Primary Cell Culture as a Model System for Evolutionary Molecular Physiology.

Primary cell culture is a powerful model system to address fundamental questions about organismal physiology at the cellular level, especially for species that are difficult, or impossible, to study under natural or semi-natural conditions. Due to their ease of use, primary fibroblast cultures are the dominant model system, but studies using both somatic and germ cells are also common. Using these models, genome evolution and phylogenetic relationships, the molecular and biochemical basis of differential longevities among species, and the physiological consequences of life history evolution have been studied in depth. With the advent of new technologies such as gene editing and the generation of induced pluripotent stem cells (iPSC), the field of molecular evolutionary physiology will continue to expand using both descriptive and experimental approaches.

Exploring the role of primary fibroblast cells in comparative physiology: a historical and contemporary overview.

With the advent of tissue culture, and eventually the in vitro growth and maintenance of individual cell types, it became possible to ask mechanistic questions about whole organism physiology that are impractical to address within a captive setting or within the whole organism. The earliest studies focused on understanding the wound-healing response while refining cell growth and maintenance protocols from various species. In addition to its extensive use in biomedical research, this approach has been co-opted by comparative physiologists interested in reductionist/mechanistic questions related to how cellular physiology can help explain whole organism function. Here, we provide a historical perspective on the emergence of primary cell culture with an emphasis on fibroblasts followed by an overview of applying this method to ask questions about the role of life-history evolution in shaping organismal physiology at the cellular level, as well as the effect of exogenous factors (i.e., temperature, and oxygen availability) on cellular function. Finally, we propose future uses for primary fibroblasts to address questions in conservation biology and comparative physiology.

Testosterone, cortisol, and criminal behavior in men and women.

Only two studies to date have considered the joint effects of testosterone and cortisol on direct measures of criminal behavior. The current study extends this earlier work by incorporating the direct and interactive effects of baseline hormone measures and hormone change scores in response to social stress. The current study also extends prior work by considering distinct measures of different criminal behavior types and sex differences. Analyses based on a large sample of undergraduates indicated that testosterone had a positive and statistically significant association with impulsive and violent criminal behavior. The interaction of testosterone with cortisol had a negative association with income generating crime. Simple slopes analyses of this interaction indicated testosterone had a positive association with income generating crime when cortisol was low (-1 SD). Associations between hormones and criminal behavior were not moderated by sex.

The resistance of domestic canine skin-derived fibroblasts to oxidative and non-oxidative chemical injury: implications of breed and body size.

Small-breed dogs live significantly longer lives than large-breed dogs, while having higher mass-specific metabolic rates and faster growth rates. Underlying this observed physiological difference across domestic dogs, there must also be differences at other levels of organization that could lead to elucidating what accounts for the disparity in aging rates and life span within this species. At the cellular level, a clear mechanism underlying whole animal traits has not been fully elucidated. Here, we cultured dermal fibroblasts from large and small breed dogs from both young and old age categories and examined the degree of resistance to multiple sources of cytotoxic stress. This included heat (42 °C), paraquat, cadmium, and hydrogen peroxide for increasing amounts of time (heat) or increasing concentrations (chemical stressors). We hypothesized that small breed dogs, with longer lifespans, would have greater cellular resistance to stress compared with large breed dogs. Final sample sizes include small puppies (N = 18), large puppy (N = 32), small old (N = 11), and large old (N = 23) dogs. Using a 2 (donor size) by 2 (donor age) between-subjects multivariate analysis of variance, we found that the values for the dose that killed 50% of the cells (LD50) were not significantly different based on donor size (p = 0.45) or donor age (p = 0.20). The interaction was also not significant (p = 0.47). Interestingly, we did find that the degree of resistance to cadmium toxicity was significantly correlated with the degree of resistance to both heat and hydrogen peroxide, but not paraquat (p < 0.01 for both). These data suggest that cellular stress resistance does not differ among domestic dogs as a function of size or age, pointing to other cellular pathways as the mechanistic basis for the observed differences in lifespan.

Testosterone, cortisol, and psychopathy: Further evidence with the Levenson self-report psychopathy scale and the inventory of callous unemotional traits.

