this model in vivo, we looked for interactions between the luminal domain of nesprin-2 (mCherry-SR-KASH2) and two SS-EGFP-tagged SUN2 luminal domain constructs. We previously showed that the EGFP-tagged luminal domain of SUN2 (SS-EGFP-SUN2261–731) forms homotrimers in the NE, whereas the SUN domain of SUN2 (SS-EGFP-SUN2595–731) remains monomeric (
). As expected, we were unable to detect a heterotypic interaction between SS-EGFP-SUN2595–731 and mCherry-SR-KASH2 using DC tsMSQ. However, DC tsMSQ did detect a concentration-dependent heterotypic interaction between SS-EGFP-SUN2261–731 and mCherry-SR-KASH2 in the NE. The limiting HSP brightness values measured for these constructs suggest the formation of a SUN2-nesprin-2 heterohexamer in vivo, which agrees with the in vitro results described above (
Kutay U.
Structural insights into LINC complexes.
These initial results provide a promising starting point for future quantitative studies of the mechanisms underlying the in vivo assembly of functional LINC complexes and their regulation. Combining quantitative modeling of the DC tsMSQ brightness data with targeted mutations that perturb the known SUN2-nesprin-1/2 binding sites should provide a comprehensive approach for investigating the assembly of SUN2-containing LINC complexes. In addition, DC tsMSQ offers a tool to investigate the formation of LINC complexes composed of lesser studied SUN proteins, such as SUN1, which forms higher-order oligomers than SUN2 (
). This article focused on luminal domains of SUN2 and nesprin-2 due to the low mobility of the full-length proteins, which prevents us from performing point FFS measurements (
Ostlund C.
Folker E.S.
Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins.
Quantitative measurement of brightness from living cells in the presence of photodepletion.
). Future work combining DC tsMSQ with an imaging- or scanning-based approach may allow for measurements of LINC complex assembly with full-length proteins. In addition, by combining scanning with the data segmentation inherent to tsMSQ, techniques such as intensity-sorted FCS may be incorporated (
Di Bona M.
Mancini M.A.
Lanzanò L.
Measuring mobility in chromatin by intensity-sorted FCS.
) to look at the assembly of full-length SUN2 and nesprin-2 and investigate the presence of spatial heterogeneity in the assembly process. Although this article has focused on the application of DC tsMSQ for characterizing the heterotypic interactions of LINC complex proteins, the development of DC tsMSQ offers a promising and general platform for future studies of heteroprotein complex formation in the NE of living cells.
J.H., G.W.G.L., and J.D.M. were responsible for experimental design. J.H. performed experiments. K.-H.H. developed analytical tools. J.H., K.-H.H., J.K., S.R.K., I.A., and J.D.M. performed analysis. J.H., K.-H.H., G.W.G.L., and J.D.M. wrote the manuscript.
We thank Cosmo A. Saunders for his help generating the DNA constructs used in this study.
This work was supported by the National Institutes of Health GM064589 (J.D.M, J.H., K.-H.H., J.K., S.R.K., and I.A.) and GM129374 (G.W.G.L., J.D.M., and K.-H.H.).
Document S1. Supporting Materials and Methods, Figs. S1–S8, and Table S1
Editor: Jason Swedlow.
Received: July 19, 2019
Jared Hennen and Kwang-Ho Hur contributed equally to this work.
DOI: https://doi.org/10.1016/j.bpj.2019.11.020
© 2019 Biophysical Society.


The module can be found within the various additional modules, this module depreciates on a straight line basis only.
To start out, create at least one Asset Group in order to register your items and establish a set of entry numbers.
The asset groups are also where you specify the accounts that you would like to make additions, disposals and depreciation within.
You can register the addition of an asset in two ways. You can either enter it in the supplier invoice or, if the asset has been depreciated in part (e.g. in a different accounting system), on the active list.
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text:
Q: Combine multiple varchar rows into one Col1        Col2        Col3        Col4    Col5        Col6    Col7    Col8
CH-0000001  IN-0000001  IN-0000001  ALL     NULL        NULL    NULL    NULL
CH-0000001  PR-0000001  NULL        NULL    PR-0000001  ALL     NULL    NULL
CH-0000001  PU-0000001  NULL        NULL    NULL        NULL    PU-0000001  ALL

Hey guys I am trying to squish all this information into a single row based on a key Col1. I have seen a trick using MAX and SUM but it only seems to work for numbers. Here is the desired result.
Col1        Col3        Col4    Col5        Col6    Col7         Col8
CH-0000001  IN-0000001  ALL     PR-0000001  ALL     PU-0000001   ALL

