The evolutionary theory of aging demonstrates that the force of natural selection declines with age in multicellular organisms. Two population genetic mechanisms are consistent with the evolutionary theory: mutation accumulation and antagonistic pleiotropy. These in turn account for the physiological, cellular, and molecular mechanisms associated with aging. Spaceflight provides an opportunity to examine how organisms physiologically acclimate to environmental conditions. Mitochondria and the gut microbiome are central regulators of host metabolism, redox homeostasis, immune function, and physiological resilience, and both are impacted by host adaptations and acclimations. Mitochondrial dysfunction and oxidative stress have consequently emerged as important candidate mechanisms linking biological aging and spaceflight-associated physiological change. This review examines evidence surrounding mitochondrial dysfunction, oxidative stress, and gut microbiome dysbiosis across humans, mice, and fruit flies under spaceflight and corresponding terrestrial control conditions. This review reports further on the progression of theory and recent evidence from spaceflight missions for how biomarker genes most associated with mutation accumulation and antagonistic pleiotropy appear to have a core role in natural aging and extreme physiological acclimation.

Serotonin (5-HT) is a neurotransmitter involved in diverse neurophysiological functions, and dietary fats may influence serotonergic pathways. However, the specific effects of different fat sources on serotonin regulation in poultry remain unclear. This study investigated the impact of five dietary fats, including coconut oil, lard, olive oil, flaxseed oil, and canola oil, on serotonergic molecules in one-day-old broiler chicks over 56 days. Levels of 5-HT, 5-HTR1B, SERT, and 5-HIAA were measured in the dorsal raphe nucleus, hypothalamus, and liver using enzyme-linked immunosorbent assays (ELISA). No significant differences were detected in the dorsal raphe nucleus. In the hypothalamus, coconut oil and lard increased 5-HT compared to the control group, and in the liver, lard elevated 5-HIAA relative to the control (p < 0.05). These findings suggest that dietary fats may influence serotonin metabolism rather than signaling in broiler chicks, but further research using normalized measures and larger sample sizes is needed to clarify these effects.

Electro-acupuncture (EAP) has been proposed as a potential intervention for decreased gastrointestinal (GI) motility in domestic rabbits (Oryctolagus cuniculus); however, prospective studies evaluating this approach in clinical settings are lacking. Twelve juvenile New Zealand White rabbits (6 male, 6 female) underwent two sedation events in a complete crossover study conducted during 2 veterinary teaching laboratories 4 weeks apart. Following recovery from sedation, rabbits received one of two treatments: EAP or control (no acupuncture). Total pre- and post-treatment pellet consumption and fecal output were measured and compared between study groups. Gastrointestinal performance was evaluated at 24 hours post-sedation using cumulative pellet consumption, cumulative fecal output, and the difference between cumulative pellet consumption and fecal output. Apparent digestibility was also calculated and compared between groups and stratified by food intake, defined as heavy consumption (>181 g) or light consumption (<181 g). Electro-acupuncture treatment did not significantly alter cumulative pellet consumption or cumulative fecal output compared with controls. Apparent digestibility values, however, demonstrated significantly lower digestibility in rabbits consuming larger amounts of food compared with those consuming less (p=0.048). The lower values reflect that a greater proportion of consumed food was recovered as fecal material and may suggest accelerated GI transit in the acupuncture group (heavy food consumption rabbits). Although electro-acupuncture demonstrated some potential to influence GI motility in this pilot study, additional better controlled studies with larger sample sizes are needed to support the validity of findings from this study and to further evaluate clinical relevance.

