Background: Although exposure to endocrine disruptor compounds (EDCs) has been suggested as a contributing factor to a range of women’s health disorders including infertility, polycystic ovaries and the early onset of puberty, considerable challenges remain in attributing cause and effect on gynaecological cancer. Until recently, there were relatively few epidemiological studies examining the relationship between EDCs and endometrial cancer, however, in the last years the number of these studies has increased. Methods: A systematic MEDLINE (PubMed) search was performed and relevant articles published in the last 23 years (from 1992 to 2016) were selected. Results: Human studies and animal experiments are confirming a carcinogenic effect due to the EDC exposure and its carcinogenesis process result to be complex, multifactorial and long standing, thus, it is extremely difficult to obtain the epidemiological proof of a carcinogenic effect of EDCs for the high number of confusing factors. Conclusions: The carcinogenic effects of endocrine disruptors are plausible, although additional studies are needed to clarify their mechanisms and responsible entities. Neverthless, to reduce endocrine disruptors (ED) exposure is mandatory to implement necessary measures to limit exposure, particularly during those periods of life most vulnerable to the impact of oncogenic environmental causes, such as embryonic period and puberty. According to the definition of the World Health Organization (WHO), an endocrine disrupting chemical (EDC) is an exogenous substance or mixture that alters the function(s) of the endocrine system and consequently causes adverse effects in an intact organism, or its progeny, or (sub)populations . EDCs arAlthough exposure to EDCs has been suggested as a contributing factor to a range of women’s health disorders, including infertility ,3, polycThe most significant EDCs are environmental pollutants or major constituents of preservatives and industrial plasticizers and approximately 800 chemicals are known or suspected to have the potential to function as EDCs . This wiEDCs are compounds with mostly estrogenic activities and considering that recently, natural estrogens have been classified as known human carcinogens  it is plAs a matter of fact, the steroid hormone 17β-estradiol (E2) is a key regulator of growth, differentiation, and function in a wide array of target tissues, including the male and female reproductive tracts and mammary gland . In addiOriginally thought to exert their actions primarily through nuclear hormone receptors, such as estrogen (ERs) and progesterone receptors (PRs), research now clearly demonstrates that the mechanisms of action of EDCs are much broader than originally recognized, including also epigenomic changes .Although several environmental compounds exert their carcinogenic action inducing genetic mutations ,13, the In the event an environmental toxicant such as an endocrine disruptor modifies the epigenome of a somatic cell, this may promote disease in the exposed individual, but not be transmitted to the next generation. In the event a toxicant modifies the epigenome of the germ line permanently, then the disease promoted can become transgenerationaly transmitted to subsequent progeny .Environmental epigenetics provides direct molecular mechanisms for factors or toxicants to influence the genetic cascade of events involved in the development. Critical windows of susceptibility exist where these factors modify and affect important stages of development. Generally, these critical windows are early in development when the developing organ system are sensitive to alterations in the epigenome. Modification of the epigenome may continue throughout development, subsequently affecting the adult transcriptome and making tissue, such as the breast, susceptible to developing disease. Importantly, in mammals exposure of gestating females to environmental factors or toxicants during the period of gonadal sex determination can alter epigenetic transgenerational inheritance. The epigenetic reprogramming and imprinting of germ cells can thus allow transgenerational transmission of adult-onset disease phenotypes .A systematic MEDLINE (PubMed) search was performed using the following keywords: “endocrine disruptors”, “endometrial cancer”, “bisphenol A”, “polychlorineted biphenyls”, “dioxin” and “cadmium”.Relevant articles published in the last 23 years (from 1992 to 2016) were selected. We considered experimental studies, epidemiological studies and previous reviews. Criteria for inclusion and exclusion were established before the bibliographic search.Studies published in languages other than English were considered only if an abstract was available. Searching for endocrine disruptors and endometrial cancer only 15 studies were found. Two were excluded because they regarded only the effects of endocrine disruptors on breast cancer genesis and one because it was in the French language. Therefore only 12 studies were determined to be useful .Clinically, endometrial cancer have been divided into estrogen-dependent type I and the less common but clinically more aggressive, estrogen-independent type II. In women, the majority of endometrial cancers are estrogen-dependent type I cancers with increased risk associated with exposure to excess estrogens. It is well established that type I endometrial cancer usually occurs due to unopposed estrogen stimulation  and therEstrogens regulate miRNA transcription through estrogen receptors α and β in a tissue-specific and cell-dependent manner and it would lead to the fact that endocrine disruptors could be involved in cancerogenesis also through this mechanism . Neverthp-dioxin (TCDD).Polychlorinated biphenyls (PCBs) are chemically stable, lipid soluble pollutants. These compounds were used widely in industry for a wide variety of purposes, ranging from plastic resins to carbonless copy paper. Despite their utility, PCBs are linked to environmental and human health risks ,33 that Although their adverse impacts on the risk of cancer are of great concern and studies on PCB mixtures have been extensive over the last several decades—including those on endometrial cancer , a mechaPCBs have been proposed to act through a variety of cellular pathways. As previously mentioned, PCBs are suspected to promote cancer through affecting the cellular redox environment.Regarding endometrial cancer, molecular epidemiological studies have shown how the activity of superoxide dismutase (SOD) would be lower in the endometrial cancer tissue than in the normal endometrium ,44 and POf particular interest is the potential estrogenic activity of these compounds that have been demonstrated in vitro and in vivo ,48,49. TTherefore studies in the 90s ,53 suggeOn the other hand, Yoshikawa et al.  performeIn the studies of PCB 126, the tertiary mixture, and the binary mixture of PCB 126 and PCB 118, no increased incidence of any change occurred in the reproductive systems. The range of changes seen with the different compounds suggested that more than one mechanism may be involved in promoting the female reproductive pathology.Actually PCBs dioxin-like are known to interact with the aryl hydrocarbon receptor (AhR); endometrial cancer cells are both Ah- and E2- responsive and express the AhR and ER and in cell proliferation and transcriptional activation studies, and several different AhR agonists inhibit E2-mediated responses. These results would demonstrate inhibitory crosstalk between the AhR and ER signalling pathways in endometrial cells  and woulBisphenol A (BPA) is one of the most widely produced chemicals worldwide and is often used in the production of food and beverage containers. As a result of BPA contact with food, drink and toiletries, its ingestion and absorption by humans has been growing. The tolerable daily intake (TDI) of 50 μg/kg bw/day in the USA and 10 μg/kg bw/day in the European Union is considered to be higher than the daily human uptake of BPA (<1 μg/kg bw/day). However, this setting did not take into account the vulnerability to BPA exposure during prenatal and neonatal development .As a matter of factsin an vivo animal study where outbred female CD-1 mice were treated on days 1–5 with subcutaneous injections of BPA , and examined at 18 months, an increase in cystic ovaries and cystic endometrial hyperplasia (CEH) was demonstrated in the BPA-100 group as compared to controls, suggesting that BPA causes long-term adverse effects if exposure occurs during critical periods of differentiation . FurtherMany studies have highlighted the correlation between the increase of BPA level in the environment and the incidence of tumor in humans. In human carcinogenesis, the overexpression of cyclooxygenase-2 (COX-2) and epithelial-mesenchymal transition (EMT) are closely related with tumor development and it had been demonstrated that BPA can induce EMT and COX-2 expression in human endometrial carcinoma cells line (RL95-2): BPA increased growth rate and colony-forming efficiency in a dose-dependent manner, induced EMT and COX-2 gene expression and promoted the migration and invasion ability of RL95-2 cells  Table 3Table 3.In a study where the ability of BPA to affect human estrogen receptor (ER) binding, expression of progesterone receptor (PR) mRNA and protein, and cell proliferation in the human endometrial cell line was evaluated, although less potent than 17β-estradiol, BPA was able to bind to the human uterine ER. BPA also induced both mRNA and protein to levels similar to E2, suggesting an ER-mediated pathway .BPA is able to induce thousands of estrogen receptor1 (ESR1) binding sites and change the expression of a subset of genes (more often up-regulated) affected by E2 . FurtherAlthough BPA is identified as an estrogenic substance and may activate both ERα and ERβ, this activation would be both cell-type- and concentration- dependent . FurtherFurthermore, in addition to the capacity to bind to nuclear estrogen receptors (ERs) α- and β- ,69, otheNotwithstanding, serum concentrations of BPA measured in both pre- and postmenopausal women were significantly lower in patients with complex endometrial hyperplasia or endometrial cancer than in healthy controls . These fBPA has structural similarities to E2 and is considered mainly as an ERα and ERβ agonist  but it cIn an in vitro study , using aIn a study investigating the effect of BPA on human endometrial stromal cell (ESC) differentiation, BPA was found to decrease proliferation of ESC and decrease expression of mRNAs encoding CYP11A1, HSD17B1 and HSD17B2; however, this effect was only observed with high (50–100 μM) doses .Aside from the receptor mediated mechanism, BPA exerts its action through epigenetic mutations, altering developmental pathways and cell processes ,82. In fp-dioxins (PCDD), dibenzofurans (F) and dioxin-like polychlorinated biphenyls (PCBs). Many of the congeners are proved to be highly toxic, but 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener, and probably the most toxic compound ever synthesized by man, being widespread and highly persistent in the environment, and accumulating in living organisms, especially in fat tissue. The dioxins are known to adversely affect the reproductive system in part due to their ability to alter hormone levels in many species [The term dioxins is used to specify polychlorinated dibenzo- species ,85.TCDD and related compounds induce a broad spectrum of biochemical and toxic responses and disrupt multiple endocrine pathways.Reproductive cancer risks were first reported following the “Seveso Accident” in 1976, which caused exposure to TCDD of a large residential population at a chemical plant in Seveso, Italy ,87,88.NeAlthough the exact mechanism of TCDD-induced toxicity is not completely understood, many studies indicate that the most of TCDD effects are mediated through the aryl hydrocarbon receptor (AhR), a ligand-activated member of the bHLH-PAS family of transcription factors ,91,92. TLaboratory research has focused on characterizing aryl hydrocarbon receptor (AhR)-mediated antiestrogenicity in the rodent uterus and mammary and in human breast cancer cells . TCDD waIndeed TCDD has been reported to cause anticarcinogenic effects in hormone-dependent tissues such as mammary, uterus and the pituitary of female rats fed the compound for a period of two years . SeveralFurthermore in ER-positive human breast cancer cell lines, TCDD inhibits 17β-estradiol(E2)-dependent proliferation , secretiThis is a relevant evidence because demonstrate that TCDD does not interact directly with ER or PR and therefore, the antiestrogenic effects cannot be explained by direct interaction of TCDD with those receptors . Rather,Although these studies indicate that dioxins may be protective against estrogenic stimulation and development of hormonal dependent cancer, many in vitro studies demonstrate other mechanisms which would correlate TCDD to endometrial cancer. The exposure to TCDD also modulates the immune response by influencing the production and action of endometrial cytokines and chemokines, destroying mucosal immunity of the reproductive tract and re-directing the tissue distribution and behavior of leukocytes . Over thCharles et al.  investigp-dioxin (TCDD). RL95-2 cells were highly responsive to TCDD in terms of cytochrome P4501A1 (CYP1A1), cytochrome P4501B1 (CYP1B1), and plasminogen activator inhibitor-2 (PAI-2), whereas KLE cells showed little stimulatory effects only at high doses.Jana et al.  investigp-dioxin (TCDD) resulting in a marginally or significantly increasing of uterine squamous cell carcinoma, respectively, in the 6 ng/kg core and 100 ng/kg stop-exposure groups  might, therefore, be also due to a disruption of retinoid action, leading to altered growth and differentiation of the endometrial epithelium resulting in squamous metaplasia (SM) and squamous cell carcinoma (SCC).On this point TCDD and DLCs are documented to induce epithelial SM and SCC in other organs, including gingival squamous-cell hyperplasia and/or SCC, SM, and/or SCC in the lung, and squamous hyperplasia in the forestomach ,129.At last but not least, a possible association between dioxin and endometriosis  has beenAccording to a number of histopathological studies endometriosis-associated ovarian cancer (EAOC) may arise from atypical endometriosis of the ovary and this heterogeneous condition is histologically characterized by hyperplasia of endometrial glands with cytological atypia or presence of atypical hobnail cells within ovarian endometriosis . Thus, iCadmium (Cd) belongs to the heavy metals, and is very common both in the industry and in the natural environment. It is classified as the 7th substance on the Agency for Toxic Substances and Disease Registry Priority List of Hazardous Substances .The main source of exposure to cadmium for the general population is smoking tobacco or consumption of food containing cadmium ,139. InhDue to the endocrine-disrupting properties of cadmium , it has Multiple mechanisms potentially link cadmium to cancer, including oxidative stress and inflammation ,149, intAlthough the cadmium concentration in cancerous endometrial samples might not be different than that found in noncancerous tissue  or only Prospective studies in Sweden showed an association between estimated dietary cadmium and endometrial cancer  and postCho et al.  conducteOf interest is the association between cadmium and endometrial cancer depending on the BMI: Eriksen et al. estimated dietary cadmium intake in the 1993–1997 Diet, Cancer and Health cohort at enrollment. A positive association between cadmium and endometrial cancer for the women with BMI < 25 was found, whereas an inverse association was seen for the women with BMI ≥ 25 .Several studies found that stronger associations between cadmium and cancer risk were observed among individuals with low bioavailable estrogen such as low body mass index (BMI) ,164,165 Difficulty in the evaluation of the lifelong exposure ;Lipophilic EDCs are stored in the fat tissue with a bio-accumulation of minimal daily doses, therefore its rapid mobilization during a drastic diet can expose the person to high doses of the chemical which are not well evaluated in the studies;EDCs can have effects at low doses that are not predicted by effects at higher doses (it cannot be assumed that there is a threshold because hormones can regulate the hormone receptors expression resulting in an inverted U dose-response curve); NMDR can arise from numerous molecular mechanisms such as opposing effects induced by multiple receptors differing by their affinity, receptor desensitization, negative feedback with increasing dose, or dose-dependent metabolism modulation;Individuals can be concurrently exposed to different EDCs (cocktail effect) generating unpredictable interactions, because the epigenetic effects can also affect future generations especially if the exposure had been in vulnerable developmental periods ; EDCs are associated with declining human reproductive health, as well as an increasing incidence of cancers of the reproductive system. Verifying such links requires animal models exposed to “real-life”, environmentally relevant concentrations/mixtures of EDC, particularly in utero, when sensitivity to EDC exposure is maximal;Some individuals can have a particular susceptibility to ED carcinogenesis;It’s not always evaluated that EDCs can also have indirect effects on carcinogen metabolism, immune system, oxidation and inflammation;The competing interests may limit research and public information on ED effects.The environment has a large biological impact on human health and disease. During decades, the long-term and persistent effects of chemical compounds to which humans are exposed daily have been ignored because of the lack of scientific information and public awareness. The carcinogenic effects of EDCs are hard to define for the following reasons:Generally exposures at critical windows of early development  have the most dramatic impact on later life disease development or abnormal physiology. This developmental concept is referred to as the developmental origins of health and disease . Since eIn conclusion, the carcinogenic effects of endocrine disruptors is plausible although additional studies are needed to clarify their mechanisms and entities. Nevertheless, to reduce ED exposure is mandatory to implement necessary measures to limit exposure, particularly during those periods of life most vulnerable to the impact of oncogenic environmental causes, such as embryonic period and puberty.