The current study explored associations between testosterone, cortisol, and both the Levenson Self-Report Psychopathy Scale (LSRPS) and the Inventory of Callous Unemotional (ICU) traits. Data were gathered from a relatively large sample of university students (n = 522) and analyses considered direct and interactive associations between hormones and psychopathic traits, as well as interactions between these associations and the time of day at which samples were gathered and the sex of participants. Baseline cortisol had a negative association with LSRPS primary psychopathy scores. In addition, baseline cortisol interacted with the time of day in association with LSRPS total scores. Simple slopes analyses indicated cortisol had a negative association with LSRPS total scores in the morning but not the afternoon. Interactions among hormone measures were not statistically significant. There was also no evidence for the moderation of associations between hormones and psychopathic traits by sex.

Linkages between the life-history evolution of tropical and temperate birds and the resistance of cultured skin fibroblasts to oxidative and non-oxidative chemical injury.

A fundamental challenge facing physiological ecologists is to understand how variation in life history at the whole-organism level might be linked to cellular function. Thus, because tropical birds have higher annual survival and lower rates of metabolism, we hypothesized that cells from tropical species would have greater cellular resistance to chemical injury than cells from temperate species. We cultured dermal fibroblasts from 26 tropical and 26 temperate species of birds and examined cellular resistance to cadmium, H(2)O(2), paraquat, thapsigargin, tunicamycium, methane methylsulfonate (MMS) and UV light. Using ANCOVA, we found that the values for the dose that killed 50% of cells (LD(50)) from tropical birds were significantly higher for H(2)O(2) and MMS. When we tested for significance using a generalized least squares approach accounting for phylogenetic relationships among species to model LD(50), we found that cells from tropical birds had greater tolerance for Cd, H(2)O(2), paraquat, tunicamycin and MMS than cells from temperate birds. In contrast, tropical birds showed either lower or no difference in tolerance to thapsigargin and UV light in comparison with temperate birds. These findings are consistent with the idea that natural selection has uniquely fashioned cells of long-lived tropical bird species to be more resistant to forms of oxidative and non-oxidative stress than cells from shorter-lived temperate species.

Heightened induction of proapoptotic signals in response to endoplasmic reticulum stress in primary fibroblasts from a mouse model of longevity.

Previous work from our laboratory has shown that primary fibroblasts from long-lived Snell dwarf mice display a higher sensitivity to the lethal effects of endoplasmic reticulum (ER) stressors, such as thapsigargin, than cells from normal mice. Here we show that thapsigargin induces higher expression of CHOP, enhanced cleavage of caspase-12, higher caspase-3 activity, and increased phosphorylation of c-JUN, all indicators of enhanced apoptosis, in dwarf fibroblasts. Dwarf and normal fibroblasts show no genotypic difference in up-regulating BiP, GRP94, and ERp72 proteins after exposure to thapsigargin. However, dwarf fibroblasts express lower basal levels of a number of putative XBP1 target genes including Armet, Edem1, Erdj3, p58(IPK) and Sec61a1, as well as Ire1α itself. Furthermore, when exposed to thapsigargin, dwarf fibroblasts display attenuated splicing of Xbp1, but similar phosphorylation of eIF2α, in comparison to normal fibroblasts. These data support the notion that IRE1/XBP1 signaling is set at a lower level in dwarf fibroblasts. Diminished Xbp1 splicing in dwarf-derived fibroblasts may tilt the balance between prosurvival and proapoptotic signals in favor of apoptosis, thereby leading to higher induction of proapoptotic signals in these cells and ultimately their increased sensitivity to ER stressors. These results, together with recent findings in Caenorhabditis elegans daf-2 mutants, point to a potential interplay between insulin/IGF-1 signals and unfolded protein response signaling.

Walking the walk: the moderating role of proactive parenting on adolescents' value-congruent behaviors.

The current study longitudinally examined adolescents' internalization of values in four domains (drug use, prosocial, school, friends), and how these values were differentially related to behaviors (delinquency, prosocial behavior, school engagement, and deviant peer association) as a function of proactive parenting. Participants included 335 adolescents (M age of child at Time 2 = 12.28, SD = .99, 51% female) and their parents. Analyses examined associations between adolescents' values at Time 3 and corresponding behaviors at Time 4, and the moderating role of proactive parenting (assessed at Time 2 using cluster analysis). Results suggested that adolescents with the most consistent value-congruent behavior had parents who used deference or reasoned deference as an approach to proactive parenting, highlighting the importance of autonomy supportive parenting in the encouragement of self-generated values and corresponding behaviors. Discussion focused on value-congruent behaviors and associations between values and behaviors across domains.

Uplifts, Respite, Stress, and Marital Quality for Parents Raising Children With Down Syndrome or Autism.