Background:The triple-negative breast cancer (TNBC) microenvironment (TME) undergoes progressive reprogramming, transitioning from an early immune-active state to a late immune-suppressed state. While tumor cell plasticity has been extensively studied, the molecular plasticity of T cells in vivo remains poorly defined. Objectives:To characterize transcriptional changes in T cells during TNBC progression and identify stage-specific shifts in T cell function, polarization, and antigen-presenting cell (APC)-T cell interactions. Results:Transcriptional analysis of T cells from BALB/c mice bearing 4T1 tumors at 1, 3, and 6 weeks revealed a decline in T cell-associated genes from 194 at 1 week to 156 at 6 weeks, with a significant late-stage loss of TCR diversity and contraction of natural killer T (NKT)- and γδ T cell-related transcripts. Cytokine and transcription factor dynamics reflected temporal T cell polarization: early (1 week) IL-12α/β-STAT4 signaling supports CD4+ type 1 T helper cell (Th1) and type 1 CD8+ cytotoxic T cell (Tc1) responses; intermediate (3 weeks) IL-21 and BCL6 expression suggest transient CD8+ cytotoxic follicular T cell (Tfc) skewing; and late (6 weeks) AhR and IL-1β induction reflect interleukin 17/22 producing CD8+ T cell (Tc17/Tc22) transition. Pro-inflammatory cytokines and chemokines increased over time, while immunosuppressive mediators (e.g., IL-10) declined significantly. Antigen-presenting cell (APC)-T cell crosstalk deteriorated at 6 weeks, characterized by a reduction in the expression of co-stimulatory and APC genes. Despite an early dominance of M1-like macrophage signals (e.g., IL-12α/β), persistent expression of arginase 1 (ARG1) and other M2-associated genes indicated a stable tolerogenic niche. Conclusions:TNBC progression is characterized by progressive T cell functional decline, narrowing of TCR diversity, impaired APC-T cell interactions, and sustained macrophage-driven immunosuppression. These temporally coordinated immune shifts suggest tumor-driven adaptation toward immune evasion and identify potential windows for stage-specific immunotherapeutic intervention.

Background and Objectives Cancer evolves via interconnected mechanisms, including changes in extrachromosomal DNA (ecDNA), genetic instability, and interactions with the tumor microenvironment (TME). These mechanisms allow for some clones to evolve metastatic traits, evade the immune system, and resist chemotherapy. However, how cancer cells evolve in vivo remains poorly understood. This study investigates the in vivo changes in gene expression of triple-negative breast cancer (TNBC) cells implanted in BALB/c mice. Methodology We analyzed RNA-seq data from 4T1 TNBC cells and tumors at different growth stages (1-, 3-, and 6-week) to identify differentially expressed genes, protein-protein interactions, and ecDNA alterations. We also assessed how ecDNA and genomic instability proteins interact with anti-TNBC drugs Results Our results reveal early transcriptional shifts within one week of tumor implantation, showing rapid acclimation. Changes in gene expression continued over time, with significant molecular reprogramming observed at six weeks under in vivo environmental pressures, including ecDNA alterations and immune evasion. The shift from the earlier generation (1 week) to the later generation (6 weeks) suggests cumulative alterations in key oncogenic pathways related to tumor progression. Additionally, we found that mutations in ecDNA and genomic instability proteins influence drug binding affinity, suggesting that adaptive changes may impact chemotherapy response. Conclusions and Implications This study provides new insights into how TNBC tumors may evolve over time and novel ecDNA-related mechanisms of possible tumor adaptation, highlighting potential biomarkers for tumor aggression and immune evasion, which could help develop more effective therapeutic strategies against TNBC. ### Competing Interest Statement The authors have declared no competing interest.

Diet has been reported to impact the diversity and function of gut microbiota. Our study investigated the effect of dietary fat types on cecal microbial composition and predicted function in broiler chickens at days 41 and 55 of age. Four dietary fat sources were evaluated and compared to a control dietary fat source of poultry fat. These were for two diets rich in omega-3 polyunsaturated fatty acids (PUFA) - fish oil and flaxseed oil, a diet rich in long-chain saturated fatty acid (SFA) - lard, and a diet rich in medium-chain saturated fatty acid - coconut oil. At day 55, broiler chickens fed a PUFA-rich diet maintained cecal microbial diversity while broiler chickens fed a SFA-rich diet exhibited a significant reduction in diversity compared to the control diet-fed chickens. More specifically, PUFA intake was associated with elevated levels of microbial carbohydrate metabolizing capability, contributing to efficient energy utilization and enhanced short-chain fatty acid production capability. In contrast, SFA-rich diets lowered abundances for key microbial families like Lachnospiraceae and Bifidobacteriaceae hampering nutrient digestibility and pathogen resistance. The microbiomes for chickens fed lard and coconut oil diets showed a significant reduction in SCFA-producing microbial taxa abundance while the microbial functional profile indicated reduced carbohydrate metabolism. Our findings underscore the contrasting effects of SFA-rich fat and PUFA-rich fat on the cecal microbiota of broiler chickens. The results suggest that incorporating PUFA-rich dietary fats into broiler feed may offer potential benefits by modulating the cecal gut microbiota toward outcomes associated with elevated carbohydrate utilization without hampering nutrient digestibility and pathogen resistance.

