National Couples' Health and Time Study: Sample, Design, and Weighting.

The National Couples' Health and Time Study (NCHAT) is the first fully powered, population-representative study of couples in America containing large samples of sexual, gender, and racial and ethnic diverse individuals. Drawn from the Gallup Panel and the Gallup Recontact Sample, when weighted, the data are population representative of individuals in the United States who (1) are married or cohabiting, (2) are between 20 and 60, (3) speak English or Spanish, and (4) have internet access. The data were collected between September 2020 and April 2021 in the midst of a global pandemic as well as racial and political upheaval. NCHAT includes surveys of 3,642 main respondents and 1,515 partners along with time diaries. We describe the sampling process, challenges weighting a diverse population-representative samples, and sociodemographic characteristics of the NCHAT study. These data will provide opportunities for new research on the health and well-being of American families.

Lack of polyamines leads to cotranslational degradation of the general stress factor RpoS in Escherichia coli.

The specialized sigma factor RpoS mediates a general stress response in Escherichia coli and related bacteria, activating promoters that allow cells to survive stationary phase and many stresses. RpoS synthesis and stability are regulated at multiple levels. Translation of RpoS is positively regulated by multiple small RNAs in response to stress. Degradation of RpoS, dependent upon the adaptor protein RssB, is rapid during exponential growth and ceases upon starvation or other stresses, increasing accumulation of RpoS. E. coli carrying mutations that block the synthesis of polyamines were previously found to have low levels of RpoS, while levels increased rapidly when polyamines were added. We have used a series of reporters to examine the basis for the lack of RpoS in polyamine-deficient cells. The polyamine requirement was independent of small RNA-mediated positive regulation of RpoS translation. Mutations in rssB stabilize RpoS and significantly bypassed the polyamine deficit, suggesting that lack of polyamines might lead to rapid RpoS degradation. However, rates of degradation of mature RpoS were unaffected by polyamine availability. Codon optimization in rpoS partially relieved the polyamine dependence, suggesting a defect in RpoS translation in the absence of polyamines. Consistent with this, a hyperproofreading allele of ribosomal protein S12, encoded by rpsL, showed a decrease in RpoS levels, and this decrease was also suppressed by either codon optimization or blocking RpoS degradation. We suggest that rpoS codon usage leads it to be particularly sensitive to slowed translation, due to either lack of polyamines or hyperproofreading, leading to cotranslational degradation. We dedicate this study to Herb Tabor and his foundational work on polyamines, including the basis for this study.

Seroprevalence of SARS-CoV-2-Specific IgG Antibodies Among Adults Living in Connecticut: Post-Infection Prevalence (PIP) Study.

BACKGROUND: A seroprevalence study can estimate the percentage of people with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in the general population; however, most existing reports have used a convenience sample, which may bias their estimates. METHODS: We sought a representative sample of Connecticut residents, ages ≥18 years and residing in noncongregate settings, who completed a survey between June 4 and June 23, 2020, and underwent serology testing for SARS-CoV-2-specific immunoglobulin G (IgG) antibodies between June 10 and July 29, 2020. We also oversampled non-Hispanic black and Hispanic subpopulations. We estimated the seroprevalence of SARS-CoV-2-specific IgG antibodies and the prevalence of symptomatic illness and self-reported adherence to risk-mitigation behaviors among this population. RESULTS: Of the 567 respondents (mean age 50 [± 17] years; 53% women; 75% non-Hispanic white individuals) included at the state level, 23 respondents tested positive for SARS-CoV-2-specific antibodies, resulting in weighted seroprevalence of 4.0 (90% confidence interval [CI] 2.0-6.0). The weighted seroprevalence for the oversampled non-Hispanic black and Hispanic populations was 6.4% (90% CI 0.9-11.9) and 19.9% (90% CI 13.2-26.6), respectively. The majority of respondents at the state level reported following risk-mitigation behaviors: 73% avoided public places, 75% avoided gatherings of families or friends, and 97% wore a facemask, at least part of the time. CONCLUSIONS: These estimates indicate that the vast majority of people in Connecticut lack antibodies against SARS-CoV-2, and there is variation by race and ethnicity. There is a need for continued adherence to risk-mitigation behaviors among Connecticut residents to prevent resurgence of COVID-19 in this region.