Also, this problem arose from LEFT JOINing 3 tables from one master table of keys. Let me know if my approach to getting to this state is the wrong way to approach this problem. Here is the query
SELECT * FROM GCL_Blackboard A
LEFT JOIN GCL_Investors B ON A.Adjective_ID = B.Investor_ID
LEFT JOIN GCL_Loan_Programs C ON A.Adjective_ID = C.Program_ID
LEFT JOIN GCL_Loan_Purposes D ON A.Adjective_ID = D.Purpose_ID

A: I think this is the logic you want:
SELECT a.col1,
       max(coalesce(b.col2, c.col2, d.col2)) as col2,
       max(coalesce(b.col3, c.col3, d.col3)) as col3,
       max(coalesce(b.col4, c.col4, d.col4)) as col4,
       max(coalesce(b.col5, c.col5, d.col5)) as col5,
       max(coalesce(b.col6, c.col6, d.col6)) as col6,
       max(coalesce(b.col7, c.col7, d.col7)) as col7,
       max(coalesce(b.col8, c.col8, d.col8)) as col8
FROM GCL_Blackboard A LEFT JOIN
     GCL_Investors B
     ON A.Adjective_ID = B.Investor_ID LEFT JOIN
     GCL_Loan_Programs C
     ON A.Adjective_ID = C.Program_ID LEFT JOIN
     GCL_Loan_Purposes D
     ON A.Adjective_ID = D.Purpose_ID
GROUP BY a.col1;

The query is a bit of a guess, because I don't know where the columns are coming from.

A: You can do:
SELECT A.Col1,
       MIN(Col3) Col3,
       MIN(Col4) Col4 ....
FROM GCL_Blackboard A
LEFT JOIN GCL_Investors B ON A.Adjective_ID = B.Investor_ID
LEFT JOIN GCL_Loan_Programs C ON A.Adjective_ID = C.Program_ID
LEFT JOIN GCL_Loan_Purposes D ON A.Adjective_ID = D.Purpose_ID
GROUP BY A.Col1

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contact@hokellc.com
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Insurance Bytes
Commercial Litigationshoke20132022-05-05T14:07:09+00:00
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Successfully argued appeals in the Illinois Supreme Court and the Illinois Appellate Court on behalf of a major retailer involved in a class action lawsuit that arose out of the retailer's practice of collecting sales tax on shipping charges for internet purchases. See Kean v. Wal-Mart Stores, Inc., 231 Ill. 2d 669 (2009).
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"Lesestart", the nationwide literacy program for children funded by the Federal Ministry of Education and Research and carried out by Stiftung Lesen, supports children and their parents in the crucial pre-school years as well as at their school enrolment. Every step includes a "Lesestart" pack of a book suitable for the relevant age-group as well as information for parents in four languages: German, Polish, Russian and Turkish. The associated website www.lesestart.de offers this information also in many other relevant languages.
Families received the first "Lesestart" pack for infants at the doctor's on the occasion of the regular check-up visit for one-year-olds. During the second stage from November 2013 until October 2016, parents with their three-year-olds were able to get their personal pack from their local library for free. With the beginning of the third milestone after school entry in 2016, the "Lesestart" packs for first graders are distributed to every primary school in Germany where the teachers hand them on to the children. And the program continues: 2017 and 2018 there will be "Lesestart" packs for every school starter.
The first years of a child`s life are fascinating. Suddenly, he or she begins to walk, speaks its first words and sentences, finds friends at kindergarten or in the neighborhood and eagerly awaits the first day of school. Children will hardly learn as much as they do at this stage of life later on. In the process, the driving force is always childlike curiosity: Children ask questions, always eager to learn something new and discover their own abilities. As parents, you play an important role in promoting your child in his or her versatile development process. This includes in particular sparking joy in reading early on.
For reading will open up horizons – at all stages of life, from childhood through adolescence into adulthood. By reading, children will also learn to focus, comprehend larger contexts and, incidentally, expand their vocabulary. They cherish the time spent reading together as moments of comfort, peace and affection. Thereby, you quite playfully lay the foundation for their course of education.
With "Lesestart – Three Reading Milestones", we would like to provide you with inspiration. The program is funded by the Federal Ministry of Education and Research and implemented by Stiftung Lesen (German Reading Foundation). I wish you and your children lots of (reading) joy and success.
The findings of the first stage's evaluation show that "Lesestart" is an effective tool in the advancement of early childhood education: Virtually all parents who received "Lesestart" packs deal with the materials. 62 percent of the parents seize the information given in the "Lesestart" packs and read to their children more frequently. One fourth of the parents notice a positive change in their attitude towards reading. On behalf of the Federal Ministry of Education and Research, the program is evaluated by an independent group of experts from the Berlin InterVal GmbH, in cooperation with the Institute for German Language and Literature II, Cologne University and AG Kinder- und Jugendpsychiatrische Epidemiologie und Evaluation, (Child and Youth Psychiatric Epidemiology and Evaluation) University Clinic Hamburg-Eppendorf.
Information on the nationwide literacy program are available on www.lesestart.de and on www.facebook.com/Lesestart.