In the rodent hippocampus in vitro, the so-called giant depolarizing potentials (GDPs) constitute a primordial form of neuronal synchrony preceding more organized forms of activity such as oscillations in the theta and gamma frequency range. GDPs are generated at the network level by the interaction of the neurotransmitters glutamate and GABA which, immediately after birth, exert both a depolarizing and excitatory action on their targets. GDPs are triggered by GABAergic interneurons, which in virtue of their extensive axonal branching operate as functional hubs to synchronize large ensembles of cells. Intrinsic bursting activity, driven by a persistent sodium conductance and facilitated by the low expression of Kv7.2 and Kv7.3 channel subunits, responsible for IM, exerts a permissive role in GDP generation. Here, we discuss how GDPs are generated in a probabilistic way when neuronal excitability within a local circuit reaches a certain threshold and how GDP-associated calcium transients act as coincident detectors for enhancing synaptic strength at emerging GABAergic and glutamatergic synapses. We discuss the possible in vivo correlate of this activity. Finally, we debate recent data showing how, in several animal models of neuropsychiatric disorders including autism, a GDPs dysfunction is associated to morphological alterations of neuronal circuits and behavioral deficits reminiscent of those observed in patients.Synchronized neuronal activity occurring at different developmental stages in various brain structures represents a hallmark of developmental circuits. This activity, which differs in its specific patterns among animal species may play a crucial role in  This in turn stimulates downstream cascades essential for several developmental functions  that in adulthood controls spike after-depolarization and burst generation  gap junctions. They constitute a way of signaling particularly well developed in immature neurons. In the hippocampus, neuronal coupling via gap junctions, contributes to trigger GDPs as demonstrated by their disruption upon gap junctions uncoupling with octanol  the low expression of Kv7.2 and Kv7.3 channels, responsible for the non-inactivating, low-threshold M current , the natural ligand for chemokine motif receptor 4 (CXCR4), known to play an important role in brain development, decreases GDPs frequency, an effect prevented by T140, a CXCR4 receptor antagonist, suggesting that SDF-1 alpha modulates GDPs via CXCR4. The inhibitory action of SDF-1 alpha on GDPs may reflect a potential mechanism for chemokine regulation of neural development early in postnatal life  that follows GDPs  oscillations occurring at ~0.1 Hz, followed by multiunit bursts lasting from 0.5 s to 3 s  and epilepsy . During maturation, silent periods between bursts decrease and, starting from the 30th week post-conception, the “tracé discontinue” evolves into the “tracé alternant” , found in a family with two autistic children  may impair GDPs expression and circuit formation. These alterations are often present in animal models of neurodevelopmental disorders including ASDs  of the γ2 subunit of GABAFurthermore, GDPs are sensitive to psychoactive agents as alcohol, particularly deleterious for babies, if assumed during pregnancy (Riley et al., It is important to stress that a causal link between GDPs disruption and behavioral deficits observed in animal models on neuropsychiatric disorders has not been proven yet and the possibility that alterations of GDPs expression are the consequence and not the cause of the above mentioned disorders cannot be excluded. Obviously, these are not mutually exclusive alternatives.in vivo counterpart of GDPs? Which circuits are involved? Combined optogenetic tools and in vivo recordings from the hippocampus and neocortex of freely moving animals may contribute to unveil the spatial and temporal contribution of selective glutamatergic and GABAergic pathways to SPW/GDP generation. In addition, optical imaging techniques may help elucidating, at mesoscale levels, brain activity in vivo (McVea et al., Although in the last years significant progresses have been made to understand the mechanisms of GDPs generation and their functional role in modifying synaptic strength at emerging GABAergic and glutamatergic connections (Ben-Ari et al., MG and EC wrote the article and MG generated the Figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Their applications have established the role of broadly neutralizing antibodies to control HCV infection. However, the virus has immunogenic regions in the viral envelope glycoproteins that are associated with viral escape or non-neutralizing antibodies. These regions serve as immunologic decoys that divert the antibody response from less prominent conserved regions mediating virus neutralization. This review outlines the immunogenic regions on E2, which are roughly segregated into the hypervariable region 1 (HVR1), and five clusters of overlapping epitopes designated as antigenic domains A-E. Understanding the molecular architecture of conserved neutralizing epitopes within these antigenic domains, and how other antigenic regions or decoys deflect the immune response from these conserved regions will provide a roadmap for the rational design of an HCV vaccine.The ability to use structure-based design and engineering to control the molecular shape and reactivity of an immunogen to induce protective responses shows great promise, along with corresponding advancements in vaccine testing and evaluation systems. We describe in this review new paradigms for the development of a B cell-based HCV vaccine. Advances in test systems to measure  Chronic hepatitis C virus (HCV) infection often leads to chronic hepatitis, liver failure and hepatocellular carcinoma  targets  derived from B cells of subjects with chronic HCV infections have identified at least seven clusters of overlapping epitopes on HCV E1E2. Four clusters, designated as antigenic domains A-D, are composed of conformational epitopes on E2 Table . Two addGlobal alanine scanning of E2 has recently been reported using E2-binding HMAbs and site-directed mutagenesis of the E1E2 coding sequence, and ELISA readout assays to compare mutant binding levels to wild-type Figure . This anNot all broadly neutralizing antibodies share the same anti-viral characteristics. In virus co-culture studies, some antigenic domain B antibodies are associated with viral escape, with and without compromising viral fitness, and others are resistant to viral escape. For instance, at a critical antibody concentration, HC-1, a domain B HMAb, can eliminate infectious virions in cell culture with no viral escape mutants detected. Other antibodies, such as CBH-2 and HC-11 that target the same antigenic domain, permitted viral escape through specific mutations in a region of domain B after repeated passages in the presence of increasing concentrations of the antibodies  structures of short helical regions (Op De Beeck et al., Due to the absence of experimentally determined structures of the E1E2 heterodimer, two recent studies have used computational modeling methods to predict the structure of this assembly (Castelli et al., Mechanisms of HCV evasion from the immune system have been described in several recent reviews (Cashman et al., Recent studies have highlighted the role of E2 co-receptor recognition in the evasion of the antibody response. Using an analysis of monoclonal antibody resistance among a large set of genotype 1 isolates, El-Diwany et al. identified key sites on E2 that permitted escape through modulation of E2 binding to SR-B1 (El-Diwany et al., Approaches for HCV vaccine development have included production of HCV E2 and E1E2 recombinant envelope proteins and use of immuno-adjuvant systems to complex engineering of viral vectors expressing multiple antigens. The rationale for these approaches has been driven by a requirement to elicit multiple broadly neutralizing antibodies to achieve sterilizing immunity to prevent HCV infection and how best to achieve such immunity. Extensive human and animal studies have been performed with a recombinant genotype 1a E1E2 vaccine (Frey et al., The correlation of host antibody response to HCV with viral clearance and recent successes in structure-based vaccine design for other viruses, such as HIV (Jardine et al., Given recent evidence of mobility of key E2 epitopes associated targeted by neutralizing antibodies (Deng et al., The medical burden of hepatitis C has decreased by the introduction of effective antiviral therapies. However, control of this insidious disease will require the successful development of an effective preventative vaccine. To achieve this goal, the complex interplay between virus and host during acute infection that leads to spontaneous clearance needs to be better understood to provide clues of protective immunity. The criteria for what constitutes a successful immune response leading to viral clearance in designing at vaccine that is broadly cross protective across the genotypes and long lasting will be the holy grail of such vaccine development efforts. Recent insights from deep sequencing, antibody-antigen interactions, structural biology, and immunogenicity studies provide the basis for novel vaccine designs for this challenging target. While accounting for the primary mechanisms of viral escape, namely sequence variability within the viral envelope glycoproteins including prominent decoy epitopes such as HVR1, immunogens can be designed to enhance their capacity to induce potent neutralizing antibodies to conserved epitopes on the viral envelope. These approaches, combined with novel adjuvant formulations capable of eliciting robust and long-lasting humoral and cellular responses, will significantly advance vaccine development efforts to successfully address this daunting medical challenge.