Background:The triple-negative breast cancer (TNBC) microenvironment undergoes progressive reprogramming, transitioning from an early immune-active state to a late immune-suppressed state. While tumor cell plasticity has been extensively studied, the temporal molecular remodeling of T cells in vivo remains poorly defined. Results:Transcriptional analysis of T cells within 4T1 TNBC tumors, harvested at one-, three-, and six-weeks post-tumor implantation in the mammary fat pads of BALB/c mice, revealed a decline in transcriptomic signatures associated with T cells from 194 at one week to 156 at six weeks, with a significant late-stage loss or reduction of transcripts related to T cell receptors (TCR), natural killer T, and gamma delta T cells. Furthermore, changes in various temporal signature genes specific to T cell cytokines and transcription factors reflected temporal T cell polarization to CD4+ type 1 T helper and type 1 CD8+ cytotoxic T cell responses at one week, CD8+ cytotoxic follicular T cell skewing at three weeks, and interleukin 17/22 producing CD8+ T cell transition at six weeks. The antigen-presenting cell (APC) transcripts deteriorated at six weeks, characterized by reduced expression of co-stimulatory and APC genes. Despite an early dominance of M1-like macrophage genes (e.g., IL-12α/β), persistent expression of arginase 1 (ARG1) and other M2-associated genes indicated a stable tolerogenic niche. Conclusions:The temporally coordinated immune shifts, such as progressive decline in transcripts associated with T cell functions, TCRs, APCs, and sustained macrophage-driven immunosuppression, suggest tumor-driven adaptation toward immune evasion and identify potential windows for stage-specific immunotherapeutic intervention.

Serotonin is a critical neurotransmitter that regulates a wide range of physiological, neurological, and behavioral functions. While peripheral serotonin, primarily produced in the gut, modulates gastrointestinal motility and vascular tone, central serotonin that is synthesized in the brain governs processes such as food intake, emotion regulation, memory, learning, and sexual behavior. Chronic consumption of a high-fat diet (HFD) disrupts serotonin signaling across the gut, brain, and the gut-brain axis, which supports bidirectional communication between these systems. Although the underlying mechanisms remain incompletely understood, this review explores how HFD alters serotonin signaling in both the gut and the brain. We report that HFD triggers pathway-specific changes that elevate serotonin levels in the gut while eliciting region-specific effects in the brain. HFD increases serotonin biosynthesis in the brain's raphe nuclei; however, enhanced 5-HT1A autoreceptor activity within these nuclei inhibits serotonin release to downstream projection areas. Coupled with increased serotonin degradation in these regions, this results in reduced serotonin levels in the hippocampus and hypothalamus. Additionally, our findings highlight a central role for microbial metabolites in mediating HFD-induced serotonergic dysfunction. Notably, short-chain fatty acids produced by gut microbiota, significantly contribute to the dysregulation of serotonin release and signaling under HFD conditions. Understanding these mechanisms may reveal new therapeutic strategies for managing serotonergic dysfunctions associated with gastrointestinal disorders, mood disturbances, and obesity-related complications.

This study evaluated neoplasia in fish using medical records from zoos, aquariums, and exotic animal veterinarians. The parameters evaluated included geographic location, habitat type, signalment, anatomic location of neoplasia, type of neoplasia as confirmed with histologic examination, survival time, and treatments provided for each patient. These data were entered into the Exotic Species Cancer Research Alliance (ESCRA) database. Out of 455 cases from across the United States and England, most animals submitted were from zoologic parks or aquariums (62.9%), followed by private ownership (1.5%). The percent of female (19.3%) and male (17.8%) patients were similar, and the mean age at the time of diagnosis was 99.45 months, with a range of 12 to 300 months. The species with the highest neoplasia prevalence was koi (18.5%), followed by goldfish (10.8%). The eye was the most commonly reported site for a primary neoplasm (8.4%), and the most prevalent diagnosis across all organ systems was soft tissue sarcoma (26.2%). Only 13 patients in this study (2.9%) received any form of treatment, with a mean survival time of 8.85 months post-treatment. These data demonstrate that while information related to clinical therapy of cancer in fish species is lacking, surgical excision of tumors in fish, when feasible for the patient and client, may improve patient outcomes.

