Association of Urinary Metals With Cardiovascular Disease Incidence and All-Cause Mortality in the Multi-Ethnic Study of Atherosclerosis (MESA).

BACKGROUND: Exposure to metals has been associated with cardiovascular disease (CVD) end points and mortality, yet prospective evidence is limited beyond arsenic, cadmium, and lead. In this study, we assessed the prospective association of urinary metals with incident CVD and all-cause mortality in a racially diverse population of US adults from MESA (Multi-Ethnic Study of Atherosclerosis). METHODS: We included 6599 participants (mean [SD] age, 62.1 [10.2] years; 53% female) with urinary metals available at baseline (2000 to 2001) and followed through December 2019. We used Cox proportional hazards models to estimate the adjusted hazard ratio and 95% CI of CVD and all-cause mortality by baseline urinary levels of cadmium, tungsten, and uranium (nonessential metals), and cobalt, copper, and zinc (essential metals). The joint association of the 6 metals as mixture and the corresponding 10-year survival probability was calculated using Cox Elastic-Net. RESULTS: During follow-up, 1162 participants developed CVD, and 1844 participants died. In models adjusted by behavioral and clinical indicators, the HR (95% CI) for incident CVD and all-cause mortality comparing the highest with the lowest quartile were, respectively: 1.25 (1.03, 1.53) and 1.68 (1.43, 1.96) for cadmium; 1.20 (1.01, 1.42) and 1.16 (1.01, 1.33) for tungsten; 1.32 (1.08, 1.62) and 1.32 (1.12, 1.56) for uranium; 1.24 (1.03, 1.48) and 1.37 (1.19, 1.58) for cobalt; 1.42 (1.18, 1.70) and 1.50 (1.29, 1.74) for copper; and 1.21 (1.01, 1.45) and 1.38 (1.20, 1.59) for zinc. A positive linear dose-response was identified for cadmium and copper with both end points. The adjusted HRs (95% CI) for an interquartile range (IQR) increase in the mixture of these 6 urinary metals and the corresponding 10-year survival probability difference (95% CI) were 1.29 (1.11, 1.56) and -1.1% (-2.0, -0.05) for incident CVD and 1.66 (1.47, 1.91) and -2.0% (-2.6, -1.5) for all-cause mortality. CONCLUSIONS: This epidemiological study in US adults indicates that urinary metal levels are associated with increased CVD risk and mortality. These findings can inform the development of novel preventive strategies to improve cardiovascular health.

Opportunities, Risks, Strengths and Weaknesses of Robotic Systems in Early Neurological Rehabilitation.

Given the conference's focus on innovative healthcare solutions, our investigation into robotic assistance systems highlights crucial advancements in early motor rehabilitation, aligning closely with emerging healthcare priorities. In combination with conventional therapy, the assistance systems offer new possible therapy programs. They can be used to mobilize and move patients as early as possible. The paper discusses the possibilities that arise from their use and considers the obstacles that arise. As part of a qualitative survey, nine expert interviews from different fields were conducted to guide them on robotic assisted living systems. The results obtained were summarized by coding into categories and evaluated. Our analysis of 148 coding points from nine expert interviews reveals significant insights into the strengths and weaknesses of robotic systems in neurorehabilitation. Each point was meticulously categorized to reflect its impact on both practice and patient outcomes, highlighting the practical implications of our findings. The results of the survey and the literature indicate a positive effect of robotic assistance systems in early rehabilitation. Their use requires intensive monitoring and studies on the long-term application of the systems.

Gut microbiota drives age-related oxidative stress and mitochondrial damage in microglia via the metabolite N6-carboxymethyllysine.

Microglial function declines during aging. The interaction of microglia with the gut microbiota has been well characterized during development and adulthood but not in aging. Here, we compared microglial transcriptomes from young-adult and aged mice housed under germ-free and specific pathogen-free conditions and found that the microbiota influenced aging associated-changes in microglial gene expression. The absence of gut microbiota diminished oxidative stress and ameliorated mitochondrial dysfunction in microglia from the brains of aged mice. Unbiased metabolomic analyses of serum and brain tissue revealed the accumulation of N6-carboxymethyllysine (CML) in the microglia of the aging brain. CML mediated a burst of reactive oxygen species and impeded mitochondrial activity and ATP reservoirs in microglia. We validated the age-dependent rise in CML levels in the sera and brains of humans. Finally, a microbiota-dependent increase in intestinal permeability in aged mice mediated the elevated levels of CML. This study adds insight into how specific features of microglia from aged mice are regulated by the gut microbiota.