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pseudomonadales, Vibrionales, and Rhodobacterales. Molecular ecological networks and phylogenetic trees showed various adaptive abilities of bacteria to the different organic compounds. For instance, some bacterial OTUs could be found only in particular organic compound-treated groups while some other OTUs could tolerate stresses from different organic compounds. Furthermore, the results indicated that some new phylotypes were emerged under stresses of different organic pollutions and these new phylotypes could adapt to the contaminated environments and contribute significantly to the microbial community shifts. Overall, this study demonstrated a crucial role of the community succession and the acclimation of functional bacteria in the adaptive responses to various environmental disturbances.The phylogenetic diversity of bacterial communities in response to environmental disturbances such as organic pollution has been well studied, but little is known about the way in which organic contaminants influence the acclimation of functional bacteria. In the present study, tolerance assays for bacterial communities from the sediment in the Pearl River Estuary were conducted with the isolation of functional bacteria using pyrene and different estrogens as environmental stressors. Molecular ecological networks and phylogenetic trees were constructed using both 16S rRNA gene sequences of cultured bacterial strains and 16S rRNA gene-based pyrosequencing data to illustrate the successions of bacterial communities and their acclimations to the different organic compounds. A total of 111 bacterial strains exhibiting degradation and endurance capabilities in response to the pyrene estrogen-induced stress were successfully isolated and were mainly affiliated with three orders,  Skeletonema costatum) used to counteract crude oil pollution. The results demonstrated that hydrocarbonoclastic bacteria in the phycosphere of phytoplankton could contribute significantly to biodegradation of hydrocarbon contaminants in marine surface water. Bacteria can adapt to a range of different habitats, including contaminated and extreme environments, and they can perform a wide diversity of physiological activities . In termIn the natural environment, microbial communities are not only a simple collection of independent individuals but also exhibit a complex interconnection between different microbial taxa found in the vicinity. These complex inter-taxa interactions make the collective microbiome more significant and effective than the function of any individual constituent species . It is oDiversity in microbial communities could significantly promote stability, productivity, and sustainability in an ecosystem . In a stPseudomonadales, Vibrionales, and Rhodobacterales, were conducted by examining culturable bacterial communities in cooperation with analyzing the next generation sequencing (NGS) data. We hope the current study would enlighten a better understanding of the bacterial acclimation to the changing environments.In the present study, we aimed to investigate the changes of bacterial communities and their acclimation to the different organic compounds (pyrene and estrogens) as environmental stressors. To this end, phylogenetic analysis and network re-construction of three bacterial phylotypes, affiliated into in situ, with a value of 26.5°C for water temperature, 21.6 g/L for salinity, and 7.9 for pH. All samples were kept in 4°C coolers and transferred to the laboratory immediately for further analyses.Bacterial strains were isolated from the subsurface sediments collected from the estuary of the Pearl River  at a depth of 8 m during our summer cruise trip . The estuary of the Pearl River was selected for sampling due to the abundance of organic pollutants in this region, which has been the result of industrialization and urban development in conjunction with fishing and aquacultural activities in the surrounding areas . PhysiolAll organic pollutants used in the present study were of analytical grade or better. A typical PAH pyrene and four different estrogens consisting of estrone (E1), 17β-estradiol (E2), estriol (E3), and 17α-ethinyl estradiol (EE2) were used as environmental stresses   were spread onto MSM agar plates pretreated with pyrene or the different estrogens . The MSM agar plates were prepared by adding 2% of agar powder  into MSM liquid medium. All plates were incubated at 25°C for the next 3 days in order to examine microbial growth. The colonies having different morphological features were streaked individually onto MSM agar plates, pre-supplemented with the different organic pollutants, and incubated again for another 3 days at 25°C for growth. A single colony of each isolate was further cultured in marine broth 2216E  overnight to cultivate enough bacterial cells for cryopreservation (-80°C with addition of 30% glycerol) and DNA extraction. Genomic DNA of each individual isolate was extracted by using an Ultra-Clean microbial DNA isolation kit . The PCR amplification of each isolate’s 16S rRNA gene was carried out using universal primers 27F and 1492R . A total of 10 g of sediments were inoculated into 100 mL of mineral salt medium   and incund 1492R  and thenTo determine the composition of cultured bacterial communities without environmental pressure, marine broth 2216E  was used to isolate pure cultures from original sediments using the procedures described above, except that original sediments were enriched in marine broth 2216E for 1 day without the addition of organic pollutants, instead of the MSM medium.To detect the degrading ability of cultured bacteria with the stress of pyrene, representative bacterial strains were selected different bacterial phylotypes based on phylogenetic analysis. Selected bacterial strains were, respectively, inoculated into 100 mL MSM medium with the supplement of 100mg/L pyrene and incubated in a constant-temperature shaker at 25°C, 150 rpm. Three experimental setups were stopped for culturing at different incubation time points  and used for extract residual pyrene while other experimental setups were kept incubation. Residual pyrene was extracted by dichloromethane analyzed by GC-MS (Agilent 7890-5975c) as the method described before .Pseudomonadales, Vibrionales, and Rhodobacterales, were selected from all sequencing for the following analysis.Aliquots (0.25 g) of original sediments and 1.5 ml of liquid cultures at different incubation time points  under the stress of pyrene were collected. Genomic DNA was extracted using an Ultra-Clean PowerSoil DNA isolation kit . DNA concentrations and purity were measured using a NanoDrop 2000 spectrophotometer . A total amount of 100 ng (with a concentration of 10 ng/μl) of each sample were sent for sequencing by Illumina HiSeq 2500 platform at the BGI. For the analysis of NGS data, Metaxa2  was usedPseudomonadales, Vibrionales, and Rhodobacterales) and the reference sequences downloaded from the NCBI for these three orders were further clustered into different OTUs by using the clustering algorithm, CD-HIT  based on sequence similarities . In the , CD-HIT . The sel1    with refPseudomonadales, Vibrionales, Rhodobacterales, Alteromonadales, Bacillales, Rhizobiales, and Sphingomonadales . A large number of significant structural differences among all of the cultured bacterial isolates that were isolated from different experimental setups with the addition of different organic pollutants and normal 2216E medium were observed. The number of bacterial strains belonging to the order Pseudomonadales and Vibrionales significantly increased under stress conditions (presence of pyrene and estrogens), whereas they were relatively low in proportion comparing to the whole cultured community found in simple 2216E medium (without any organic pollutants). In contrast, bacteria from the Bacillales order showed a sharp quantitative decrease under pyrene- and estrogens-treated incubation conditions.The sediment samples were added to MSM, and incubated for 30 days with pyrene and estrogens. It was observed that pyrene was almost completely degraded within 30 days of incubation , Vibrionales (mainly the genus Vibrio and Photobacterium), and Rhodobacterales. The 16S rRNA gene sequences of these strains have been deposited in GenBank under accession numbers MF948916–MF948993. These selected bacterial isolates (78 isolates) were further successfully divided into different 37 OTUs using CD-HIT software   were selected for further investigation in the present study, on the basis of both their potential degradation/toleration capabilities and their large proportions in bacterial communities. These strains were affiliated with three orders, including Vibrionales; strains PrPl070 and PrPl084 affiliated into the order Pseudomonadales; and strains PrRy136 and PrRr052 affiliated into the order Rhodobacterales were used to test the capability of degrading pyrene  and heatmap  analyses were done.To illustrate the responses and successions of functional bacterial phylotypes to pyrene- and estrogens-induced stress and non-stress environments (2216E medium), molecular ecological network  were found only in the pyrene-treated groups, while OTU5 (Vibrio spp.) was detected only in the E1-treated samples. Other estrogen-treated (E2) samples also had their own specific OTUs . Nevertheless, some functional bacterial species also exhibited extensive interactions with different pollutants, which indicated a positive response from these bacterial species toward the different organic pollutants, and hence they might play a significant role in the degradation of different pollutants. For example, OTUs 1 and 15  were found to be common in both pyrene- and estrogens-treated samples. OTUs 1, 15, and 26  were shared by pyrene- and E1-treated samples. In addition, the OTUs under pyrene tolerance were different from those isolated from MSM plates with added estrogens. For example, OTU15 (Pseudomonas spp.) could only be detected after E1 and E3 treatments. The heatmap profile analysis indicated that quantitative changes in different bacterial phylotypes in pyrene- and estrogens-treated samples had occurred. Results from the analysis indicated that some of the functional bacteria emerged in both pyrene- and estrogens-treated groups. The presence of some new bacterial phylotypes was also observed in pyrene-treated samples after 30 days of incubation. Results from the above analysis indicated that, during ecological diversity investigation, certain specific functional phylotypes should also be considered along with whole local communities.The correlation interpretation between functional groups of different organic compounds without considering the abundance of different bacterial phylotypes was carried out using molecular ecological network analysis. The pyrene-treated samples appeared to generate more OTUs compared to the estrogen-treated samples. Some OTUs showed an obvious bias to the different substrates. For instance, OTUs 12 and 16  analysis of the OTUs from NGS data demonstrated that the continuous shift in pyrene-degrading community structure. The NMDS plots showed that relatively large changes in bacterial communities occurred at 12 and 24 days, while small community changes were observed before 6 days. The initial large changes in bacterial community at 12 days could be attributed to adaptation of the seeded bacterial communities to the stress environment. The community profiles from 12 days were most widely dispersed and distantly located from the profiles from the early communities. This seemed to be mainly affected by the maintenance of highly dominant Alteromonadales populations and the decrease of Vibrionales and Desulfobacterales until the end of incubation . An obvious decrease of OTUs was observed after 12 days for all three bacterial groups which might due to the redundancy reduction. Like composition of culturable bacterial communities, the bacterial communities after 12 days revealed by NGS were also dominated by key functional OTUs, such as OTU P50 (Acinetobacter spp.), OTU V0 (Photobacterium