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text:
Q: CKEditor: toolbar dropdowns open out of line I use inline ckeditor on my webpage. This works perfectly everywhere besides in a modal.
What happen is that dropdowns in the toolbar show 50-200px above the rest of the toolbar when clicked. ex styles or font selection.
I can not figure out WHY! and the error is only in this modal, not anywhere else.
Does anyone have any idea why? Is it CSS inheritance? Is it that I render elements above the ckeditor after the ckeditor is rendered?
My initialization looks like this:

   <div id="<%= UID %>-cktool" class="ckeditor-toolbar-div hidden">

   <div class="text-field" id="<%= UID %>" data-fieldid="<%= FieldId %>">
   <div class="ckEditor read-only-content" contenteditable="true" id="<%= UID %>-textarea"><%= Content %>

var toolbar = self.$("#"+self.$(".text-field").attr("id") + "-cktool");
editor = (this.editor = CKEDITOR.inline(this.textarea[0],
{
      sharedSpaces:{
                     top: self.toolbar.attr("id")
      }
}));

toolbar in config.js:
config.toolbar =
[
    { name: 'clipboard', items: ['Cut', 'Copy', 'Paste', '-', 'Undo', 'Redo'] },
    { name: 'editing', items: ['Find', 'Replace', '-', 'SelectAll'] },
    { name: 'colors', items: ['TextColor'] },
    { name: 'insert', items: ['Table', App.currentUser.isSuperAdmin ? 'Image' : ''] },
    { name: 'basicstyles', items: ['Bold', 'Italic', 'Underline', 'Strike', '-', 'RemoveFormat'] },
    { name: 'paragraph', items: ['NumberedList', 'BulletedList', '-', 'Outdent', 'Indent', '-', 'JustifyLeft', 'JustifyCenter', 'JustifyRight', 'JustifyBlock'] },
    { name: 'links', items: ['Link', 'Unlink', 'Anchor'] },
    { name: 'plugin', items: ['SpellChecker', 'Scayt', 'Sourcedialog'] },
    { name: 'styles', items: ['Styles', 'Format', 'Font', 'FontSize'] }
];

UPDATE:
So I figured out why, but not how to fix it.
The modal has a scrollbar (the body-content is longer than the modal).
When editing the content in the browser to be as long as the modal, it works.

A: Fixed: Had to set the modal css FROM
 position: absolute;

to:
 position: static;


text:
Хосе́ Миа́ха Мена́нт (; , , Астурия — , ) — испанский военачальник, участник Гражданской войны в Испании в 1936—1939 годах, генералиссимус армии республики (1939).

Военная служба
Окончил пехотное училище в Толедо. Начал офицерскую службу в Астурии, позднее по собственной инициативе был переведён в Мелилью (Марокко), где в это время шла война против местных племён. За военные заслуги был произведён в майоры в 1911 году.

Однако при королевской власти Миаха не поднялся в военной иерархии выше полковника, а чин генерала получил уже при республике в 1932 году. Он входил в состав правого Испанского военного союза, но, несмотря на это, правоцентристское правительство страны не испытывало к нему доверия. Поэтому военный министр Хосе Мария Хиль-Роблес направил его командующим войсками в провинциальную Лериду. После победы на выборах левого Народного фронта в феврале 1936 года Миаха был переведён в Мадрид, где назначен командиром первой бригады, что являлось существенным повышением.

Участие в Гражданской войне
Во время антиправительственного выступления военных в июле 1936 года многие подчинённые Миахи присоединились к восставшим. Сам он некоторое время пребывал в нерешительности, видимо, из-за того, что его семья находилась в зоне, контролируемой националистами. Однако он принял решение остаться верным правительству и получил пост военного министра в кабинете Диего Мартинеса Баррио, просуществовавшего только одни сутки (18-19 июля 1936 года). В следующее правительство Хосе Хираля Миаха не вошёл.

Однако уже 25 июля он был назначен начальником операций на Юге. По словам историка Хью Томаса, среди тех, кто поддерживал правительство скорее в силу случайности, чем по убеждениям, оказался вялый, добродушный генерал Миаха, который в своё время входил в антиреспубликанский военный союз. 28 июля войска под командованием Миахи начали наступление на позиции восставших и осадили Кордову, но из-за недисциплинированности подчинённых и нерешительности самого генерала успеха не добились. 23 августа под давлением войск генерала Хосе Энрике Варелы осада Кордовы была снята.

Несмотря на это, Миаха получил повышение, став командиром 3-й органической дивизии со штабом в Валенсии, а в октябре переведён на должность командира 1-й органической дивизии со штабом в Мадриде.