Correction to: Analysis of the Fungal Flora in Environmental Dust Samples by PCR-SSCP Method.

The original version of this article contained a mistake in the co-author's conflict of interest statement. Erika von Mutius wishes to add the following disclosures: "E. von Mutius is listed as an inventor on the following patents: publication number EP 1411977, composition containing bacterial antigens used for the prophylaxis and the treatment of allergic diseases, granted on 18 April 2007; publication number EP1637147, stable dust extract for allergy protection, granted on 10 December 2008; publication number EP 1964570, pharmaceutical compound to protect against allergies and inflammatory diseases, granted on 21 November 2012. E. von Mutius is listed as inventor and has received royalties on the following patent: publication number EP2361632, specific environmental bacteria for the protection from and/or the treatment of allergic, chronic inflammatory and/or autoimmune disorders, granted on 19 March 2014".

Identification of fungal candidates for asthma protection in a large population-based study.

BACKGROUND: Exposure to molds has been related to asthma risk both positively and negatively, depending on the environmental setting. The pertinent results are based on generic markers or culturing methods although the majority of present fungi cannot be cultured under laboratory conditions. The aim of the present analysis was to assess environmental dust samples for asthma-protective fungal candidates with a comprehensive molecular technique covering also non-cultivable and non-viable fungi. METHODS: Mattress dust samples of 844 children from the GABRIELA study were analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) of the fungus-specific internal transcribed spacer (ITS) region. Known asthma candidate species were tested for their associations with asthma, and further gel positions were sought to explain the above. As a second, data-driven, analysis, we tested the association of each individual gel position with asthma. RESULTS: In the hypothesis-driven approach, Penicillium chrysogenum emerged with an odds ratio of 0.80 (95% confidence interval 0.66-0.96; p = 0.020). The effect size was changed by 39% toward the null when adjusting for the two bands 683 (DNA of Metschnikowia sp., Aureobasidium spp.) and 978 (DNA of Epicoccum spp., Galactomyces spp., uncultured Penicillium). The data-driven approach yielded an additional band (containing DNA of Pseudotaeniolina globosa) with reduced risk of asthma (OR = 0.80 [0.66-0.96], p = 0.012). CONCLUSIONS: A large population-based study revealed several fungal taxa with inverse associations with childhood asthma. Molds produce a variety of bioactive compounds with detrimental but also beneficial immunoregulatory capacities, which renders them promising targets for further asthma research.

Toward a synthesis of developmental biology with evolutionary theory and ecology.

The evolutionary conservation of developmental mechanisms is a truism in biology, but few attempts have been made to integrate development with evolutionary theory and ecology. To work toward such a synthesis, we summarize studies in the nematode model Pristionchus pacificus, focusing on the development of the dauer, a stress-resistant, alternative larval stage. Integrative approaches combining molecular and genetic principles of development with natural variation and ecological studies in wild populations have identified a key role for a developmental switch mechanism in dauer development and evolution, one that involves the nuclear hormone receptor DAF-12. DAF-12 is a crucial regulator and convergence point for different signaling inputs, and its function is conserved among free-living and parasitic nematodes. Furthermore, DAF-12 is the target of regulatory loops that rely on novel or fast-evolving components to control the intraspecific competition of dauer larvae. We propose developmental switches as paradigms for understanding the integration of development, evolution, and ecology at the molecular level.

The Orphan Gene dauerless Regulates Dauer Development and Intraspecific Competition in Nematodes by Copy Number Variation.