Direct, indirect, and partner effects estimated among uplifts, respite care, stress, and marital quality across mothers and fathers of children with autism spectrum disorder (n = 102) and Down syndrome (n = 111) were examined in this cross-sectional study. Parents of children with ASD who reported more uplifts and less stress individually reported better marital quality; these wives reported better marital quality as their husbands reported more uplifts and less stress. Wives with children with DS who reported more uplifts, individually along with their husbands reported less stress and better marital quality. Respite was directly associated with marital quality for parents of children with ASD and indirectly associated with marital quality for parents of children with DS with reduced individual stress. Implications are discussed.

Macrophage migration inhibitory factor-knockout mice are long lived and respond to caloric restriction.

Macrophage migration inhibitory factor (MIF) affects inflammation, glucose homeostasis, and cellular proliferation in mammals. Previously, we found that MIF was significantly elevated in multiple long-lived mouse models, including calorie restriction (CR), which led us to hypothesize that MIF might be important in the control of mammalian life span and be necessary for the life-extending effects of CR. To test this hypothesis, we examined the life span of mice with a targeted deletion of the Mif gene on a segregating B6 x 129/Sv background (MIF-KO) under ad libitum (AL) feeding and CR conditions. Control mice were generated by mating C57BL/6J females with 129/SvJ males to make an F1 hybrid, and crossing F1 males to F1 females to produce segregating F2 mice homozygous for the normal MIF allele. Not only did MIF-KO mice show a life span extension in response to CR, they were, unexpectedly, longer lived than controls under standard AL conditions. MIF-KO mice were significantly protected against lethal hemangiosarcoma, but more likely than controls to die of disseminated amyloid, an age-related inflammatory syndrome. Overall, these data refute the suggestion that MIF is required for the CR effect on life span, but raise the possibility that MIF may limit life span in normal mice.

Early life growth hormone treatment shortens longevity and decreases cellular stress resistance in long-lived mutant mice.

Hypopituitary Ames dwarf mice were injected either with growth hormone (GH) or thyroxine for a 6-wk period to see whether this intervention would reverse their long life span or the resistance of their cells to lethal stresses. Ames dwarf mice survived 987 ± 24 d (median), longer than nonmutant control mice (664 ± 48), but GH-injected dwarf mice did not differ from controls (707 ± 9). Fibroblast cells from Ames dwarf mice were more resistant to cadmium than cells from nonmutant controls (LD(50) values of 9.98 ± 1.7 and 3.9 ± 0.8, respectively), but GH injections into Ames dwarf mice restored the normal level of cadmium resistance (LD(50)=5.8 ± 0.9). Similar restoration of normal resistance was observed for fibroblasts exposed to paraquat, methyl methanesulfonate, and rotenone (P<0.05 in each case for contrast of GH-treated vs. untreated dwarf mice; P<0.05 for dwarf vs. nonmutant control mice.) T4 injections into Ames dwarf mice, in contrast, did not restore normal life span. We conclude that the remarkable life-span extension of Ames dwarf mice, and the stress resistance of cells from these mice, depends on low levels of GH exposure in juvenile and very young adult mice.

Fibroblasts from long-lived bird species are resistant to multiple forms of stress.

Evolutionary senescence theory postulates that aging results from the declining force of natural selection with increasing chronological age. A goal of comparative studies in the biology of aging is to identify genetic and biochemical mechanism(s) driving species-specific differences in the aging process that are the end product of life history trade-offs. We hypothesized that cells from long-lived bird species are more resistant to stress agents than are cells from short-lived species, and that cells from birds are more resistant to stress than are cells from relatively short-lived mammals of similar size. We tested primary fibroblast cultures from 35 species of free-living birds for their resistance to multiple forms of cellular stress and found that cell lines from longer-lived species were resistant to death caused by cadmium (R(2)=0.27, P=0.002), paraquat (R(2)=0.13, P=0.03), hydrogen peroxide (R(2)=0.09, P=0.07) and methyl methanesulfonate (R(2)=0.13, P=0.03), as well as to the metabolic inhibition seen in low-glucose medium (R(2)=0.37, P<0.01). They did not differ in their resistance to UV radiation, or to thapsigargin or tunicamycin, inducers of the unfolded protein response. These results were largely consistent even after accounting for the influence of body mass and phylogeny. Cell lines from longer-lived bird species also proliferate more rapidly than cells from short-lived birds, although there was no relationship between proliferation and stress resistance. Finally, avian fibroblasts were significantly more resistant than rodent fibroblasts to each of the tested stressors. These results support the idea that cellular resistance to injury may be an important contributor to the evolution of slow aging and long lifespan among bird species, and may contribute to the relatively long lifespan of birds compared with rodents of the same body size.