Руководитель обороны Мадрида
6 ноября 1936 года в условиях наступления на столицу войск националистов правительство эвакуировалось из Мадрида, а генерал Миаха был назначен главой хунты обороны Мадрида. На этом посту он вновь не проявил большой решительности, но в то же время оказывал большое моральное влияние на подчинённых — сам факт того, что во главе обороны города, казалось, обречённого на сдачу, стоял генерал, демонстрировавший спокойствие и уверенность в своих силах, давал им дополнительную надежду на победу. Не будучи ангажирован ни одной из конкурировавших партий, входивших в состав Народного фронта, беспартийный военачальник выступал в качестве сплачивающего фактора для защитников Мадрида.

Кроме того, начальником штаба Миахи стал способный офицер Висенте Рохо, который фактически руководил военными действиями, причём Миаха ему в этом не мешал. По словам историка Пола Престона, прямой и добродушный Миаха сразу собрал вокруг себя штат из высокопрофессиональных помощников, наиболее видным из которых был его начальник штаба подполковник Висенте Рохо. Пока Рохо занимался организацией обороны, Миаха поднимал моральный дух защитников столицы.

Будущий маршал Советского Союза Кирилл Мерецков, являвшийся во время гражданской войны советником Миахи, в своих мемуарах дал противоречивую характеристику генералу, отмечая, однако, его профессиональные знания: Совместная работа с ним была делом сложным. В Миахе жило два человека: военный и политик. В качестве политика Хосе Миаха, официально беспартийный, был на деле очень далёк от коммунистов. Это сильно мешало упорядочить руководство боевыми операциями в «красной зоне», как называли тогда район Мадрида за откровенно левые настроения большей части его населения и за ту выдающуюся роль в его обороне, которую играла испанская компартия. А в качестве военного Миаха оказался человеком знающим. Так, он хорошо разбирался в боевых возможностях марокканских войск — основной силы Франко под Мадридом. Оказалось, что он имел опыт колониальной войны в Марокко… (С точки зрения советского советника, находившегося на стороне коммунистов, «равноудалённость» Миахи от различных политических сил была не плюсом, а минусом).

После тяжёлых боёв в районе Университетского городка республиканские войска смогли остановить войска националистов. Миаха лично выезжал на передовую для того, чтобы поддержать своих подчинённых. Руководство обороной Мадрида сделала Миаху одним из наиболее популярных военачальников республики; его имя получило международную известность.

Командующий армией Центра
В феврале 1937 года Миаха сменил генерала Себастьяна Посаса на посту командующего армией Центра. Уже в марте войска под его командованием смогли отбить наступление итальянских войск — союзников националистов — под Гвадалахарой и затем перейти в контрнаступление. Это сражение стало одним из основных успехов республиканцев за всё время гражданской войны, сильно поднявших их моральный дух. В конце мая — начале июня того же года он осуществлял общее руководство наступлением республиканских войск на Сеговию, которое закончилось с незначительными территориальными приобретениями (Сеговию взять не удалось) и большими потерями (около 30 % личного состава наступавших войск).

В июне 1937 года Миаха получил наиболее почётную награду республики — Мадридский знак отличия с лавровым венком.

В июле 1937 года армия Центра добилась тактического успеха во время наступления на Брунете, но националисты смогли остановить армию Миахи и затем перешли в контрнаступление. Фактически планированием этих и других операций занимались Рохо и начальник штаба Миахи Мануэль Матальяна.

Главнокомандующий сил Центра и Юга
В апреле 1938 года генерал Миаха стал главнокомандующим сил Центра и Юга на суше, на море и в воздухе. Впрочем, по мнению российского историка С. Ю. Данилова в это время генерал уже давно
жил прежней славой. В оперативные дела генерал с весны 1937 года почти не вмешивался, тайно пьянствовал и даже вызывал подозрения в наркомании. Его втихомолку прозвали «свадебным генералом». «Я командую только сушей, морем и воздухом, — ответил Миаха однажды советскому офицеру, старавшемуся что-то выяснить, — будьте добры, обратитесь к Негрину или к Матальяне». Миаха сыграл негативную роль во время весеннего сражения в Леванте, срывая выделение резервов на помощь Восточному фронту. Но он заблаговременно вступил во все партии Народного фронта. И в результате ни одна из них не потребовала сместить неработоспособного «свадебного генерала»…

24 января 1939 года премьер-министр республики Хуан Негрин присвоил Миахе высший чин генералиссимуса, однако в условиях военных поражений (26 января войска националистов вошли в Барселону) это решение не привлекло большого внимания. К тому времени Миаха уже утратил надежду на победу. В марте 1939 года он поддержал выступление командующего армией Центра полковника Сехисмундо Касадо против правительства Негрина. В ночь с 5 на 6 марта в Мадриде был совершён переворот, в результате которого власть перешла к Совету национальной обороны (), формальным лидером которого был Касадо, а официальным председателем — Миаха. После того, как Франсиско Франко отказался вести с советом (в советской литературе — «хунтой») переговоры о мире, Миаха выехал в Гандию, где сел на английский корабль, на котором 26 марта покинул страну.