Many nematodes form dauer larvae when exposed to unfavorable conditions, representing an example of phenotypic plasticity and a major survival and dispersal strategy. In Caenorhabditis elegans, the regulation of dauer induction is a model for pheromone, insulin, and steroid-hormone signaling. Recent studies in Pristionchus pacificus revealed substantial natural variation in various aspects of dauer development, i.e. pheromone production and sensing and dauer longevity and fitness. One intriguing example is a strain from Ohio, having extremely long-lived dauers associated with very high fitness and often forming the most dauers in response to other strains' pheromones, including the reference strain from California. While such examples have been suggested to represent intraspecific competition among strains, the molecular mechanisms underlying these dauer-associated patterns are currently unknown. We generated recombinant-inbred-lines between the Californian and Ohioan strains and used quantitative-trait-loci analysis to investigate the molecular mechanism determining natural variation in dauer development. Surprisingly, we discovered that the orphan gene dauerless controls dauer formation by copy number variation. The Ohioan strain has one dauerless copy causing high dauer formation, whereas the Californian strain has two copies, resulting in strongly reduced dauer formation. Transgenic animals expressing multiple copies do not form dauers. dauerless is exclusively expressed in CAN neurons, and both CAN ablation and dauerless mutations increase dauer formation. Strikingly, dauerless underwent several duplications and acts in parallel or downstream of steroid-hormone signaling but upstream of the nuclear-hormone-receptor daf-12. We identified the novel or fast-evolving gene dauerless as inhibitor of dauer development. Our findings reveal the importance of gene duplications and copy number variations for orphan gene function and suggest daf-12 as major target for dauer regulation. We discuss the consequences of the novel vs. fast-evolving nature of orphans for the evolution of developmental networks and their role in natural variation and intraspecific competition.

Nematode orphan genes are adopted by conserved regulatory networks and find a home in ecology.

Nematode dauer formation represents an essential survival and dispersal strategy and is one of a few ecologically relevant traits that can be studied in laboratory approaches. Under harsh environmental conditions, the nematode model organisms Caenorhabditis elegans and Pristionchus pacificus arrest their development and induce the formation of stress-resistant dauer larvae in response to dauer pheromones, representing a key example of phenotypic plasticity. Previous studies have indicated that in P. pacificus, many wild isolates show cross-preference of dauer pheromones and compete for access to a limited food source. When investigating the genetic mechanisms underlying this intraspecific competition, we recently discovered that the orphan gene dauerless (dau-1) controls dauer formation by copy number variation. Our results show that dau-1 acts in parallel to or downstream of steroid hormone signaling but upstream of the nuclear hormone receptor daf-12, suggesting that DAU-1 represents a novel inhibitor of DAF-12. Phylogenetic analysis reveals that the observed copy number variation is part of a complex series of gene duplication events that occurred over short evolutionary time scales. Here, we comment on the incorporation of novel or fast-evolving genes into conserved genetic networks as a common principle for the evolution of phenotypic plasticity and intraspecific competition. We discuss the possibility that orphan genes might often function in the regulation and execution of ecologically relevant traits. Given that only few ecological processes can be studied in model organisms, the function of such genes might often go unnoticed, explaining the large number of uncharacterized genes in model system genomes.

Natural variation in dauer pheromone production and sensing supports intraspecific competition in nematodes.

Dauer formation, a major nematode survival strategy, represents a model for small-molecule regulation of metazoan development [1-10]. Free-living nematodes excrete dauer-inducing pheromones that have been assumed to target conspecifics of the same genotype [9, 11]. However, recent studies in Pristionchus pacificus revealed that the dauer pheromone of some strains affects conspecifics of other genotypes more strongly than individuals of the same genotype [12]. To elucidate the mechanistic basis for this intriguing cross-preference, we compared six P. pacificus wild isolates to determine the chemical composition of their dauer-inducing metabolomes and responses to individual pheromone components. We found that these isolates produce dauer pheromone blends of different composition and respond differently to individual pheromone components. Strikingly, there is no correlation between production of and dauer response to a specific compound in individual strains. Specifically, pheromone components that are abundantly produced by one genotype induce dauer formation in other genotypes, but not necessarily in the abundant producer. Furthermore, some genotypes respond to pheromone components they do not produce themselves. These results support a model of intraspecific competition in nematode dauer formation. Indeed, we observed intraspecific competition among sympatric strains in a novel experimental assay, suggesting a new role of small molecules in nematode ecology.