Absolute requirement for polyamines for growth of Escherichia coli mutants (mnmE/G) defective in modification of the wobble anticodon of transfer-RNA.

The genes mnmE and mnmG are responsible for the modification of uridine 34, 'the wobble position' of many aminoacyl-tRNAs. Deletion of these genes affects the strength of the codon-anticodon interactions of the aminoacyl-tRNAs with the mRNAs and the ribosomes. However, deletion of these genes does not usually have a significant effect on the growth rate of the standard Escherichia coli strains. In contrast, we have found that if the host E. coli strain is deficient in the synthesis of polyamines, deletion of the mnmE or mnmG gene results in complete inhibition of growth unless the medium contains polyamines. The finding of an absolute requirement for polyamines in our current work will be significant in studies on polyamine function, in studies on the function of the mnmE/G genes, and in studies on the role of aminoacyl-tRNAs in protein biosynthesis.

Polyamines Stimulate the Level of the σ38 Subunit (RpoS) of Escherichia coli RNA Polymerase, Resulting in the Induction of the Glutamate Decarboxylase-dependent Acid Response System via the gadE Regulon.

To study the physiological roles of polyamines, we carried out a global microarray analysis on the effect of adding polyamines to an Escherichia coli mutant that lacks polyamines because of deletions in the genes in the polyamine biosynthetic pathway. Previously, we have reported that the earliest response to polyamine addition is the increased expression of the genes for the glutamate-dependent acid resistance system (GDAR). We also presented preliminary evidence for the involvement of rpoS and gadE regulators. In the current study, further confirmation of the regulatory roles of rpoS and gadE is shown by a comparison of genome-wide expression profiling data from a series of microarrays comparing the genes induced by polyamine addition to polyamine-free rpoS(+)/gadE(+) cells with genes induced by polyamine addition to polyamine-free ΔrpoS/gadE(+) and rpoS(+)/ΔgadE cells. The results indicate that most of the genes in the E. coli GDAR system that are induced by polyamines require rpoS and gadE. Our data also show that gadE is the main regulator of GDAR and other acid fitness island genes. Both polyamines and rpoS are necessary for the expression of gadE gene from the three promoters of gadE (P1, P2, and P3). The most important effect of polyamine addition is the very rapid increase in the level of RpoS sigma factor. Our current hypothesis is that polyamines increase the level of RpoS protein and that this increased RpoS level is responsible for the stimulation of gadE expression, which in turn induces the GDAR system in E. coli.

Polyamines are critical for the induction of the glutamate decarboxylase-dependent acid resistance system in Escherichia coli.

As part of our studies on the biological functions of polyamines, we have used a mutant of Escherichia coli that lacks all the genes for polyamine biosynthesis for a global transcriptional analysis on the effect of added polyamines. The most striking early response to the polyamine addition is the increased expression of the genes for the glutamate-dependent acid resistance system (GDAR) that is important for the survival of the bacteria when passing through the acid environment of the stomach. Not only were the two genes for glutamate decarboxylases (gadA and gadB) and the gene for glutamate-γ-aminobutyrate antiporter (gadC) induced by the polyamine addition, but the various genes involved in the regulation of this system were also induced. We confirmed the importance of polyamines for the induction of the GDAR system by direct measurement of glutamate decarboxylase activity and acid survival. The effect of deletions of the regulatory genes on the GDAR system and the effects of overproduction of two of these genes were also studied. Strikingly, overproduction of the alternative σ factor rpoS and of the regulatory gene gadE resulted in very high levels of glutamate decarboxylase and almost complete protection against acid stress even in the absence of any polyamines. Thus, these data show that a major function of polyamines in E. coli is protection against acid stress by increasing the synthesis of glutamate decarboxylase, presumably by increasing the levels of the rpoS and gadE regulators.

Escherichia coli glutathionylspermidine synthetase/amidase: phylogeny and effect on regulation of gene expression.