Diet has been found to significantly influence gut microbiota throughout various life stages, and gut microbiota have been increasingly shown to influence host physiology, health, and behavior. This study uses 16S rRNA sequencing to examine the effects of six different fat-supplemented diets (canola oil, coconut oil, fish oil, flaxseed oil, lard, and olive oil) on broiler chicken cecal microbial composition and predicted function in comparison with a common and inexpensive fat source (poultry fat). Groups of broilers were fed each of these diets and then evaluated on day 41 and day 55 of age. For both 41- and 55-day samples, Firmicutes and Bacteroidetes phyla were the dominant bacteria in the ceca accounting for 99% of the microbial community. Across the 41- and 55-day samples, treatment time was associated with a stronger and more significant microbiota shift (p < 0.001) than differences in dietary treatment alone (p = 0.117), but dietary treatment combined with treatment time is a significant factor as well (p = 0.047). Sparse partial least squares discriminant analysis was used to explore the more discriminating taxa for each treatment group. For identified species, butyrate production appears to be affected in a diet-specific manner, with many butyrate-producing species being evident for the fish-based diet at day 41 and a few of these species for the flaxseed-based diet at day 55. Predicted functions, as conducted with PICRUSt2, were significant for comparisons between the control and the flaxseed-based dietary treatment group at day 55, with indications of host health benefit for the flaxseed-based diet. Predicted functions found to be significant were for enzymes and pathways such as propionate CoA ligase, aminobutyraldehyde dehydrogenase, vitamin B12-transporting ATPase, thiamine kinase, acetylneuraminate epimerase, and L-tryptophan biosynthesis. This study provides insight surrounding specific dietary fat-based treatments to be investigated further and highlights the importance of polyunsaturated fat sources in poultry feed that may offer a favorable cecal microbial modulation compared to saturated fat sources.

Neoplasia has been reported in lizards, but more research is needed to accurately document the prevalence and prognosis of the various known neoplasms that affect lizards. This study reviewed medical records from an online database, the Exotic Species Cancer Research Alliance (ESCRA), and reviewed published literature to determine the prevalence of neoplasia, malignancy, metastasis, treatment strategies, and outcomes by species and sex. Records from 55 individual lizards, 20 different species, and 37 different tumors were identified. In the literature, 219 lizards, 59 species, and 86 unique tumors were identified from 72 published case reports. Potential signalment factors such as age, sex, and species were evaluated to see if they affected case outcome. Additional factors including neoplasia type, presence of metastasis, and types of pursued treatments were also evaluated. Statistical analysis was performed to determine whether a factor was significantly associated with animal death due to the identified neoplasia or with animal survival or death due to other causes (non-neoplastic outcomes). Komodo dragons and savannah monitors were more likely to die from neoplasia compared to other lizard species. Cases where the status of metastasis was unknown were significantly associated with death due to neoplasia. Having an unknown status of male versus female was significantly associated with non-neoplastic outcomes of death. Leukemia and islet cell carcinoma were significantly associated with death due to neoplastic causes. Chondrosarcoma, myxosarcoma, osteosarcoma, and squamous cell carcinoma were significantly associated with non-neoplastic outcomes of death. Surgery alone and radiation therapy alone each were significantly associated with non-neoplastic outcomes of death, while lizards not receiving treatment were significantly associated with death due to neoplasia. Benign neoplasia was significantly associated with non-neoplastic outcomes of death. These results will aid in the improved diagnosis and management of neoplasia in lizard species, as well as expanding our understanding of prognostic indicators of neoplasia in lizards.