Multiple regulatory safeguards confine the expression of the GATA factor Serpent to the hemocyte primordium within the Drosophila mesoderm.

serpent (srp) encodes a GATA-factor that controls various aspects of embryogenesis in Drosophila, such as fatbody development, gut differentiation and hematopoiesis. During hematopoiesis, srp expression is required in the embryonic head mesoderm and the larval lymph gland, the two known hematopoietic tissues of Drosophila, to obtain mature hemocytes. srp expression in the hemocyte primordium is known to depend on snail and buttonhead, but the regulatory complexity that defines the primordium has not been addressed yet. Here, we find that srp is sufficient to transform trunk mesoderm into hemocytes. We identify two disjoint cis-regulatory modules that direct the early expression in the hemocyte primordium and the late expression in mature hemocytes and lymph gland, respectively. During embryonic hematopoiesis, a combination of snail, buttonhead, empty spiracles and even-skipped confines the mesodermal srp expression to the head region. This restriction to the head mesoderm is crucial as ectopic srp in mesodermal precursors interferes with the development of mesodermal derivates and promotes hemocytes and fatbody development. Thus, several genes work in a combined fashion to restrain early srp expression to the head mesoderm in order to prevent expansion of the hemocyte primordium.

Analysis of the fungal flora in environmental dust samples by PCR-SSCP method.

Conventional microbiological techniques yield only limited information on the composition of fungal communities in dust. The aim of this study was to establish and optimize PCR-single strand conformation polymorphism (PCR-SSCP) analysis for investigation of fungal diversity in rural dust samples. Three different DNA extraction protocols were tested on 38 fungal cultures. A total of six known universal fungal primer pairs were tested targeting the 18S rRNA gene, the 28S rRNA gene and the ITS region, respectively. Objective evaluation was performed with respect to the following parameters: efficiency to amplify all 38 strains; separation of seven species from different phylogenetic groups on the SSCP gel; additional bands in PCR-SSCP analysis; possibility to classify the amplified gene fragments to species level. Primer ITS1/ITS4 and PowerSoil™ DNA isolation showed the best performance in most cases and were chosen for further analysis. The detection limit of the developed system was 200 CFU/g dust. Moreover, the reproducibility of the system could be demonstrated, leading to average profile similarities of 94.94% [SD = 2.51] within gels, 93.03% [SD = 4.69] between different days and 87.66% [SD = 6.62] between different gels when testing shed and mattress dust samples. Sequencing allowed identification on species level, in detail: Alternaria alternata, Cladosporium sphaerospermum, Cladosporium cladosporioides as well as the yeasts Candida cabralensis and Candida catenulata. This demonstrates the adaptability of the method. In this study, a standardized system for fungal community analysis was developed that provides reproducible results applicable for epidemiological purposes.

Natural variation in Pristionchus pacificus dauer formation reveals cross-preference rather than self-preference of nematode dauer pheromones.

Many free-living nematodes, including the laboratory model organisms Caenorhabditis elegans and Pristionchus pacificus, have a choice between direct and indirect development, representing an important case of phenotypic plasticity. Under harsh environmental conditions, these nematodes form dauer larvae, which arrest development, show high resistance to environmental stress and constitute a dispersal stage. Pristionchus pacificus occurs in a strong association with scarab beetles in the wild and remains in the dauer stage on the living beetle. Here, we explored the circumstances under which P. pacificus enters and exits the dauer stage by using a natural variation approach. The analysis of survival, recovery and fitness after dauer exit of eight P. pacificus strains revealed that dauer larvae can survive for up to 1 year under experimental conditions. In a second experiment, we isolated dauer pheromones from 16 P. pacificus strains, and tested for natural variation in pheromone production and sensitivity in cross-reactivity assays. Surprisingly, 13 of the 16 strains produce a pheromone that induces the highest dauer formation in individuals of other genotypes. These results argue against a simple adaptation model for natural variation in dauer formation and suggest that strains may have evolved to induce dauer formation precociously in other strains in order to reduce the fitness of these strains. We therefore discuss intraspecific competition among genotypes as a previously unconsidered aspect of dauer formation.