Glutathionylspermidine synthetase/amidase (Gss) and the encoding gene (gss) have only been studied in Escherichia coli and several members of the Kinetoplastida phyla. In the present article, we have studied the phylogenetic distribution of Gss and have found that Gss sequences are largely limited to certain bacteria and Kinetoplastids and are absent in a variety of invertebrate and vertebrate species, Archea, plants, and some Eubacteria. It is striking that almost all of the 75 Enterobacteria species that have been sequenced contain sequences with very high degree of homology to the E. coli Gss protein. To find out the physiological significance of glutathionylspermidine in E. coli, we have performed global transcriptome analyses. The microarray studies comparing gss(+) and Δgss strains of E. coli show that a large number of genes are either up-regulated (76 genes more than threefold) or down-regulated (35 genes more than threefold) by the loss of the gss gene. Most significant categories of up-regulated genes include sulfur utilization, glutamine and succinate metabolism, polyamine and arginine metabolism, and purine and pyrimidine metabolism.

Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex.

Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC:AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC:AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% ß-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (K(A)) between yeast AZ and yeast ODC is 6×10(7) (M(-1)). Using purified His-tagged AZ as a binding partner, we have purified the ODC:AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC:AZ complex shows a change in the secondary structure in the complex.

Microarray studies on the genes responsive to the addition of spermidine or spermine to a Saccharomyces cerevisiae spermidine synthase mutant.

The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well-established in vivo. In this work we have performed microarray experiments with a spermidine synthase, spermine oxidase mutant (Deltaspe3 Deltafms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine or spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10(-5) M) or spermine (10(-5) M) compared to a control culture containing 10(-8) M spermidine. 247 genes were upregulated > two-fold and 11 genes were upregulated >10-fold after spermidine addition. Functional categorization of the genes showed induction of transport-related genes and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were downregulated more than two-fold, and six genes were downregulated > eight-fold after spermidine addition. A majority of the downregulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only a few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine.

Polyamines are not required for aerobic growth of Escherichia coli: preparation of a strain with deletions in all of the genes for polyamine biosynthesis.

A strain of Escherichia coli was constructed in which all of the genes involved in polyamine biosynthesis--speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), spe D (adenosylmethionine decarboxylase), speE (spermidine synthase), speF (inducible ornithine decarboxylase), cadA (lysine decarboxylase), and ldcC (lysine decarboxylase)--had been deleted. Despite the complete absence of all of the polyamines, the strain grew indefinitely in air in amine-free medium, albeit at a slightly (ca. 40 to 50%) reduced growth rate. Even though this strain grew well in the absence of the amines in air, it was still sensitive to oxygen stress in the absence of added spermidine. In contrast to the ability to grow in air in the absence of polyamines, this strain, surprisingly, showed a requirement for polyamines for growth under strictly anaerobic conditions.

Hypusine modification for growth is the major function of spermidine in Saccharomyces cerevisiae polyamine auxotrophs grown in limiting spermidine.

Spermidine and its derivative, hypusinated eIF5A, are essential for the growth of Saccharomyces cerevisiae. Very low concentrations of spermidine (10(-8) M) are sufficient for the growth of S. cerevisiae polyamine auxotrophs (spe1Delta, spe2Delta, and spe3Delta). Under these conditions, even though the growth rate is near normal, the internal concentration of spermidine is <0.2% of the spermidine concentration present in wild-type cells. When spe2Delta cells are grown with low concentrations of spermidine, there is a large decrease in the amount of hypusinated eukaryotic initiation factor 5A (eIF5A) (1/20 of normal), even though there is no change in the amount of total (modified plus unmodified) eIF5A. It is striking that, as intracellular spermidine becomes limiting, an increasing portion of it (up to 54%) is used for the hypusine modification of eIF5A. These data indicate that hypusine modification of eIF5A is a most important function for spermidine in supporting the growth of S. cerevisiae polyamine auxotrophs.

Polyamine deficiency leads to accumulation of reactive oxygen species in a spe2Delta mutant of Saccharomyces cerevisiae.

