Genetics and epigenetics: paternal adolescent ethanol consumption in serotonin transporter knock-out rats and offspring sensitivity to ethanol.

RATIONALE: Alcohol use disorder (AUD) is shown to have an overall heritability of around 50%. One of the genes associated with AUD is SLC6A4 (solute carrier family 6 member A4) which codes for the serotonin transporter (SERT). The study looked at serotonin dysfunction on ethanol consumption in adolescents and the subsequent intergenerational effects of drinking by using a rat model: SERT+/+ (regular functioning), SERT+/- (50% transporter reduction) and SERT-/- (complete reduction). OBJECTIVES: We investigated sex and genotype differences in ethanol consumption in SERT knock-out Wistar rats (F0) followed by studying behaviour in the offspring (F1) of the male drinkers to assess effects of paternal alcohol consumption. METHODS: An intermittent access two-bottle choice paradigm (IA2BC) was used to yield ethanol drinking behaviour in F0 adolescent Wistar rats. The highest drinking males were mated to alcohol-naive females and their offspring were compared with controls. Drinking behaviour (IA2BC) and ethanol-induced motor coordination effects (via rotarod) were measured in the F1s. RESULTS: F0 drinking saw no SERT genotype differences in males. However, females consumed higher volumes of ethanol compared to males, with SERT-/- females showing the highest intake. A clearer genotype effect was seen in the F1 animals, with reduction in SERT activity leading to enhanced ethanol intake in both sexes. Importantly, paternal exposure to ethanol significantly reduced the ethanol induced motor side effects in offspring, independent of sex and genotype. CONCLUSIONS: These indicate a difference in the way genetic factors may act across sexes and suggest the involvement of epigenetic mechanisms in the intergenerational effects of alcohol.

Sex bias in the serotonin transporter knockout model: Implications for neuropsychiatric disorder research.

Serotonergic signalling is implicated in the aetiology of many neuropsychiatric disorders. The serotonin reuptake transporter (SERT) is an important regulator of synaptic serotonin, being an important pharmacological target with genetic variants implicated with risk of developing neuropsychiatric disorders. Animal models have played an important role in understanding the genetic risk and role of SERT function in brain development having highlighted sex differences in incidence, presentation, and treatment efficacy, however, sex bias due to unequal representation of sexes in research remains a significant issue. While more studies are addressing sex as a biological variable this is not reflected in studies using SERT knockout models as the proportion including sex comparisons has declined since 2000. This bias needs to be addressed if research findings from animal studies are to have translation relevance to human conditions.

The serotonin reuptake transporter modulates mitochondrial copy number and mitochondrial respiratory complex gene expression in the frontal cortex and cerebellum in a sexually dimorphic manner.

Neuropsychiatric and neurodevelopmental disorders such as major depressive disorder (MDD) and autism spectrum disorder (ASD) are complex conditions attributed to both genetic and environmental factors. There is a growing body of evidence showing that serotonergic signaling and mitochondrial dysfunction contribute to the pathophysiology of these disorders and are linked as signaling through specific serotonin (5-HT) receptors drives mitochondrial biogenesis. The serotonin transporter (SERT) is important in these disorders as it regulates synaptic serotonin and therapeutically is the target of selective serotonin reuptake inhibitors which are a major class of anti-depressant drug. Human allelic variants of the serotonin transporter-linked polymorphic region (5-HTTLPR) such as the S/S variant, are associated with reduced SERT expression and increased susceptibility for developing neuropsychiatric disorders. Using a rat model that is haploinsufficient for SERT and displays reduced SERT expression similar to the human S/S variant, we demonstrate that reduced SERT expression modulates mitochondrial copy number and expression of respiratory chain electron transfer components in the brain. In the frontal cortex, genotype-related trends were opposing for males and females, such that reduced SERT expression led to increased expression of the Complex I subunit mt-Nd1 in males but reduced expression in females. Our findings suggest that SERT expression and serotonergic signaling have a role in regulating mitochondrial biogenesis and adenosine triphosphate (ATP) production in the brain. We speculate that the sexual dimorphism in mitochondrial abundance and gene expression contributes to the sex bias found in the incidence of neuropsychiatric disorders such as MDD and ASD.