В советской исторической литературе поведение Миахи в марте 1939 года считалось предательством. В то же время в западной литературе при общем скептическом отношении к военным талантам генерала, его действия в конце войны объясняются признанием неизбежности поражения и стремлением договориться с националистами на сколько-нибудь приемлемых условиях.

Эмигрант
Первоначально Миаха выехал в Алжир, затем перебрался во Францию, а позже эмигрировал в Мексику, где и скончался.

Примечания

Литература

Ссылки
 Воспоминания К. А. Мерецкова
 Биография

Участники гражданской войны в Испании (республиканцы)
Военачальники Испании
Генералиссимусы
Иммигрировавшие в Мексику из Испании

redpajama_set_name: RedPajamaWikipedia


The researchers imagine that is the primary time anyone has filmed sand cat kittens within the wild. Any changes to the weight loss program must be made regularly, transitioning to the new food slowly over a period of days. Save and handle your pet searches and electronic mail communications.
Kittens can regulate their food consumption effectively and so meals might be offered usually however it is alright to depart a few biscuits out always for your kitten to eat. Kittens needs to be started on prevention from 12 weeks of age. After 3-four weeks for cats or 6-eight weeks for kittens (or once your cat has settled), introduce your cat to the garden in short supervised periods.
Incorporate grooming into your interactions with the kittens—especially you probably have a single kitten, or you are raising a litter with out the mother cat. It is a section of speedy growth for kittens by which they're going to have almost 3 times more energy than an grownup cat.


Tesaro's niraparib has been approved as a maintenance therapy for ovarian cancer in the US, although the company experienced a setback in its bid to develop the drug in breast cancer after patients quitting a chemotherapy arm ruined the results.
According to Evaluate Pharma, niraparib, a poly-ADP ribose polymerase (PARP) inhibitor, which will be marketed as Zejula, could generate peak sales of around $2 billion.
And Tesaro hopes to develop the drug in a host of other uses after this first approval for maintenance treatment of women with recurrent epithelial, ovarian, fallopian tube, or primary peritoneal cancer, who are in complete or partial response to platinum chemotherapy.
Zejula will also not require a patient selection with a biomarker test, a blow for Myriad Genetics, which had argued that its companion diagnostic test would be needed to select patients.
This means that Zejula is first PARP inhibitor to market in the maintenance therapy indication after AstraZeneca's Lynparza (olaparib) failed to impress an FDA expert panel in this use back in 2014.
Lynparza, the first PARP inhibitor to market, was later approved by the FDA in an advanced ovarian cancer indication, after failure of three or more lines of chemotherapy, along with a diagnostic test for defective BRCA genes.
However, it has just reported strong results in phase 3 trials, including its use as ovarian cancer maintenance therapy, that could help it establish a firmer grip on the market.
Clovis' Rubraca (rucaparib) has also been approved in advanced ovarian cancer with a BRCA test.
To counter this, Tesaro will now press on with the development of Zejula in a host of other uses, including a first line ovarian cancer maintenance treatment in combination with an anti-PD-1 antibody, and in combination with Roche's Avastin (bevacizumab) in patients with first recurrence of the disease, instead of chemo.
Tesaro also plans trials in metastatic triple-negative breast cancer in combination with Merck & Co's Keytruda, and in indications including metastatic non-small cell lung cancer, in combination with an anti PD-1 antibody.
Tesaro has already begun the BRAVO study assessing Zejula in breast cancer with BRCA mutations – but following an independent data analysis, Tesaro said the trial is unlikely to produce data suitable for registration.
A large number of patients in the chemotherapy arm did not continue with the trial long enough to receive their first radiological scan required to assess disease progression.
Tesaro said patients wanted to be treated with a PARP inhibitor rather than chemotherapy and quit to get treatment with rival PARP inhibitors.
A steering committee will decide whether enrollment will continue, although no new safety concerns emerged.


text:
Q: gcloud auth does not work out with windows WSL I tried to authenticate with gcp using my personal account, but I can able to make it work entirely.
so I tried doing it using

when I copy the same url and paste it into the browser, it is not working, see this

Not sure why this is now happening, as it is the WSL - ubuntu machine on windows does not have web browser attached to terminal, so I had to do it manually paste into web which is not working.