We have previously shown that polyamine-deficient Saccharomyces cerevisiae are very sensitive to incubation in oxygen. The current studies show that, even under more physiological conditions (i.e. growth in air), polyamine-deficient cells accumulate reactive oxygen species (ROS). These cells develop an apoptotic phenotype and, after incubation in polyamine-deficient medium, die. To show a specific effect of polyamines on ROS accumulation, uncomplicated by any effects on growth, spermine was added to spermidine-deficient spe2Delta fms1Delta cells, since spermine does not affect the growth of this strain. In this strain, spermine addition caused a marked, but not complete, decrease in the accumulation of ROS and a moderate protection against cell death. In other experiments with polyamine-deficient cells containing plasmids that overexpress superoxide dismutases (SOD1, SOD2), ROS decreased but with only a partial protection against cell death. Polyamine-deficient cells incubated anaerobically show markedly less cell death. These data show that part of the function of polyamines is protection of the cells from accumulation of ROS.

Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1delta mutant.

As part of our studies on polyamine biosynthesis in yeast, the metabolism of methylthioadenosine was studied in a mutant that lacks methylthioadenosine phosphorylase (meu1delta). The nucleoside accumulates in this mutant and is mainly excreted into the culture medium. Intracellular accumulation of the nucleoside is enough to account for the inhibition of spermidine synthase and thus to indirectly regulate the polyamine content of the meu1delta cells. By comparing the results with this mutant with a meu1delta spe2delta mutant that cannot synthesize spermidine or spermine, we showed that >98% of methylthioadenosine is produced as a byproduct of polyamine synthesis (i.e., from decarboxylated S-adenosylmethionine). In contrast, in MEU1+ SPE2+ cells methylthioadenosine does not accumulate and is metabolized through the methionine salvage pathway. Using a met15delta mutant we show that this pathway (i.e., involving polyamine biosynthesis and methylthioadenosine metabolism) is a significant factor in the metabolism of methionine, accounting for 15% of the added methionine.

Studies on the regulation of ornithine decarboxylase in yeast: effect of deletion in the MEU1 gene.

Methylthioadenosine is formed during the biosynthesis of spermidine and of spermine and is metabolized by methylthioadenosine phosphorylase, an enzyme missing in several tumor cell lines. In Saccharomyces cerevisiae, this enzyme is coded by the MEU1 gene. We have now studied the effect of the meu1 deletion on polyamine metabolism in yeast. We found that the effects of the meu1Delta mutation mostly depend on the stage of cell growth. As the cell density increases, there is a marked fall in the level of ornithine decarboxylase (ODC) in the MEU1(+) cells, which we show is caused by an antizyme-requiring degradation system. In contrast, there is only a small decrease in the ODC level in the meu1Delta cells. The meu1Delta cells have a higher putrescine and a lower spermidine level than MEU1(+) cells, suggesting that the decreased spermidine level in the meu1Delta cultures is responsible for the greater apparent stability of ODC in the meu1Delta cells. The lower spermidine level in the meu1Delta cells probably results from an inhibition of spermidine synthase by the methylthioadenosine that presumably accumulates in these mutants. In both MEU1(+) and the meu1Delta cultures, the ODC levels were markedly decreased by the addition of spermidine to the media, and thus our results contradict the postulation of Subhi et al. [Subhi, A. L., et al. (2003) J. Biol. Chem. 278, 49868-49873] of a novel regulatory pathway in meu1Delta cells in which ODC is not responsive to spermidine.

Spermidine but not spermine is essential for hypusine biosynthesis and growth in Saccharomyces cerevisiae: spermine is converted to spermidine in vivo by the FMS1-amine oxidase.