Obesity is a major health concern that poses significant risks for many other diseases, including diabetes, cardiovascular disease, and cancer. Prevalence of these diseases varies by biological sex. This study utilizes a mouse (C57BL/6J) model of obesity to analyze liver and fecal metabolic profiles at various time points of dietary exposure: 5, 9, and 12 months in control or high fat diet (HFD)-exposed mice. Our study discovered that the female HFD group has a more discernable perturbation and set of significant changes in metabolic profiles than the male HFD group. In the female mice, HFD fecal metabolites including pyruvate, aspartate, and glutamate were lower than control diet-exposed mice after both 9th and 12th month exposure time points, while lactate and alanine were significantly downregulated only at the 12th month. Perturbations of liver metabolic profiles were observed in both male and female HFD groups, compared to controls at the 12th month. Overall, the female HFD group showed higher lactate and glutathione levels compared to controls, while the male HFD group showed higher levels of glutamine and taurine compared to controls. These metabolite-based findings in both fecal and liver samples for a diet-induced effect of obesity may help guide future pioneering discoveries relating to the analysis and prevention of obesity in people, especially for females.

Diepoxybutane (DEB) is the most toxic metabolite of the environmental chemical 1,3‐butadiene. We previously demonstrated the occurrence of DEB‐induced p53‐mediated apoptosis in human lymphoblasts. The p53 protein functions as a master transcriptional regulator in orchestrating the genomic response to a variety of stress signals. Transcriptomic analysis indicated that C‐C chemokine ligand 4 (CCL4) gene expression was elevated in a p53‐dependent manner in DEB‐exposed p53‐proficient TK6 cells, but not in DEB‐exposed p53‐deficient NH32 cells. Thus, the objective of this study was to determine whether the CCL4 gene is a transcriptional target of p53 and deduce its role in DEB‐induced apoptosis in human lymphoblasts. Endogenous and exogenous wild‐type p53 transactivated the activity of the CCL4 promoter in DEB‐exposed lymphoblasts, but mutant p53 activity on this promoter was reduced by ∼80% under the same experimental conditions. Knockdown of the upregulated CCL4 mRNA levels in p53‐proficient TK6 cells inhibited DEB‐induced apoptosis by ∼45%–50%. Collectively, these observations demonstrate for the first time that the CCL4 gene is upregulated by wild‐type p53 at the transcriptional level, and this upregulation mediates apoptosis in DEB‐exposed human lymphoblasts.

The rise in antimicrobial resistant bacteria have prompted the need for antibiotic alternatives. To address this problem, significant attention has been given to the antimicrobial use and novel applications of copper. As novel applications of antimicrobial copper increase, it is important to investigate how bacteria may adapt to copper over time. Here, we used experimental evolution with re-sequencing (EER-seq) and RNA-sequencing to study the evolution of copper resistance in Escherichia coli. Subsequently, we tested whether copper resistance led to rifampicin, chloramphenicol, bacitracin, and/or sulfonamide resistance. Our results demonstrate that E. coli is capable of rapidly evolving resistance to CuSO4 after 37 days of selection. We also identified multiple de novo mutations and differential gene expression patterns associated with copper, most notably those mutations identified in the cpx gene. Furthermore, we found that the copper resistant bacteria had decreased sensitivity when compared to the ancestors in the presence of chloramphenicol, bacitracin, and sulfonamide. Our data suggest that the selection of copper resistance may inhibit growth in the antimicrobials tested, resulting in evolutionary trade-offs. The results of our study may have important implications as we consider the antimicrobial use of copper and how bacteria may respond to increased use over time.

This multi-institutional collaborative study of neoplasia in snakes reviewed medical records of snakes at each facility to determine species prevalence, survival, and methods of treatment. Complete species numbers of snakes were also collected at each facility. In total, 65 species, 133 snakes, and 149 unique neoplasias were included in this study. Affected species, age, sex, and their tumor prevalence, tumor type and location, metastasis, treatment, and survival data are reported. The highest species-specific tumor prevalence was in Common or Northern Watersnakes (Nerodia sipedon) (30.8%, n = 4 of 13), Eastern Diamond-Backed Rattlesnakes (Crotalus adamanteus) (26.3%, n = 5 of 19), and Timber rattlesnakes (Crotalus horridus) (22.7%, n = 5 of 22). Malignant tumors predominated (86.6%, n = 129 of 149) with soft tissue sarcomas being the most common (30.2%, n = 45 of 149). Snakes with malignant neoplasia, metastases, or indeterminate presence of metastases were statistically more likely to die from their neoplasms than snakes having either benign neoplasia or no diagnosed metastases (p < 0.05). Gender, taxonomic family, and species of those evaluated did not significantly affect the outcome of snakes with neoplasia. Only 27.1% (n = 36 of 133) of snakes received a reported form of treatment and, for those treated, surgical excision was the most common treatment modality. There was not a significant difference in outcome based on treatment; however, surgery and chemotherapy were associated with death from a cause other than their tumor.