A: gcloud auth login --no-launch-browser

ref: https://cloud.google.com/sdk/docs/authorizing

A: I missed, instead of gcloud auth I need to do this -
gcloud init --console-only

which works out the way I expected. (I copied the url from console paste it into console where verification code)


When designing a campaign with an overarching storyline the most important thing you as a DM can do, is create a believable villain. It is easy to get caught up in the world building, maps, and all the cool ideas. The Big Bad Evil Guy/Gal (BBEG) is the piece that ties it all together. Without a well developed BBEG and their minions, all of the world building and mapping are just soulless backdrops for rolling dice. A BBEG brings flavor and stakes to that village they are visiting or some quest to find some object or piece of information before the BBEG does.
Wait. What? I thought the PCs are the heroes. They are of their own stories.
A good BBEG thinks what they are doing is right and making the world better. Their goal might be the same as the PCs, like unifying a nation, but their methods differ. The PCs might be looking for the rightful heir to the throne or building consensus while the BBEG might be a conquer. The important thing to remember is the BBEG doesn't think he is the bad guy. Maybe she is the Spare and tries to prove to her father that she would be a more adept ruler than the Heir, who really doesn't want to rule anyway. She ends up trying so hard to prove that she can do it, but messes it up and keeps going with the plan that doesn't work.
Gul Duka, Prefect of Bajor from Deep Space 9, wanted to help the people of Bajor come to accept the occupation and being part of the Cardassian Empire. He abolished child labor, increase food rations and cut back on quotas in the camps. His actions saved lives and made the occupation more bearable for the people of Bajor. However, the resistance couldn't see what he was trying to do and sabotaged his good works, so he had to ruthless suppress the people to get them to see the light.
How many times have you watched a movie or played a game where there was a really cool bad guy that seemed like a real serious threat to the protagonists only to do something so completely moronic and out of character you wanted to punch a puppy?
If your BBEG is a methodical plotter whose emotions are not easily roused, then in the penultimate battle goes into a blind rage out of nowhere, then they are not being internally consistent. Or if you create someone who spends years wanting to tear down the world to watch it burn only to become a leader of a highly organized military state for no reason. These types of characters ruin the immersion of the story. A good DM won't let their players do things out of character, so why would you do it?
Not all of your BBEGs have to look like monsters or want to destroy everything good or pretty things just because they can. Those types of characters get boring and are more suited for lieutenants of the BBEG. This particular one is a little more subjective than that the others. For BBEGs that are looking to take over the world and rule it themselves, then no reason to go over board with the "evil acts" for shock value. Sure the occasional "object lesson" in brutality can be effective but if there is nothing left of the world when they are done, what's the point?
Some of the most insidious evil is the cultured evil. Lex Luthor, Hannibal Lector, and Admiral Thrawn are some good examples.
This one ties back to the previous topic. It's ok for your BBEG to be funny or charming or have one of those personalities that people just like to be around. A dour bad guy that scowls all the time is really one dimensional and gets old quickly. Giving a little quirk to your villain gives them depth, humanizes them and makes the world they inhabit much richer.
Think of Heath Ledger's Joker character. Even though he was batshit crazy, he had really magnetic personality with a simmering hint of menace. The Joker was blunt, there was no sugar coating what his goals were, but you were still drawn to him to see what he would do next. Negan, from The Walking Dead, is charismatic and he leads a cult of personality. He's brutally honest, does what he says and has a smile even for his defeated enemies.
Most of the times, your PC's will go up against a BBEG, who already has established them in some position of power. Which means they are competent at something. Remember that and don't muck it up.
Sometimes they are looking to expand that power or consolidate what they have. Either way, they got there somehow. Your BBEG is a warlord who conquered nation after nation and leads through force. They aren't going to fall to some 1st level characters who stumble into their throne room. If the BBEG got to her position through cunning and guile isn't going to get surprised by a group of bumbling PCs who manage to thwart scheme after scheme.
When you create your BBEG how they got to where they are is a blue print for going forward. Don't be hemmed in by the past though. If they BBEG took over a country by force 20 years ago, then its ok there to be a large spy network in the kingdom or to have some advanced weapons/tactics out of the army.
Your BBEG is the thing that ties all of your storyline together. Put as much or more time into creating the BBEG and their power structure as you would anything else in your world. The more details you have the more flavor you can to your world. A well crafted villain will be something your players remember for years.