Exposure to environmental microorganisms and childhood asthma.

BACKGROUND: Children who grow up in environments that afford them a wide range of microbial exposures, such as traditional farms, are protected from childhood asthma and atopy. In previous studies, markers of microbial exposure have been inversely related to these conditions. METHODS: In two cross-sectional studies, we compared children living on farms with those in a reference group with respect to the prevalence of asthma and atopy and to the diversity of microbial exposure. In one study--PARSIFAL (Prevention of Allergy-Risk Factors for Sensitization in Children Related to Farming and Anthroposophic Lifestyle)--samples of mattress dust were screened for bacterial DNA with the use of single-strand conformation polymorphism (SSCP) analyses to detect environmental bacteria that cannot be measured by means of culture techniques. In the other study--GABRIELA (Multidisciplinary Study to Identify the Genetic and Environmental Causes of Asthma in the European Community [GABRIEL] Advanced Study)--samples of settled dust from children's rooms were evaluated for bacterial and fungal taxa with the use of culture techniques. RESULTS: In both studies, children who lived on farms had lower prevalences of asthma and atopy and were exposed to a greater variety of environmental microorganisms than the children in the reference group. In turn, diversity of microbial exposure was inversely related to the risk of asthma (odds ratio for PARSIFAL, 0.62; 95% confidence interval [CI], 0.44 to 0.89; odds ratio for GABRIELA, 0.86; 95% CI, 0.75 to 0.99). In addition, the presence of certain more circumscribed exposures was also inversely related to the risk of asthma; this included exposure to species in the fungal taxon eurotium (adjusted odds ratio, 0.37; 95% CI, 0.18 to 0.76) and to a variety of bacterial species, including Listeria monocytogenes, bacillus species, corynebacterium species, and others (adjusted odds ratio, 0.57; 95% CI, 0.38 to 0.86). CONCLUSIONS: Children living on farms were exposed to a wider range of microbes than were children in the reference group, and this exposure explains a substantial fraction of the inverse relation between asthma and growing up on a farm. (Funded by the Deutsche Forschungsgemeinschaft and the European Commission.).

CXC chemokine receptor 3 expression by activated CD8+ T cells is associated with survival in melanoma patients with stage III disease.

Despite the presence of tumor Ag-specific CD8(+) T cells in the peripheral blood, metastatic melanoma often evades immune-mediated destruction. Even after therapeutic efforts to expand Ag-specific T-cell populations, the correlation between magnitude of response and clinical efficacy has been weak. Because the migratory phenotype of tumor Ag-specific effector T cells may determine their ability for tumor control, we hypothesized that the expression of CC or CXC chemokine receptor (CCR) molecules on activated CD8(+) T cells may define phenotypes associated with more effective control of melanoma progression and prolonged survival. In a retrospective evaluation of patient isolates, CCR expression was determined for activated CD8(+) T cells derived from the peripheral blood or tumor-involved lymph nodes of 52 patients with stage III or IV metastatic melanoma. In patients with stage III disease, expression of CXCR3 by CD8(+)CD45RO(+) cells was significantly associated with enhanced survival. This was a stage-specific effect, because it was not observed in patients with stage IV disease. In addition, CCR4 and CXCR3 were highly coexpressed and associated with enhanced survival in stage III patients; however, CXCR3 seems to be the dominant receptor associated with clinical outcome. These findings support the hypothesis that the host immune system affects cancer progression and control, and that measures of CCR status of circulating lymphocytes may have prognostic value.

Identification of protein complexes required for efficient sister chromatid cohesion.

Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae. We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes required for efficient sister chromatid cohesion. We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the genes identified in the ctf8 SGA screen. Deletion of seven genes (CHL1, CSM3, BIM1, KAR3, TOF1, CTF4, and VIK1) resulted in defective sister chromatid cohesion. Mass spectrometric analysis of immunoprecipitated complexes identified a physical association between Kar3p and Vik1p and an interaction between Csm3p and Tof1p that we confirmed by coimmunoprecipitation from cell extracts. These data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to a reference gene. Furthermore, we find that genes involved in mitotic spindle integrity and positioning have a previously unrecognized role in sister chromatid cohesion.