The chicken gastrointestinal tract has a diverse microbial community. There is increasing evidence for how this gut microbiome affects specific molecular pathways and the overall physiology, nervous system and behavior of the chicken host organism due to a growing number of studies investigating conditions such as host diet, antibiotics, probiotics, and germ-free and germ-reduced models. Systems-level investigations have revealed a network of microbiome-related interactions between the gut and state of health and behavior in chickens and other animals. While some microbial symbionts are crucial for maintaining stability and normal host physiology, there can also be dysbiosis, disruptions to nutrient flow, and other outcomes of dysregulation and disease. Likewise, alteration of the gut microbiome is found for chickens exhibiting differences in feather pecking (FP) behavior and this alteration is suspected to be responsible for behavioral change. In chickens and other organisms, serotonin is a chief neuromodulator that links gut microbes to the host brain as microbes modulate the serotonin secreted by the host's own intestinal enterochromaffin cells which can stimulate the central nervous system via the vagus nerve. A substantial part of the serotonergic network is conserved across birds and mammals. Broader investigations of multiple species and subsequent cross-comparisons may help to explore general functionality of this ancient system and its increasingly apparent central role in the gut-brain axis of vertebrates. Dysfunctional behavioral phenotypes from the serotonergic system moreover occur in both birds and mammals with, for example, FP in chickens and depression in humans. Recent studies of the intestine as a major site of serotonin synthesis have been identifying routes by which gut microbial metabolites regulate the chicken serotonergic system. This review in particular highlights the influence of gut microbial metabolite short chain fatty acids (SCFAs) on the serotonergic system. The role of SCFAs in physiological and brain disorders may be considerable because of their ability to cross intestinal as well as the blood-brain barriers, leading to influences on the serotonergic system via binding to receptors and epigenetic modulations. Examinations of these mechanisms may translate into a more general understanding of serotonergic system development within chickens and other avians.

Background: There has been an increased usage of metallic antimicrobial materials to control pathogenic and multi-drug resistant bacteria. Yet, there is a corresponding need to know if this usage leads to genetic adaptations that could produce more harmful strains. Methodology: Experimental evolution was used to adapt Escherichia coli K-12 MG1655 to excess iron (II) with subsequent genomic analysis. Phenotypic assays and gene expression studies were conducted to demonstrate pleiotropic effects associated with this adaptation and to elucidate potential cellular responses. Results: After 200days of adaptation, populations cultured in excess iron (II), showed a significant increase in 24-h optical densities compared to controls. Furthermore, these populations showed increased resistance toward other metals [iron (III) and gallium (III)] and to traditional antibiotics (bacitracin, rifampin, chloramphenicol and sulfanilamide). Genomic analysis identified selective sweeps in three genes; fecA, ptsP and ilvG unique to the iron (II) resistant populations, and gene expression studies demonstrated that their cellular response may be to downregulate genes involved in iron transport (cirA and fecA) while increasing the oxidative stress response (oxyR, soxS and soxR) prior to FeSO4 exposure. Conclusions and implications: Together, this indicates that the selected populations can quickly adapt to stressful levels of iron (II). This study is unique in that it demonstrates that E. coli can adapt to environments that contain excess levels of an essential micronutrient while also demonstrating the genomic foundations of the response and the pleiotropic consequences. The fact that adaptation to excess iron also causes increases in general antibiotic resistance is a serious concern. Lay summary: The evolution of iron resistance in E. coli leads to multi-drug and general metal resistance through the acquisition of mutations in three genes (fecA, ptsP and ilvG) while also initiating cellular defenses as part of their normal growth process.