Does Anybody Care? Podcast is the show that asks you what you want to hear! We've got a bunch of stories for you today! Do you think Nomi gives the worst parenting advice? Do you think this Uber driver was high whilst giving rides? Do you think Sparks should buy HGH from a strange old man? All of these questions are up for you to decide in Ep 221 of Does Anybody Care? Podcast! Remember, you can always interact with us LIVE on our Facebook page when we do the show on Friday's!
Share it with us by commenting or playing along when we're podcasting live!


text:
2023: PDP guber ticket zoned to Kwara North senatorial district
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Abdulrazaq Adebayo
Peoples Democratic Party (PDP) in Kwara State, at the weekend, zoned the choice of its gubernatorial flag bearer to Kwara North Senatorial District, ahead of the next general elections in 2023.
The decision was taken after a series of exhaustive sessions with stakeholders of the party, comprising party faithful from the three senatorial districts of the state.
"This development as expected elicited many positive reactions from many members of the public most especially those with concern for the future of our great state at heart," a party source stated.
"Well, it is important to state that the choice of the party was made in good faith and recourse to justice, fairness and equity, the very fundamentals of our party."
According to the spokesman of the party, Tunde Ashaolu, the main essence of democracy is inclusiveness which is the crux of the decision made by the party.
He said the party is creating a realm where everyone will feel they belong and have their roles to play irrespective of where they come from or the colour of their tongues.
DAILY POST recalls that the last time a native of Kwara North Senatorial District occupied the office of the governor of Kwara State, was during the short-lived Third Republic in the person of former governor Sha'aba Lafiagi of the defunct Social Democratic Party, SDP, who was in office from January 1992 to November 1993 before it was truncated by military rule.
"The administration was cut short by military incursion and by 2023 it will be 30 years ago, meaning it will be the third decade the last time Kwara North governed the state.
"All the organs of the party played a crucial role in assisting the party in arriving at its decision after making a series of consultations around the state under the guidance of our leader, His Excellency Sen. Dr Abubakar.Bukola Saraki.
"The decision process involved the Local government working committees of the party across the 16 local government areas and 193 wards that the state comprises.
Each local government area presented seventeen stakeholders which comprised women, youths and elders of the party in the decision-making process.
By and large, the decision has been made and has been agreed as a consensus by all the three senatorial districts that Kwara North should produce the next Governor come 2023, the spokesman added.
Related Topics:2023Kwara NorthPDP
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Kano APC crisis: Ganduje's faction denies rumours of planned mass defection to PRP
2023: Fate of Nigeria lies in hands of politicians – Abdulsalami Abubakar


text:
How to retire on just $25,000 in the bank
Tracey Samuelson Mar 19, 2013
Don Easton knows he needs to save for retirement. But he's been out of work for five years and so instead of putting money into his retirement accounts, he's been pulling it out. There's food, gas, and credit card debit.
"Dental care's a big issue," he says. "I need a new crown."
Easton hopes to return to work soon and has been picking up side work painting houses and doing handiwork. He's optimistic that he'll again be able to save. But he says if he hits retirement without sufficient savings, he's lucky to have a safety net—his family.
"I would move in with my sister," he says. "She has kids and they all enjoy having me around."
He'd also try to delay retiring by simply working longer.
Those strategies are common solutions for American workers who have not saved enough for retirement.
It's not that people don't want to save, says Nevin Adams, the director of education at the Employee Benefit Research Institute. It's just that there are often more pressing economic concerns.
"Retirement's down a really long list of things people are trying to worry about," he says.
More than half of respondents to a EBRI survey released Tuesday reported they have less than $25,000 in savings, excluding their houses and pensions.
Therefore, like Easton, people could lean on family. They can work more. But the biggest chunk of the retirement pie will be Social Security.
"For people who have their whole lives been in, let's say, the bottom third of the pay distribution, Social Security is going to replace a very large chunk of their pre-retirement earnings, thereby relieving them, potentially, of the need to save a whole lot more," says Olivia Mitchell, the executive director at Pension Research Council and a professor at the University of Pennsylvania.
They'll also likely lean on Medicaid, Medicare, and Supplemental Security Income (SSI) benefits.
For middle income households, Social Security offers a smaller percentage of what they're used to living on.
"It's really the middle class I think is potentially most in trouble," says Mitchell. "Working a few extra years could well make the difference between having enough and running out."
For those approaching retirement who haven't yet saved enough, she has this not-so-cheery mantra: "Work longer, save more, and expect less."