Genetic Knockout of the Serotonin Reuptake Transporter Results in the Reduction of Dendritic Spines in In vitro Rat Cortical Neuronal Culture.

Dysregulation of the serotonergic system has been reported to have a significant role in several neurological disorders including depression, autism and substance abuse disorders. Changes in the expression of the serotonin transporter (SERT) through polymorphisms in the regulatory regions of the SERT gene have been associated, but not yet been conclusively linked to, neuropsychiatric disorders. In turn, dendritic spine structure and function are critical for neuronal function and the disruption of dendritic spine formation at glutamatergic synapses is a hallmark of several neuropsychiatric disorders. To understand the effect of SERT depletion on dendritic spine formation, neuronal cultures were established from the cortex of postnatal day 0-1 SERT knockout (KO) rats. Cortical neurons were subsequently allowed to mature to 21 days in vitro, and dendritic spine density was assessed using immunocytochemical co-labelling of drebrin and microtubule associated protein 2. Genetic knockout of the SERT had a gene-dose effect on dendritic spine densities of cortical neurons. The results of this paper implicate SERT function with the formation of dendritic spines at glutamatergic synapses, thereby offering insight into the aetiology of several neuropathologies.

Evaluation of i-Motif Formation in the Serotonin Transporter-Linked Polymorphic Region.

Neuropsychiatric disorders such as major depressive disorder (MDD) arise from a complex set of genetic and environmental factors. The serotonin transporter (SERT) is a key regulator of synaptic serotonin (5-HT), and its inhibition is an important pharmacological target for treating MDD. The SERT-linked polymorphic region (5-HTTLPR) contains two major variants (short and long) that have been implicated in modulating susceptibility to MDD by altering the level of expression of SERT. Both variants contain C-rich repeats that conform to consensus i-motif folding sequences. i-Motifs are quadruplex DNA structures that have been proposed to have a role in transcription regulation. With spectroscopic techniques, we demonstrate that both alleles are able to form i-motifs at acidic pH, and at neutral pH under conditions of molecular crowding. This highlights the potential for i-motif formation to contribute to transcriptional regulation of the serotonin transporter, with a potential role in the pathophysiology of neuropsychiatric disorders.

Correction: A novel chemotactic factor derived from the extracellular matrix protein decorin recruits mesenchymal stromal cells in vitro and in vivo.

[This corrects the article DOI: 10.1371/journal.pone.0235784.].

A novel chemotactic factor derived from the extracellular matrix protein decorin recruits mesenchymal stromal cells in vitro and in vivo.

Soft tissue is composed of cells surrounded by an extracellular matrix that is made up of a diverse array of intricately organized proteins. These distinct components work in concert to maintain homeostasis and respond to tissue damage. During tissue repair, extracellular matrix proteins and their degradation products are known to influence physiological processes such as angiogenesis and inflammation. In this study we developed a discovery platform using a decellularized extracellular matrix biomaterial to identify new chemotrophic factors derived from the extracellular matrix. An in vitro culture of RAW.264 macrophage cells with the biomaterial ovine forestomach matrix led to the identification of a novel ~12 kDa chemotactic factor, termed 'MayDay', derived from the N-terminal 31-188 sequence of decorin. The recombinant MayDay protein was shown to be a chemotactic agent for mesenchymal stromal cells in vitro and in vivo. We hypothesize that the macrophage-induced cleavage of decorin, via MMP-12, leads to the release of the chemotactic molecule MayDay, that in turn recruits cells to the site of damaged tissue.

Delivery of antibacterial silver nanoclusters to Pseudomonas aeruginosa using species-specific DNA aptamers.

Introduction. The use of silver as an antimicrobial therapeutic is limited by its toxicity to host cells compared with that required to kill bacterial pathogens.Aim. To use aptamer targeting of DNA scaffolded silver nanoclusters as an antimicrobial agent for treating Pseudomonas aeruginosa infections.Methodology. Antimicrobial activity was assessed in planktonic cultures and in vivo using an invertebrate model of infection.Results. The aptamer conjugates that we call aptabiotics have potent antimicrobial activity. Targeted silver nanoclusters were more effective at killing P. aeruginosa than the equivalent quantity of untargeted silver nanoclusters. The aptabiotics have an IC50 of 1.3-2.6 µM against planktonically grown bacteria. Propidium iodide staining showed that they rapidly depolarize bacterial cells to kill approximately 50 % of the population within 10 min following treatment. In vivo testing in the Galleria mellonella model of infection prolonged survival from an otherwise lethal infection.Conclusion. Using P. aeruginosa as a model, we show that targeting of DNA-scaffolded silver nanoclusters with an aptamer has effective fast-acting antimicrobial activity in vitro and in an in vivo animal model.