AbstractBackgroundThere has been an increased usage of metallic antimicrobial materials to control pathogenic and multi-drug resistant bacteria. Yet, there is a corresponding need to know if this usage leads to genetic adaptations that could produce more harmful strains.MethodologyExperimental evolution was used to adapt Escherichia coli K-12 MG1655 to excess iron (II) with subsequent genomic analysis. Phenotypic assays and gene expression studies were conducted to demonstrate pleiotropic effects associated with this adaptation and to elucidate potential cellular responses.ResultsAfter 200 days of adaptation, populations cultured in excess iron (II), showed a significant increase in 24-h optical densities compared to controls. Furthermore, these populations showed increased resistance toward other metals [iron (III) and gallium (III)] and to traditional antibiotics (bacitracin, rifampin, chloramphenicol and sulfanilamide). Genomic analysis identified selective sweeps in three genes; fecA, ptsP and ilvG unique to the iron (II) resistant populations, and gene expression studies demonstrated that their cellular response may be to downregulate genes involved in iron transport (cirA and fecA) while increasing the oxidative stress response (oxyR, soxS and soxR) prior to FeSO4 exposure.Conclusions and implicationsTogether, this indicates that the selected populations can quickly adapt to stressful levels of iron (II). This study is unique in that it demonstrates that E. coli can adapt to environments that contain excess levels of an essential micronutrient while also demonstrating the genomic foundations of the response and the pleiotropic consequences. The fact that adaptation to excess iron also causes increases in general antibiotic resistance is a serious concern.Lay summary: The evolution of iron resistance in E. coli leads to multi-drug and general metal resistance through the acquisition of mutations in three genes (fecA, ptsP and ilvG) while also initiating cellular defenses as part of their normal growth process.

There has been an increased usage of metallic antimicrobial materials to control pathogenic and multi-drug resistant bacteria, yet there is a corresponding need to know if this usage may lead to genetic adaptations that produce even more dangerous bacterial strains. In this paper we examine important recent results from the literature as well as report results from a series of our own studies. In that work, we utilized experimental evolution to produce strains of Escherichia coli K-12 MG1655 resistant to silver (Ag+), excess copper (Cu2+), excess iron (II, III), and the iron analog gallium (Ga3+). Silver and gallium are toxic to bacteria, whereas iron and copper are essential micronutrients that can be toxic in excess amounts. In all cases, the evolution of metal resistance was rapid and resulted in pleiotropic effects that included resistance to other metals as well as traditional antibiotics. Genomic analysis identified mutations in several genes associated with metal resistance, falling in several broad classes: genes that prevent entry of metal into the cell, genes involved in the energy-dependent efflux of metal out of the cell, genes associated with ROS-induced membrane damage, and genes associated with transcription.

Diepoxybutane (DEB) is the most potent active metabolite of the environmental chemical 1,3-butadiene (BD). BD is a human carcinogen that exhibits multiorgan systems toxicity. Our previous studies demonstrated that the X-C motif chemokine ligand 1 (XCL1) gene expression was upregulated 3.3-fold in a p53-dependent manner in TK6 lymphoblasts undergoing DEB-induced apoptosis. The tumor-suppressor p53 protein is a transcription factor that regulates a wide variety of cellular processes, including apoptosis, through its various target genes. Thus, the objective of this study was to determine whether XCL1 is a novel direct p53 transcriptional target gene and deduce its role in DEB-induced toxicity in human lymphoblasts. We utilized the bioinformatics tool p53scan to search for known p53 consensus sequences within the XCL1 promoter region. The XCL1 gene promoter region was found to contain the p53 consensus sequences 5 '-AGACATGCCTAGACATGCCT-3 ' at three positions relative to the transcription start site (TSS). Furthermore, the XCL1 promoter region was found, through reporter gene assays, to be transactivated at least threefold by wild-type p53 promoter in DEB-exposed human lymphoblasts. Inactivation of the XCL1 promoter p53-binding motif located at -2.579 kb relative to TSS reduced the transactivation function of p53 on this promoter in DEB-exposed cells by 97%. Finally, knockdown of XCL1 messenger RNA with specific small interfering RNA inhibited DEB-induced apoptosis in human lymphoblasts by 50%. These observations demonstrate, for the first time, that XCL1 is a novel DEB-induced direct p53 transcriptional target gene that mediates apoptosis in DEB-exposed human lymphoblasts.