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Zindagi Mere Ghar Aana 24th September 2021 Upcoming Story, Spoilers, Latest News, Gossips, Twist
Over the phone, Kabir tells Meera that no one in the family knows he works for her. Meera expresses her want to say something. Meera inquires of Kabir about Amrita's best interests. She requests that he mention something unique. Kabir requests that she give her a car as a gift. He claims she'll be there, and that's the major present. Gurmeet approaches Kabir and asks him to take Amrita's clothes to the tailor.
Kabir claims to be on the phone with his supervisor. Meera teases him, and he tells her to be quiet. Gurmeet asks him not to speak to his boss in this manner. Kabir informs him that they have a meeting and asks him to leave. Meera inquires about Gurmeet's reaction to her father's visit. Kabir invites her to come meet him tomorrow, which she eagerly accepts. Kabir requests that she give her a pram as a gift. She inquires as to whether they contacted DJ, to which he responds that they did not because they are in financial distress. He hangs up the phone. Meera is concerned about how they will handle the event.
Pritam walks inside a room, holding a box. He looks for something and then asks where he hid the note. Dadi inquires as to what he is looking for under the bed. She inquires as to whether he performs repairs. He claims that he does. She hands him a torch and a mosquito bat and instructs him on how to fix it. Pritam has stated that he will do so after Amrita's baby shower.
Nimmi inquires as to what she was saying to Saroj. Saroj claims she is enraged with him because she has yet to view the items she brought. She instructs Kamli to deliver it. Pritam looks for the bag but hides when he sees Kamli. They receive the bag from Kamlie.
When Amrita appears in front of Pritam, he is still looking for the bag. She inquires as to what he is doing in the kitchen, and he responds with the same question. Amrita claims that she has come to dry her clothing. They engage in some amusing banter. Pritam tries to open the cup board after Amrita informed him that there is mirchi light inside.
A box of Aata lands on him unexpectedly. Nimmi, who was ready to open it, is startled by the noise. Everyone runs into the kitchen and bursts out laughing when they discover Pritam covered in flour. They are still laughing. Dadi makes an attempt to take a selfie with him. Amrita is asked to quit giggling by Saroj.
Pritam claims she only knows how to laugh. Someone recorded his video as he walked to his room. Kabir notices him, but he flees before he can apprehend him. The man contacts the police and claims to know where the murderer is hiding. Pritam will be apprehended, police swear.
Pritam claims that he had to put in a lot of effort for just one bag. There are Angad and Kabir. They both say they want to unwind by drinking alcohol. He refutes the allegations. They insist, so he sits down with them and drinks with them.
The three of them begin to imbibe. Gurmeet arrives, and Angad and Kabir remain hidden till Gurmeet arrives. He inquires as to why he is drinking in this location. He tries to get up and says he was going to walk inside. Gurmeet, on the other hand, requests that he take a seat. He sits next to him, his feet resting on a beer bottle. He inquires as to what this is and why there are three bottles. He speaks for himself. Gurmeet admits that he is exhausted as well, and he begins to drink with him.
Gurmeet hears his father calling and flees, pleading with him not to tell anyone. Kabir and Angad are seen by Gurmeet. They get into a witty debate. When he arrives, he notices Pritam drinking and teases him. Pritam takes everyone's names, which Gurmeet promises he will strike.
He inquires as to who the other two are. Kabir and Angad join hands before he says he brought for him. He claims that he will drink with him. They ask him to put a stop to it. He claims that he is unemployed and that he also consumes alcoholic beverages. He downs the entire bottle in one sitting, stunning Pritam.
The ladies are preparing for the Baby Shower. Sony requests that she apply mehendi to Amrita, but Saroj stops her and asks how she can do so. She inquires as to whether she has forgotten about Karan. Amrita's screen goes black.
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President Obama's fiscal year 2014 budget proposal is a mixed bag for students. The most important good news is that it fully funds Pell Grants, which help more than nine million students attend and complete college and reduce how much they need to borrow. Expanding access to work-study jobs and making the American Opportunity Tax Credit permanent will also help limit students' need to borrow. The budget improves Income-Based Repayment (IBR) for all federal loan borrowers and prevents the taxation of debt forgiven through IBR and similar repayment plans. The budget also lowers the interest rates on federal loans for all students and parents who borrow this fall. Unfortunately, the proposal pays for some of this good news by letting interest rates rise steeply for students and families who borrow in the coming years.


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package com.maschel.roomba.song;

/**
 * Class used for describing Roomba Songs
 * A RoombaSongNote consists of a note (RoombaNote) and a
 * duration (RoombaNoteDuration)
 */
public class RoombaSongNote {

    private RoombaNote note;
    private RoombaNoteDuration duration;

    public RoombaSongNote(RoombaNote note, RoombaNoteDuration duration) {
        this.note = note;
        this.duration = duration;
    }

    public RoombaNote getNote() {
        return note;
    }

    public RoombaNoteDuration getDuration() {
        return duration;
    }

    /**
     * Get the byte array of give RoombaSongNote array.
     * @param notes RoombaSongNote array
     * @param tempo Tempo in BPM at which notes should be played
     * @return byte[] of notes and note duration.
     */
    public static byte[] songNotesToBytes(RoombaSongNote[] notes, int tempo) {
        byte[] bytes = new byte[notes.length * 2];
        int cursor = 0;
        for (RoombaSongNote note: notes) {
            bytes[cursor++] = note.note.getNoteByte();
            bytes[cursor++] = note.duration.getDurationByte(tempo);
        }
        return bytes;
    }
}

redpajama_set_name: RedPajamaGithub