Selection of DNA aptamers specific for live Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes significant morbidity and mortality in immunocompromised patients, particular cystic fibrosis sufferers, burns victims, diabetics and neonates. It thrives in moist places where it forms biofilms that are exceedingly difficult to eradicate on hospital surfaces, in water supplies and implanted biomaterials. Using a live cell SELEX approach we selected DNA aptamers to P. aeruginosa grown as biofilms in microfluidic cells. From a pool of aptamer candidates showing tight binding a stem-loop structure was identified as being important for binding. Enhanced binding and increased specificity was achieved by truncating structures and generating chimeric aptamers from the pool of top candidates. The top candidates have low nanomolar binding constants and high discrimination for P. aeruginosa over other Gram-negative bacteria. The aptamers bind both planktonic grown and biofilm grown cells. They do not have intrinsic bacteriostatic or bactericidal activity, but are ideal candidates for modification for use as aptamer-drug conjugates and in biosensors.

Increased apoptosis and secretion of tryptase by mast cells in infantile haemangioma treated with propranolol.

Propranolol is increasingly used to treat problematic infantile haemangioma (IH), although its molecular mechanisms remain unclear. A key feature of propranolol therapy is the decreased deposition of fibrofatty residuum compared with spontaneously involuting IH. This study investigated the molecular consequences of propranolol treatment for IH in vivo.Immunohistochemical and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining was performed on five age matched patients with proliferative IH. Two patients (A and B) were undergoing propranolol treatment at the time of surgical resection.Propranolol treatment increased apoptosis, and induced mast cells to degranulate and secrete tryptase into the interstitium. The microvessels of patient A were immature [weak von Willibrand Factor (vWF), and strong osteoprotegerin (OPG) staining], comparable to untreated proliferative IH, while those of patient B were mature (strong vWF staining, and no OPG staining). The perivascular CD90 mesenchymal stem cell population was preserved in both propranolol treated patients.Using rarely obtained biopsies from IH patients treated with propranolol, we show increased apoptosis by propranolol for the first time in vivo. We also suggest that mast cells, through secreted proteases, may contribute to the decreased fibrofatty residuum seen with propranolol treatment.

Delta-9-tetrahydrocannabinol disrupts hippocampal neuroplasticity and neurogenesis in trained, but not untrained adolescent Sprague-Dawley rats.

Cannabis is the most widely used illicit drug, and disruption of learning and memory are commonly reported consequences of cannabis use. We have previously demonstrated a spatial learning impairment by ∆(9)-tetrahydrocannabinol (THC) in adolescent Sprague-Dawley rats (Steel et al., 2011). The molecular mechanisms underlying behavioural impairment by cannabis remain poorly understood, although the importance of adaptive changes in neuroplasticity (synaptic number and strength) and neurogenesis during learning are accepted. Here we aimed to identify any effects of THC on the early induction of these adaptive processes supporting learning, so we conducted our analyses at the mid-training point of our previous study. Both untrained and trained (15 days of training) adolescent (P28-P42) Sprague-Dawley rats were treated daily with THC (6 mg/kg i.p.) or its vehicle, and changes in the levels of markers of hippocampal neuroplasticity (CB1R, PSD95, synapsin-I, synapsin-III) and neurogenesis (Ki67, DCX, PSA-NCAM, BrdU labelling) by training were measured. Training of control animals, but not THC-treated animals increased neuroplasticity marker levels. However training of THC-treated animals, but not control animals reduced immature neuronal marker levels. Levels of hippocampal proliferation, and survival of the BrdU-labelled progeny of these divisions were unaffected by THC in trained and untrained animals. These data show a smaller neuroplastic response, and a reduction of new-born neuronal levels not attributable to effects on proliferation or survival by THC-treatment during training. Importantly no effects of THC were seen in the absence of training, indicating that these effects represent specific impairments by THC on training-induced responses.

Mast cells in infantile haemangioma possess a primitive myeloid phenotype.