spp.), and OTU R1 (Ruegeria spp.). Moreover, the molecular ecological network based on NGS data . These strains were regarded as emerging phylotypes, because of their phylogenetic distance from other strains in the tree and their absence in bacterial communities enriched in 2216E medium without any organic pollutant . In general, some of the emerged phylotypes were not easily identified from phylogenetic tree analysis, but could be easily discovered with the help of CD-HIT software in the present study. For instance, OTUs 24 and 13, including strains PrPf083 and PrPl085 were identified as emerging taxonomies through the CD-HIT analysis. Based on phylogenetic analysis, certain new phylotypes were also explored in the genus Acinetobacter, such as OTU12 , OTU11 (PrPl070), OTU18 (PrPb063 and PrPb064), and OTU21 (PrPf067)  in the genus Vibrionales ; these phylotypes included OTUs 2 and 6 . OTUs representing Photobacterium spp. (OTUs 2 and 10) and Vibrio spp. (OTUs 6 and 8) were detected; these might have been induced by the stress of pyrene and EE2. The order Rhodobacterales also exhibited some new phylotype OTUs . It was also observed that certain microbial strains belonging to the genus Yangia were present in the pyrene-treated incubations but absent in estrogen-treated cultures.Emerging phylotypes were also observed in the tree of the order Rhodobacterales positively responded to pyrene, but not estrogens. Rhodobacterales were acknowledged to be capable of degrading aliphatic and low-molecular-weight aromatic hydrocarbons  were isolated from the pyrene- and the estrogen-treated samples. The sample analysis at different exposure time intervals was considered as a key factor in microbial adaptation to the different organic contaminants. It was well known that bacterial species belonging to the genera Pseudomonas and Vibrio have efficient capabilities for degrading various organic compounds, including crude oil and estrogens  before being co-incubated with EE2, while two new OTUs (OTUs 6 and 8) emerged under EE2-induced stress. Similarly, two new phylotypes (OTUs 13 and 24) affiliated to Pseudomonas pseudoalcaligenes were also detected after the addition of the organic pollutants, while only OTU15 was identified in the original samples. These new phylotypes were capable of tolerating or degrading the additional organic pollutants and acted as a substitute for those phylotypes, which were unsuitable to the changing environment, thus led to a community shift. This shift of ecological diversity could be attributed to physiological changes in the community or to the indirect effects on other members of the community , Vibrionales (0.21–26.22%), and Rhodobacterales (0.45–4.02%), were abundant and showed dramatic degrading efficiency to pyrene. However, the dominant bacterial sequences at different time points were all affiliated into another bacterial order, Alteromonadales. Bacteria in this order were anticipated to have potential function for metabolizing different hydrocarbons . For instance, these novel branches represent OTUs 12, 18, and 21 in the order Pseudomonadales and OTUs 2, 8, and 10 in the order Vibrionales, respectively. The evolutionary changes shown in the phylogenetic trees could influence population dynamics, species interactions, and even ecosystem functioning  were used as environmental disturbances or stresses for the analysis of bacterial acclimation. The results revealed that the organic pollutant-induced stresses could significantly influence the bacterial community composition, as well as the acclimation of functional bacteria in these communities. The appearance of new phylotypes under these stress conditions was favorable for community stability as well as for the remediation of the polluted environment. The overall results indicated that the acclimation of functional bacteria was crucial for the adaptation of bacterial communities to environmental disturbances. Considering the finite number of pure cultures and limited information 16S rRNA gene based NGS, this study could not provide a complete picture of the acclimation of bacterial communities to changing environments. The future work could be addressed by (i) analyzing the genomic changes of specific bacterial species to illustrate the mechanism of their capabilities of biodegradation or tolerance; (ii) revealing the co-acclimation and co-evolution of bacterial communities based on metagenomic, metatranscriptomic, and metaproteomic data; and (iii) isolating “uncultivable” functional bacteria in pure culture for comprehensively revealing the function of bacterial community under the press of organic compounds.All 16S rRNA gene sequences of newly isolated bacterial strains and NGS data are publicly available in the GenBank database (NIH genetic sequence database). The accession numbers for the 16S rRNA gene sequences of cultured bacterial strains are MF948916–MF948993, while the accession numbers for NGS data are listed in Supplementary Table SZ, ZH, and HWa designed the experiment; SZ, XY, JX, CS, and HWe performed the experiment; SZ and HWa collected and analyzed the data and wrote the draft of the article; SZ, AP, ZH, and HWa revised the draft of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite clinical practice guidelines recommending the routine provision of nicotine dependence treatment to smokers in inpatient psychiatric facilities, the prevalence of such treatment provision is low. The aim of this study was to examine the effectiveness of a clinical practice change intervention in increasing clinician recorded provision of nicotine dependence treatment to patients in inpatient psychiatric facilities.N = 4175) over a 19 month period in two inpatient adult psychiatric facilities in New South Wales, Australia. The clinical practice change intervention comprised six key strategies: leadership and consensus, enabling systems and procedures, training and education, information and resources, audit and feedback and an on-site practice change support officer. Systematic medical record audit and segmented logistic regression was used to determine differences in proportions for each nicotine dependence treatment outcome measure between the ‘pre’, ‘during’ and ‘post-intervention’ periods.We undertook an interrupted time series analysis of nicotine dependence treatment provision before, during and after a clinical practice change intervention to increase clinician recorded provision of nicotine dependence treatment for all hospital discharges ; assessment of patient nicotine dependence status ; provision of brief advice to quit ; provision of nicotine replacement therapy ; and provision of nicotine dependence treatment on discharge .This is the first study to provide evidence that a clinical practice change intervention may increase clinician recorded provision of nicotine dependence treatment in inpatient psychiatric settings. The intervention offers a mechanism for psychiatric facilities to increase the provision of nicotine dependence treatment in accordance with clinical guidelines.The online version of this article (doi:10.1186/s12888-017-1220-7) contains supplementary material, which is available to authorized users. Smoking remains a leading global cause of death and disability . While sDespite clinical practice guidelines , 14 recoSystematic review evidence supports the effectiveness of a number of clinical practice change strategies for improving health-related practices across a range of clinical settings, including: engaging local opinion leaders , audit aTo address this evidence gap, a study was undertaken to assess the effectiveness of a multi-modal clinical practice-change intervention in increasing clinician recorded provision of five recommended elements of nicotine dependence treatment: 1) assessment of smoking status; 2) assessment of nicotine dependence; 3) provision of brief advice to quit; 4) provision of NRT; and 5) provision of nicotine dependence treatment at discharge to patients admitted to inpatient psychiatric facilities.st October 2009), ‘intervention period’ (nine months from 1st November 2009 to 31st July 2010), and ‘post-intervention’ (five months from 1st August to 31st December 2010).An interrupted time series study was undertaken. A clinical practice change intervention was implemented for nine months, with outcome data collected for three periods over 19 months: ‘pre-intervention’  consisted of a discrete psychiatric facility catering for approximately 2000 patient discharges per annum across five units: an emergency/intensive care unit, two general acute units, a dual diagnosis unit, and an older persons unit. The second psychiatric facility (125 beds) catered for approximately 750 patient discharges per annum from a single general acute psychiatric care unit. Both facilities introduced a smoke-free policy in all buildings and grounds three years prior to the study.The study participants consisted of all adult (>18 years) patient discharges, including repeat discharges for individual patients, and for both voluntary and involuntary patients.All clinical staff (approximately 170), including registered nurses, nurse managers, allied health professionals, and medical/psychiatric staff received the intervention. Based on both local and international clinical guidelines , 14, cliConsultation with senior medical, nursing and facility management staffUnit level meetings with managers, and clinical leaders and staff using the principles of motivational interviewing ;Meetings with consumer advocates, and;Appointment of local (minimum of 1 per unit) clinical champions.Leadership and consensusNSW Health Guide for the Management of Nicotine Dependent Inpatients [Distribution of state nicotine dependence treatment guidelines, the patients  (see AddModification of existing admission forms to prompt recording of smoking treatment.Enabling systems and proceduresn = 30) to increase awareness, agreement and adoption of nicotine dependence treatment guidelines;o Face-to-face staff group training sessions (n = 33), and;o Problem solving with local champions and clinician groups on ‘as needs’ basis .o Audit and feedback of unit medical records regarding prevalence of nicotine dependence treatment  was available to support delivery of the above strategies.All components of the medical record for each patient discharged during the study period were audited post-discharge by trained auditors employed by the research team , 29. AudPatient characteristicsb)Nicotine dependence treatmentThe following patient characteristics were obtained from the medical record: gender, age (determined by date of birth), admission and discharge dates, previous admissions , number of discharges during study period), treating unit at discharge, Aboriginal or Torres Strait Islander status , English speaking . Patient diagnosis was classified via ICD-10 codes .b)NicotiNSW Health Guide for the Management of Nicotine Dependent Inpatients [assessment of smoking status ;assessment of nicotine dependence ;provision of brief advice to quit ;prescription of any form of NRT  , and;provision of nicotine dependence treatment on discharge record  .Based on the patients , see AddAnalyses were conducted using SAS version 9.4 software . AdmissiDescriptive statistics were used to summarise patient characteristics, and prevalence of each of the nicotine dependence treatment measures at each of three time periods . Chi-square analyses were undertaken to assess differences in patient socio-demographic and clinical characteristics between patients with one or more admission, and between study periods.st October 2009), nine time points in the ‘intervention period’ (1st November 2009 to 31st July 2010), and five time points in the ‘post-intervention’ period (1st August to 31st December 2010). The models adjusted for differences in patient socio-demographic and clinical characteristics (including discharging unit) between study periods. All discharges were treated independently in the analyses. All statistical tests were two-tailed, and the threshold for statistical significance was p < 0.01. We used the conservative 1% threshold for statistical significance  in an attempt to correct for an increased overall type I error rate from the multiple tests  that were conducted.Similar to other studies of nicotine dependence treatment in hospital settings based on medical record audit , 33, eacn = 1277, 30.6%) were for patients readmitted to the facilities during the study period. There were no differences in socio-demographic or clinical characteristics between patients who had one or multiple admissions.There were 4,175 discharges  during the 19 month study period for 2,898 individual patients. Nearly one third of total discharges (p < .0001), and these variable were added as confounding variables to the segmented logistic regression models , had an average age of 39.2 years SD = 15.3) and an average length of stay of 17.0 days (SD = 33.4). The most prevalent primary diagnoses on admission were schizophrenia and related psychosis (25%), adjustment disorders (18%) and unipolar affective disorders (17%). Primary diagnosis on admission, and discharging unit were identified to vary significantly between study periods . As shown in Fig. Stratified results for the 100-bed (~2000 discharges per annum) and 125-bed facility (~750 discharges per annum) are shown in Tables This is the first study to examine the effectiveness of a clinical practice change intervention in increasing the recorded provision of nicotine dependence treatment in inpatient psychiatric facilities. The findings suggest that the intervention was effective in increasing clinician-recorded provision of nicotine dependence treatment, with increases identified for all five nicotine dependence treatment measures during the intervention period, and continuing for up to five months post-intervention.The magnitude of the observed improvement in recording of nicotine dependence treatment in our study are similar to , and in The observed rates of recorded provision of the five nicotine dependence treatment variables were stable across the pre-intervention period, and increased in accordance with the timing of the delivery of the clinical practice change intervention. Critically, these increases continued into the post-intervention period, suggesting not only the efficacy of the clinical practice change intervention, but some degree of sustainability of effect. This finding adds to the growing body of evidence that multimodal nicotine dependence treatment interventions show efficacy in increasing nicotine dependence treatment provision in hospital settings both during, and after implementation of the intervention . FurtherNotwithstanding the consistent increases in all outcomes, at post-intervention, an assessment of smoking status occurred for only 52% of patients, with even lower rates for other elements of nicotine dependence treatment. This suggests that additional strategies may be required to ensure that all patients receive nicotine dependence treatment. Previous research has indicated that interventions that are integrated with, and address existing hospital systems and procedures may maximise nicotine dependence treatment delivery , 39, 43.This study has multiple strengths, including its’ large sample size, inclusion of a number of patient diagnostic groups, and use of systematically independently collected medical record audit data. However, a number of study characteristics may have impacted on its findings. Firstly, we relied on medical record audit as a measure of clinical staff provision of nicotine dependence treatment. It is possible that clinical staff may have recorded care delivery in the absence of it occurring, and hence overestimated actual delivery of nicotine dependence treatment. However, systematic review evidence suggests that medical record audit is more likely to underestimate clinical staff behaviour . AlthougSecondly, given that the recording of smoking status did not occur systematically for all patients at any time point during the study, the true sample of smokers for whom nicotine dependence treatment should be delivered was unknown. Previous research conducted among patients in the same facility within the same timeframe indicated that smoking rates were approximately 53% , 48. HenThird, given our focus on increasing clinician-recorded nicotine dependence treatment according to the standard hospital care guidelines, we did not collect data on other factors that may have had an impact on the likelihood of clinical staff providing and recording such treatment to patients. This may have included the voluntary or involuntary status of patients, which has shown to be important in the implementation of smoke-free policies in locked psychiatric facilities, given the relative abilities of these patients to leave the grounds to smoke. While we did not collect such data in our study, in Australia in 2014–15 it was estimated that 31.1% of all hospitalisations to specialised psychiatric care services were for patients who had an involuntary admission . These tFurther, utilising a controlled trial design was not feasible, and the use of a non-controlled design constrains our ability to directly attribute the observed increases in recording of nicotine dependence treatment to the intervention component. Given the limited resources available, the study facilities were required to be within close geographical proximity to the administering University, and were too dissimilar in size and annual patient admissions in order for one site to act as a comparator to the other. As such, the interrupted time series design was adopted, as it has been demonstrated to be appropriate in evaluating the efficacy of clinical practice change interventions in hospital settings where whole-of-system practice change is the focus of the intervention . FurtherFinally, although the study data were collected from 2009–2010, more recent evaluations of the study facilities by the authorship team indicate that prevalence of smoking among patients, and clinician provision of nicotine dependence treatment  are unlikely to have changed in this time , 47, 54.This study is the first to examine the effectiveness of a multi-modal clinical practice change intervention in increasing recorded nicotine dependence treatment in inpatient psychiatric facilities. The use of inpatient psychiatric facilities as a setting for supporting and encouraging smokers to change behaviour may make an important contribution to addressing the recognised need to reduce smoking rates for this population group. Our results provide evidence that improvement in the routine recording of nicotine dependence treatment is achievable on a whole-of hospital basis and are sustainable in the short to medium term. However additional strategies are required to ensure all smokers are systematically identified and offered nicotine dependence treatment.


The aims of the present study were to explore the expression pattern of haem biosynthesis enzymes in circulating cells of patients affected by two types of porphyria , together with the antioxidant enzyme pattern in AIP in order to identify a possible situation of oxidative stress. Sixteen and twelve patients affected by AIP and VP, respectively, were analysed with the same numbers of healthy matched controls. Erythrocytes, neutrophils and peripheral blood mononuclear cells (PBMCs) were purified from blood, and RNA and proteins were extracted for quantitative real time PCR (qRT-PCR) and Western-blot analysis, respectively. Porhobilinogen deaminase (PBGD) and protoporphyrinogen oxidase (PPOX) gene and protein expression was analysed. Antioxidant enzyme activity and gene expression were additionally determined in blood cells, together with protein carbonyl content in plasma. PBMCs isolated from AIP patients presented low mRNA levels of PBGD when compared to controls, while PBMCs isolated from VP patients presented a decrease in PPOX mRNA. PPOX protein content was higher in AIP patients and lower in VP patients, compared to healthy controls. Regarding antioxidant enzymes, PBMCs and erythrocyte superoxide dismutase (SOD) presented statistically significant higher activity in AIP patients compared to controls, while catalase activity tended to be lower in these patients. No differences were observed regarding antioxidant gene expression in white blood cells. Circulating cells in AIP and VP patients present altered expression of haem biosynthetic enzymes, which could be useful for the differential diagnosis of these two types of porphyria in certain difficult cases. AIP patients present a condition of potential oxidative stress similar to VP patients, evidenced by the post-transcriptional activation of SOD and possible catalase impairment. Porphyrias are a group of metabolic disorders where haem biosynthesis is affected, thus resulting in the accumulation of porphyrins or their precursors in different tissues ,2. AlthoIt has been well described that the chronic accumulation of haem precursors could enhance reactive oxygen species (ROS) production and generate an unbalanced oxidative situation, especially in patients with low exogenous antioxidant defences such as porphyria patients –9. In faThe aim of this study was to compare the expression levels of PBGD and PPOX in circulating blood cells of AIP and VP patients, and, in the case of AIP patients, to analyse the antioxidant enzyme activity in erythrocytes, peripheral blood mononuclear cells (PBMCs) and neutrophils, compared to matched controls. As for the samples taken from VP patients, these were compared with previously published data.A total of 16 AIP patients  from Murcia (Spain) recruited through Health Centres of endemic areas were contacted and agreed to participate in the study. The AIP condition had been clinically diagnosed to all the patients included in the study. AIP patients carried a mutation in the 12 exon (30-bp deletion) 669–698 position, 11 g 23Æ3 locus, and presented