A comparison of attachment outcomes in enactment-based versus therapist-centered therapy process modalities in couple therapy.

Attachment issues are viewed by many therapists as lying at the heart of couple distress. It is critical to empirically validate therapy processes that facilitate couples in responding to each other's attachment needs. This study examined enactments as a therapy process and change mechanism to promote secure attachment in couple therapy. Sixteen couples were randomly assigned to 1 of 2 experimental groups--1 group received 3 therapist-centered sessions followed by 3 enactment-based sessions, and a second group received 3 enactment-based sessions followed by 3 therapist-centered sessions. To measure between-session and within-session change, each spouse completed presession and postsession measures of attachment security each week. Results showed that couples who received enactment-based sessions first reported greater increases in attachment security than those receiving therapist-centered sessions first. These same couples continued to show improvement after switching to the therapist-centered sessions. Conversely, couples who received therapist-centered sessions first did not increase attachment after switching to enactment-based sessions. For wives, enactment-based sessions produced the greatest improvement in attachment, yet both therapy process modalities led to some improvement. Conversely, for husbands, attachment improved only when they received enactment-based sessions first. Enactment-based sessions may therefore be more important for husbands than wives. Overall, with some qualification it appears that enactment-based therapy process may improve attachment more than a therapist-centered process. These observed trends and findings are generally consistent with previous research supporting use of enactments in couple therapy.

New Perspectives on Avian Models for Studies of Basic Aging Processes.

Avian models have the potential to elucidate basic cellular and molecular mechanisms underlying the slow aging rates and exceptional longevity typical of this group of vertebrates. To date, most studies of avian aging have focused on relatively few of the phenomena now thought to be intrinsic to the aging process, but primarily on responses to oxidative stress and telomere dynamics. But a variety of whole-animal and cell-based approaches to avian aging and stress resistance have been developed-especially the use of primary cell lines and isolated erythrocytes-which permit other processes to be investigated. In this review, we highlight newer studies using these approaches. We also discuss recent research on age-related changes in neural function in birds in the context of sensory changes relevant to homing and navigation, as well as the maintenance of song. More recently, with the advent of "-omic" methodologies, including whole-genome studies, new approaches have gained momentum for investigating the mechanistic basis of aging in birds. Overall, current research suggests that birds exhibit an enhanced resistance to the detrimental effects of oxidative damage and maintain higher than expected levels of cellular function as they age. There is also evidence that genetic signatures associated with cellular defenses, as well as metabolic and immune function, are enhanced in birds but data are still lacking relative to that available from more conventional model organisms. We are optimistic that continued development of avian models in geroscience, especially under controlled laboratory conditions, will provide novel insights into the exceptional longevity of this animal taxon.

Relational aggression in marriage.

Drawing from developmental theories of relational aggression, this article reports on a study designed to identify if spouses use relationally aggressive tactics when dealing with conflict in their marriage and the association of these behaviors with marital outcomes. Using a sample of 336 married couples (672 spouses), results revealed that the majority of couples reported that relationally aggressive behaviors, such as social sabotage and love withdrawal, were a part of their marital dynamics, at least to some degree. Gender comparisons of partner reports of their spouse's behavior revealed that wives were significantly more likely to be relationally aggressive than husbands. Structural equation modeling demonstrated that relational aggression is associated with lower levels of marital quality and greater marital instability for both husbands and wives. Implications are drawn for the use of relational aggression theory in the future study of couple conflict and marital aggression.

Depression and communication processes in later life marriages.