In our earlier work we showed that either spermidine or spermine could support the growth of spe2Delta or spe3Delta polyamine-requiring mutants, but it was unclear whether the cells had a specific requirement for either of these amines. In the current work, we demonstrate that spermidine is specifically required for the growth of Saccharomyces cerevisiae. We were able to show this specificity by using a spe3Delta fms1Delta mutant that lacked both spermidine synthase and the FMS1-encoded amine oxidase that oxidizes spermine to spermidine. The polyamine requirement for the growth of this double mutant could only be satisfied by spermidine; i.e., spermine was not effective because it cannot be oxidized to spermidine in the absence of the FMS1 gene. We also showed that at least one of the reasons for the absolute requirement for spermidine for growth is the specificity of its function as a necessary substrate for the hypusine modification of eIF5A. Spermine itself cannot be used for the hypusine modification, unless it is oxidized to spermidine by the Fms1 amine oxidase. We have quantified the conversion of spermine in vivo and have shown that this conversion is markedly increased in a strain overexpressing the Fms1 protein. We have also shown this conversion in enzymatic studies by using the purified amine oxidase from yeast.

Polyamines protect Escherichia coli cells from the toxic effect of oxygen.

Wild-type Escherichia coli cells grow normally in 95% O(2)/5% CO(2). In contrast, cells that cannot make polyamines because of mutations in the biosynthetic pathway are rapidly killed by incubation in 95% O(2)/5% CO(2). Addition of polyamines prevents the toxic effect of oxygen, permitting cell survival and optimal growth. Oxygen toxicity can also be prevented if the growth medium contains an amino acid mixture or if the polyamine-deficient cells contain a manganese-superoxide dismutase (Mn-SOD) plasmid. Partial protection is afforded by the addition of 0.4 M sucrose or 0.4 M sorbitol to the growth medium. We also report that concentrations of H(2)O(2) that are nontoxic to wild-type cells or to mutant cells pretreated with polyamines kill polyamine-deficient cells. These results show that polyamines are important in protecting cells from the toxic effects of oxygen.

Absolute requirement of spermidine for growth and cell cycle progression of fission yeast (Schizosaccharomyces pombe).

Schizosaccharomyces pombe cells that cannot synthesize spermidine or spermine because of a deletion-insertion in the gene coding for S-adenosylmethionine decarboxylase (Deltaspe2) have an absolute requirement for spermidine for growth. Flow cytometry studies show that in the absence of spermidine an overall delay of the cell cycle progression occurs with some accumulation of cells in the G(1) phase; as little as 10(-6) M spermidine is sufficient to maintain normal cell cycle distribution and normal growth. Morphologically some of the spermidine-deprived cells become spherical at an early stage with little evidence of cell division. On further incubation in the spermidine-deprived medium, growth occurs in most of the cells, not by cell division but rather by cell elongation, with an abnormal distribution of the actin cytoskeleton, DNA (4', 6-diamidino-2-phenylindole staining), and calcofluor-staining moieties. More prolonged incubation in the spermidine-deficient medium leads to profound morphological changes including nuclear degeneration.

Veteran identity and race/ethnicity: influences on VA outpatient care utilization.

BACKGROUND: "Veteran identity" is defined as veterans' self-concept that derives from his/her military experience within a sociohistorical context. Veteran identity may vary by race/ethnicity because the sociohistorical context of the military experience varies by race. OBJECTIVES: To explore veteran identity and how it varies by race/ethnicity, and to identify aspects of veteran identity that significantly influence preferences for, and use of, VA outpatient care. RESEARCH DESIGN: Focus groups were conducted at community sites to explore concepts related to veteran identity, race/ethnicity, military experience, and health services use. The focus groups informed the development of a telephone survey, which was administered to veterans of four racial/ethnic groups in Southern California and Southern Nevada. SUBJECTS: One hundred seventy-eight veterans participated in the focus groups, and 3,227 veterans completed the telephone survey. MEASURES: Dependent variables include: (1) preference for VA health services, (2) VA-only outpatient use, (3) Any VA outpatient use, and (4) number of outpatient visits within the previous 12 months. Independent variables include veteran identity, sociodemographic, and health-related characteristics. RESULTS: All veteran identity variables were significantly associated with race/ethnicity. Race/ethnicity, eg, being black or Hispanic, in addition to veteran identity factors, significantly influenced preferences for VA outpatient care. Veteran identity factors, however, had less influence on VA outpatient service utilization than socioeconomic factors. CONCLUSIONS: Minority veterans who highly identify with their veteran status may prefer the VA to other systems of care. Factors associated with veteran identity may be useful for incorporation into interventions to improve access to VA care.