PBGD enzyme activity decreased by 50%. A complete clinical, biochemical and anthropometric characterisation of these patients has already been published [A total of 12 women affected by VP, as well as 12 pair-matched healthy control women, were also recruited for the study using the Balearic Association of Porphyria, which registers all the porphyric patients in Balearic Islands (Spain). The subjects included in the VP group had been previously diagnosed by specialized doctors on the basis of different parameters such as plasma fluorescence peak at 626 nm, levels of excreted urinary and faecal porphyrins and clinical manifestations such as abdominal pain during attacks . ControlThis study was conducted according to guidelines written in the Declaration of Helsinki and all procedures involving human subjects/patients were approved by the Ethical Committee of Clinical Investigation of the Balearic Islands and the Ethics Committee of Clinical Investigation of the University of Murcia. Written informed consent was obtained from all subjects.Venous blood samples were obtained from the antecubital vein of control and porphyric patients in resting conditions after overnight fasting and placed in EDTA vacutainers. PBMCs, neutrophils and erythrocytes were purified following an adaptation of the method described by Ferrer  using FimRNA expressions were determined by quantitative real time PCR (qRT-PCR). For this purpose, mRNA was isolated from PBMCs using Tripure Isolation Reagent . cDNA was synthesized from 1 μg total RNA using reverse transcriptase with oligo-dT primers. q-RT-PCR was performed using the LightCycler instrument  with DNA-master SYBR Green I . All PCRs were performed with the same basic program, with one cycle at 95°C for 10 min, followed by 40 cycles at the conditions shown in (-ΔΔCt). Basal mRNA levels at the beginning of the stage were arbitrarily referred to as 1. The expression of the target gene was normalized with respect to ribosomal 18S.The relative quantification was performed by standard calculations considering 2PPOX and PBGD protein levels were determined by Western blot. PBMCs samples were treated with RIPA (radioimmunoprotection assay) buffer and total protein concentrations were measured by the method of Bradford . ProteinProtein carbonyl derivatives were determined in plasma by immunological methods using the OxiSelectTM Protein Carbonyl Immunoblot Kit  by following the manufacturer’s details. Total plasma protein concentrations were measured by the Bradford method  using thSuperoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) activities were measured in PBMCs, neutrophils and erythrocytes as previously published ,13,15,16Statistical analysis was performed using the statistical package for social sciences (SPSS 19.0 for Windows). Results are expressed as mean ± SEM (standard error of the mean) and p<0.05 was considered statistically significant. The statistical significance was assessed by Student’s t-test for unpaired data.Gene expression and protein content of both PBGD and PPOX enzymes were analysed in PBMCs from AIP and VP patients and their respective controls in order to verify alterations in the haem biosynthetic pathway . AIP patOn the other hand, PBMCs isolated from VP patients presented decreased PPOX gene expression (about 75% of control) and similar PBGD expression compared to control subjects . Again, Although PBGD and PPOX protein levels were similar to controls in the PBMCs of both types of porphyria, it is very likely that the enzymatic activities are altered, leading to accumulation of haem biosynthesis intermediates and causing oxidative stress and defective function of haem-containing enzymes, such as catalase. For that reason, antioxidant enzyme activities were analysed in PBMCs, erythrocytes and neutrophils of AIP patients and compared to control subjects  and to pPBMCs SOD activity was significantly higher in AIP patients than in controls Catalase activity was approximately half normal levels in patients, although this difference was not statistically significant (p = 0.063). GPX activity was slightly, but not significantly, higher in AIP patients. Neutrophils presented an antioxidant enzyme activity pattern similar to that observed in PBMCs, although the differences between groups were not statistically significant . In addiAntioxidant gene expression was additionally assessed in white blood cells in order to explain the possible mechanisms behind the antioxidant response observed in enzyme activity. Antioxidant gene expression did not differ between AIP patients and controls . NeutropThe present study indicates that PBMCs isolated from AIP and VP patients possess different expression patterns of the enzymes that cause the disease. Specifically, while in AIP patients there are lower PBGD gene expression levels in PBMCs, VP patients present lowered PPOX gene expression. Although more than 130 mutations in the PBGD gene have been identified in relation to AIP, all patients manifest the same symptoms and the residual PBGD enzyme activity is approximately half normal levels, independent of the mutation carried. The present study is the first to demonstrate that AIP patients have lower PBGD gene expression in PBMCs compared to healthy individuals. This decrease in expression is not attributable to a technical artefact of the qRT-PCR technique, because the primers used in this study were not targeted to the mutated sequence, which could have impaired a correct annealing and resulted in a lower number of amplicons. Therefore, the most likely explanation is that the AIP patients analysed in this study actually presented lower PBGD mRNA levels in PBMCs than their respective controls. In accordance with our observation, a previous study reported that PBGD mRNA levels were found to be less than half in brains of Alzheimer’s disease patients than in controls . The lowA similar rationale can be assessed for the PPOX gene in PBMCs from VP patients, although the decrease in expression seems to be less prominent than in the case of the PBGD gene for AIP patients (only 20%). Furthermore, the determination of PBGD and PPOX protein levels in PBMCs does not present the differences observed using mRNA . The exp2O2 by the complex III of the mitochondrial respiratory chain than control women after ROS production stimulation [In any case, the impaired enzyme activities lead to the accumulation of porphyrin intermediates that can be easily oxidized and thus result in increased oxidative stress in affected tissues ,21. The mulation .2O2 produced by SOD during the reduction of the superoxide anion. In this context, and compared to controls, catalase activity tends to present a slightly lower activity in AIP patients. These results are in accordance with those previously reported in VP patients, who presented lower catalase activity in PBMCs and neutrophils [In addition to the higher ROS production presented by porphyria patients, the accumulation of porphyrins could also affect the functionality of antioxidant enzymes such as catalase, thus impairing the ROS detoxifying capacity. Catalase is a major antioxidant enzyme that presents a tetrameric structure containing one haem group in each subunit. This enzyme detoxifies the Htrophils ,16, evidRegarding the remaining antioxidant enzymes in circulating blood cells, SOD also seems to present a similar pattern of activity in AIP and VP patients, mainly in erythrocytes. AIP patients present higher SOD activity in circulating cells  when compared to controls, and erythrocyte SOD activity in VP patients is also higher than in their respective controls . Concern2O2, an important cytotoxic intermediate. Hypothetically, AIP patients should produce higher amounts of H2O2 since they present higher SOD activity compared to controls. However, and independently from the H2O2 levels produced in circulating blood cells, this toxic compound cannot be properly detoxified by catalase activity, a haem-containing enzyme which tends to be defective in porphyria patients. Therefore, GPX could be postulated as the main pathway to eliminate H2O2. Although GPX activity in porphyria patients seems to be normal, glutathione is necessary for this enzyme to function properly. In this context, intracellular levels of glutathione are dependent of many environmental factors which include diet antioxidants that in many cases are deficient in AIP patients, such is the case for vitamin E [2O2 than healthy controls with a potential impairment in the detoxifying mechanism for these compound, facing these cells with a potential situation of oxidative stress which is apparently not manifested in basal, non-prompted conditions, as evidenced by the similar protein carbonyl content between AIP patients and controls. This picture is very similar to that previously described in VP patients, who also do not present protein or DNA oxidative damage in basal conditions but are more susceptible to develop oxidative damage in situations of increased ROS production [Although a precise picture of the antioxidant pattern observed in porphyria patients is complicated, our results allow us to hypothesize on a first model that might explain this antioxidant response. SOD activity is the first line of defence against ROS and is activated by the presence of increased levels of ROS, transforming superoxide anion into Hitamin E . Taking oduction ,15. Theroduction , pointin2O2 production by post-transcriptionally activated SOD and impaired H2O2 detoxifying mechanisms, such as catalase, pointing out that the supplementation of the diet of these patients with higher intakes of antioxidants could ameliorate its pathological condition.In conclusion, patients affected by AIP present reduced mRNA expression of PBGD and normal expression of PPOX in circulating PBMCs, while patients affected by VP present with the exact opposite picture. The determination of PPOX protein levels in erythrocytes could be a potential diagnostic tool to discriminate AIP from VP in difficult cases in which the gene sequencing and other diagnostic factors are not conclusive. Patients affected by AIP present a situation of potential oxidative stress similar to those affected by VP and characterized by increased H