OBJECTIVE: About six hundred and fourteen elderly people married to each other, average ages 66 and 63 respectively, in long term, mature marriages, lasting on the average 36 years, completed the Marital Satisfaction Inventory, Revised-MSIr (Snyder, D.K. 1999) and the short version of the Center for Epidemiological Studies Depression Scale (Kohout, F.J., Berkman, L.F., Evans, D.A., & Cornoni-Huntley, J. (1993). The purpose of this study was to determine whether depression in one or both spouses and poor affective and problem solving communication occur together. METHODS: Husbands and wives were divided into nine groups based on their levels of depression (hlow/wlow, hmed/wmed, hhigh/whigh, hhigh/wlow, hhigh/wmed, wlow/hhigh, and wlow/hmed, hmed/wlow, and hmed/whigh). Analysis of Variance was used to examine the difference in couple affective communication and problem solving scores from the MSIr (1999). RESULTS: The findings indicated that when husbands or wives are more depressed, both affective communication and problem solving processes are impaired for the couple. When both are depressed, affective communication and problem solving are worse than when only one is depressed, and both husband and wife communication scores are worse when one or both partners is depressed than when neither husband nor wife is depressed. CONCLUSION: While these findings do not point to cause, implications for providing mental health services (including marital therapy) or couple based education groups as supports to the depressed elderly and their spouses are recommended.

The impact of marital conflict on health and health care utilization in older couples.

This study tested the hypothesis that marital distress has a significant effect on health care utilization for older couples. Results from 536 intact couples in long term marriages showed men in dissatisfying marriages are more likely to utilize health care services; women in satisfying marriages are more likely to use health care services. There were no significant partner effects of marital quality on health problems or health care utilization. The association between husbands' marital quality and wives' health care utilization approached significance. Implications for future research and clinical practice, including gender differences, are discussed.

Facilitated disclosure versus clinical accommodation of infidelity secrets: an early pivot point in couple therapy. Part 1: couple relationship ethics, pragmatics, and attachment.

A critical and potentially polarizing decision in treating infidelity is whether facilitating partner disclosure or accommodating nondisclosure is most beneficial following private disclosure of infidelity to the therapist. Given couple distress and volatility following disclosure, understandably some therapists judge accommodating an infidelity secret both efficient and compassionate. Employing Western ethics and an attachment/intimacy lens, we consider ethical, pragmatic, and attachment intimacy implications of accommodating infidelity secrets. Issues bearing on the decision to facilitate disclosure or accommodate nondisclosure include (a) relationship ethics and pragmatics; (b) attachment and intimacy consequences; and (c) prospects for healing. We conclude that facilitating voluntary disclosure of infidelity, although difficult and demanding, represents the most ethical action with the best prospects for renewed and vital attachment intimacy.

Coping strategies used by hypersexual patients to defend against the painful effects of shame.

This article reports the findings of a study investigating coping strategies used by hypersexual patients (n = 71), compared with a control group (n = 73), in their attempts to defend against shame. Coping strategies were measured using the Compass of Shame Scale (CoSS) and hypersexual behaviour was measured by the Hypersexual Behavior Inventory (HBI). A multivariate analysis of variance of between-group differences was significant, and examination of post hoc univariate tests revealed that the sample of hypersexual patients defended against shame with higher levels of withdrawal and higher tendencies to attack self and others when compared with the control group. The effect sizes of these differences were moderate to large. A categorical analysis of the patient group indicated that the greatest percentages of elevated shame scores were clustered on the Withdrawal and Attack Self subscales of the CoSS. Between-group differences on the Avoidance subscale of the CoSS were not significant. The results of this study are discussed as they pertain to clinical practice, and future recommendations for research are offered.

Skin-derived fibroblasts from long-lived species are resistant to some, but not all, lethal stresses and to the mitochondrial inhibitor rotenone.

Fibroblast cell lines were developed from skin biopsies of eight species of wild-trapped rodents, one species of bat, and a group of genetically heterogeneous laboratory mice. Each cell line was tested in vitro for their resistance to six varieties of lethal stress, as well as for resistance to the nonlethal metabolic effects of the mitochondrial inhibitor rotenone and of culture at very low glucose levels. Standard linear regression of species-specific lifespan against each species mean stress resistance showed that longevity was associated with resistance to death induced by cadmium and hydrogen peroxide, as well as with resistance to rotenone inhibition. A multilevel regression method supported these associations, and suggested a similar association for resistance to heat stress. Regressions for resistance to cadmium, peroxide, heat, and rotenone remained significant after various statistical adjustments for body weight. In contrast, cells from longer-lived species did not show significantly greater resistance to ultraviolet light, paraquat, or the DNA alkylating agent methylmethanesulfonate. There was a strong correlation between species longevity and resistance to the metabolic effects of low-glucose medium among the rodent cell lines, but this test did not distinguish mice and rats from the much longer-lived little brown bat. These results are consistent with the idea that evolution of long-lived species may require development of cellular resistance to several forms of lethal injury, and provide justification for evaluation of similar properties in a much wider range of mammals and bird species.