Diabetes strongly predisposes to cardiovascular disease (CVD), the leading cause of mortality in these patients, as well as in the entire population. Hyperglycemia is an important cardiovascular risk factor as shown by the observation that even transient periods of hyperglycemia, despite return to normoglycemia during follow-up, increase the risk for CVD, a phenomenon termed ‘hyperglycemic memory’. The molecular mechanisms underlying this phenomenon remain largely unknown. As inflammation plays an important role in the pathogenesis of atherosclerosis, we propose that long-term functional reprogramming of monocytes and macrophages, induced by hyperglycemia, plays an important role in the phenomenon of hyperglycemic memory leading to cardiovascular complications in patients with diabetes. In this review, we discuss recent insights showing that innate immune cells possess the capacity to reprogram their function through epigenetically mediated rewiring of gene transcription, a process termed ‘trained immunity’. The long-term reprogramming of monocytes can be induced by microbial as well as metabolic products, and involves a shift in cellular metabolism from oxidative phosphorylation to aerobic glycolysis. We hypothesize that hyperglycemia in diabetes patients induces long-term activation of monocytes and macrophages through similar mechanisms, thereby contributing to plaque development and subsequent macrovascular complications. There are 422 million people with diabetes worldwide and probably many more individuals with undiagnosed diabetes. The presence of diabetes severely augments the risk for atherosclerosis and associated cardiovascular diseases (CVD), making cardiovascular disease the leading cause of morbidity and mortality in individuals diagnosed with type 1 diabetes (T1D) or type 2 diabetes mellitus (T2D), as well as the entire population. Multiple risk factors have been described to contribute to diabetic complications, such as genetic predisposition , which cThe importance of hyperglycemia as a risk factor is also demonstrated by the observation that a period of elevated HbA1c levels in the past translates into a higher future incidence of CVD despite return to normoglycemia during follow-up , 8. ThusEpigenetic modifications refer to modifications in chromatin state or non-coding RNAs that may lead to long-term reprogramming in gene expression without changing the nucleotide sequence of the DNA itself. Prior episodes of hyperglycemia associate with epigenetic changes in various vascular cell types in vitro and ex vivo  conferring cell memories and may be of critical importance in the development and progression of vascular complications, as reviewed elsewhere –13. It hIn the current review, we address the current knowledge on the activation of monocytes and macrophages in patients with diabetes in general and by hyperglycemic conditions specifically. Subsequently, we discuss recent novel insights into mechanisms implicated in long-term activation and memory of monocytes and macrophages, defined as trained immunity. We finally propose that reprogramming of monocytes by hyperglycemia may contribute to the hyperglycemic memory as an inducer of vascular complications in patients with diabetes.Initially viewed as primarily the result of lipid accumulation, atherosclerotic development is currently considered as a low-grade inflammatory disease of the arterial wall. Altered monocyte and macrophage numbers, function, and skewed proinflammatory signaling are considered to facilitate vascular inflammation . In the hi monocytes, whereas non-inflammatory Ly6-Clo monocytes were largely unaffected [In high-risk populations, the amount of circulating monocytes independently predicts the risk for coronary artery disease –20. In daffected .Functional alterations have been observed in monocytes isolated from both T2D and T1D patients. Monocyte activation in T2D patients has been associated ex vivo with enhanced inflammasome activation, known to activate caspase-1 ultimately leading to proteolytic cleavage and activation IL-1β and IL-18 . Indeed,The proinflammatory phenotype of monocytes with increased binding to endothelium is expected to increase monocyte migration in atherosclerotic plaques . AutopsyMycobacterium tuberculosis [Experimental murine and in vitro cell culture studies have shed more light on possible mechanisms that may explain the effect of hyperglycemia on monocyte activation. Peritoneal macrophages isolated from T1D and T2D mouse models show increased proinflammatory cytokine expression . Recent rculosis , 40. ThiThus, there is strong evidence that chronic hyperglycemia, the hallmark of diabetes, is associated with a (low-grade) activation of the innate immune system, which, in turn, is associated with accelerated atherosclerosis. The phenomenon of hyperglycemic memory, i.e., the observation that even transient periods of hyperglycemia enhance development of atherosclerosis, suggests that elevated glucose may be somehow memorized and induce long-term activation of the innate immune system, which eventually accelerates atherosclerosis development. However, knowledge on the molecular mechanisms controlling these processes is lacking. Identifying these pathways may help in our efforts to find new therapeutic targets to treat or prevent atherosclerosis.Candida or Bacillus Calmette–Guérin (BCG) is associated with changes in H3K4me3 at promoters of proinflammatory cytokines [Recent discoveries in the field of innate immunity may shed light on how monocytes and macrophages adopt a long-term proinflammatory phenotype in patients with risk factors for atherosclerosis. In recent years, it has been revealed that innate immune cells exhibit an immunological (non-specific) memory of past insults, which has been named “trained immunity” or “innate immune memory” , 15. Witytokines , as wellytokines , 45. In Although this phenomenon of trained immunity can be beneficial in the context of infections, long-term activation of the innate immune system might be detrimental in chronic inflammatory diseases including atherosclerosis . ImportaClinical evidence for the concept of trained immunity in coronary artery disease has been recently provided by the study of Shirai et al. This study evaluated whether monocytes derived from patients with coronary artery disease are primed differently, resulting in proinflammatory cytokine production upon entering the tissue space, by comparing ex vivo-differentiated macrophages from patients and controls . PatientCollectively, the long-term memory of innate immune cells mediated by epigenetic reprogramming has important implications for our understanding of the pathogenesis of atherosclerosis, although solid clinical evidence is still needed. In addition, further studies are required to evaluate the concept of trained immunity in response to other metabolites associated with metabolic disease. AGEs and high glucose levels that are increased in patients with diabetes represent interesting candidates to evaluate whether trained immunity could be an underlying mechanisms involved in the phenomenon of hyperglycemic memory.The identification of oxLDL as an inducer of trained immunity suggests a role for metabolic endogenous compounds in training of monocytes, potentially unraveling epigenetic reprogramming of monocytes as a novel pathogenic pathway contributing to atherosclerotic development. Importantly, this opens new avenues for future research to explore the contribution of trained immunity to the phenomenon of hyperglycemic memory in patients with diabetes. Shirai et al. revealed that various risk factors for coronary events, i.e., hypertension, hyperlipidemia, and T2D all correlated with IL-6 release by monocytes ex vivo, showing considerable potential for exploration of other atherogenic risk factors that could induce training of monocytes . A previWe hypothesize that hyperglycemic conditions in diabetes patients induces training of monocytes, functionally reprogramming the monocytes to display a more ‘aggressive’ proatherosclerotic response to subsequent encounter with other stimuli . We propose that this process contributes to the phenomenon of hyperglycemic memory, resulting in enhanced plaque development and subsequent macrovascular complications Fig. . EndogenActivation of recognition receptors on the surface of innate immune cells is likely to play a central role in innate immune memory , 54. SevCollectively, PRRs and especially CLRs could perhaps translate hyperglycemic conditions into an elevated risk for the development of atherosclerosis via induction of trained immunity in monocytes. Involvement of individual PRRs, such as Dectin-1, as well as potential endogenous ligands that are known to be present under hyperglycemic conditions, such as AGEs, needs further evaluation.Immunological studies have shown that long-term reprogramming of monocytes is mediated by epigenetic changes , 43. WheWhile there is evidence that certain epigenetic changes occur in monocytes of patients with diabetes and can persist over time, it remains to be proved that these are causally related to a proinflammatory, proatherogenic phenotype of monocytes, and subsequent cardiovascular complications. Moreover, the mechanisms by which epigenetic changes are induced in monocytes exposed to high glucose levels remain to be established.−/− mice in vivo [Recent interest and research focus on the direct relation between metabolism and chromatin dynamics , which w in vivo , suggest in vivo . PossiblRecent data show that monocytes and macrophages of patients with atherosclerotic coronary artery disease display overutilization of glucose, promoting excessive and prolonged production of proinflammatory cytokines . These dAn important molecular switch that drives cellular metabolism in macrophages is AMP-activated protein kinase (AMPK) . ImportaIn sum, the long-term reprogramming of monocytes by pathogenic stimuli involves a shift in cellular metabolism from oxidative phosphorylation to aerobic glycolysis. We hypothesize that hyperglycemia in diabetes patients induces long-term activation of monocytes and macrophages through similar mechanisms, thereby contributing to plaque development and subsequent macrovascular complications.Chronically elevated glucose levels are one of the main risk factors for CVD, rendering blood glucose control of utmost importance. However, the hyperglycemic memory induced by previous episodes of uncontrolled blood glucose levels limits subsequent effectiveness of glucose control to prevent long-term CVD complications. High glucose levels might prime innate immune cells and change their epigenetic profile to a predisposed proinflammatory state, referred to as “trained immunity” ultimately promoting atherosclerotic plaque development. Effectiveness of glucose lowering therapies in diabetes patients may suffer from such training effects, reducing effectiveness because of an imprinted proinflammatory response.While compelling new data suggest that this effect of hyperglycemia on long-term functional reprogramming of monocytes and macrophages is important in the pathophysiology of atherosclerosis in patients with diabetes, many crucial factors remain to be explored. First, the life span of circulating monocytes ranges from days to weeks, while deleterious effects of exposure to hyperglycemia persist for years after implementation of improved glycemic control. It may well be that elevated glucose levels already induce ‘training’ and (epigenetic) changes in hematopoietic stem cells (HSCs) within the bone marrow. Recent evidence from diabetes animal models characterized by high glucose levels indeed shows alterations in HSCs—including epigenetic changes and a prolonged intrinsic proinflammatory phenotype , 76, 77.