AIMS: Recent reports on infantile haemangioma (IH) have demonstrated a primitive population of interstitial cells expressing the embryonic transcription factor, Nanog, with decreasing abundance during involution. In this report we investigated the expression of Nanog on mast cells in all three phases of IH progression. METHODS: Paraffin-embedded sections of six proliferating, six involuting and six involuted IH lesions were used to investigate the expression of tryptase, Nanog, CD45, CD34 and GLUT-1 by immunostaining. RESULTS: Mast cells, identified by their expression of tryptase, were located in the interstitium of IH lesions. 93%, 42% and 0% of these tryptase(+) cells also expressed Nanog, in proliferating, involuting and involuted IH, respectively. CONCLUSIONS: The identification of an abundant population of tryptase(+)/Nanog(+) cells in IH is novel. The relative loss of Nanog expression as IH involutes may be a result of maturation and/or proliferation of these cells. This report supports the primitive nature of IH.

Impact of primary biliary cirrhosis on perceived quality of life: the UK-PBC national study.

UNLABELLED: Primary biliary cirrhosis (PBC) has a complex clinical phenotype, with debate about the extent and specificity of frequently described systemic symptoms such as fatigue. The aim of this study was to use a national patient cohort of 2,353 patients recruited from all clinical centers in the UK to explore the impact of disease on perceived life quality. Clinical data regarding diagnosis, therapy, and biochemical status were collected and have been reported previously. Detailed symptom phenotyping using recognized and validated symptom assessment tools including the PBC-40 was also undertaken and is reported here. Perception of poor quality of life and impaired health status was common in PBC patients (35% and 46%, respectively) and more common than in an age-matched and sex-matched community control group (6% and 15%, P < 0.0001 for both). Fatigue and symptoms of social dysfunction were associated with impaired perceived quality of life using multivariate analysis. Fatigue was the symptom with the greatest impact. Depression was a significant factor, but appeared to be a manifestation of complex symptom burden rather than a primary event. Fatigue had its greatest impact on perceived quality of life when accompanied by symptoms of social dysfunction, suggesting that maintenance of social networks is critical for minimizing the impact of fatigue. CONCLUSION: The symptom burden in PBC, which is unrelated to disease severity or ursodeoxycholic acid response, is significant and complex and results in significant quality of life deficit. The complexity of symptom burden, and its lack of relation to disease severity and treatment response, suggest that specific approaches to symptom management are warranted that address both symptom biology and social impact.

The impact of liver transplantation on the phenotype of primary biliary cirrhosis patients in the UK-PBC cohort.

BACKGROUND & AIMS: Liver transplantation improves survival in end-stage primary biliary cirrhosis (PBC), but the benefit for systemic symptoms including fatigue is less clear. The aim of this study was to utilise the comprehensive UK-PBC Research Cohort, including 380 post-transplant patients and 2300 non-transplanted patients, to answer key questions regarding transplantation for PBC. METHODS: Cross-sectional study of post-transplant PBC patients and case-matched non-transplanted patients. Detailed clinical information was collected, together with patient systemic symptom impact data using validated assessment tools. RESULTS: Over 25% of patients in the transplant cohort were grafted within 2 years of PBC diagnosis suggesting advanced disease at presentation. Transplanted patients were significantly younger at presentation than non-transplanted (mean 7 years) and >35% of all patients in the UK-PBC cohort who presented under 50 years had already undergone liver transplantation at the study censor point (>50% were treatment failures (post-transplant or unresponsive to UDCA)). Systemic symptom severity (fatigue and cognitive symptoms) was identical in female post-transplant patients and matched non-transplanted controls and unrelated to disease recurrence or immunosuppression type. In males, symptoms were worse in transplanted than in non-transplanted patients. CONCLUSIONS: Age at presentation is a major risk factor for progression to transplant (as well as UDCA non-response) in PBC. Although both confirmatory longitudinal studies, and studies utilising objective as well as subjective measures of function, are needed if we are to address the question definitively, we found no evidence of improved systemic symptoms after liver transplantation in PBC and patients should be advised accordingly. Consideration needs to be given to enhancing rehabilitation approaches to improve function and life quality after liver transplant for PBC.

Verrucous hemangioma expresses primitive markers.

BACKGROUND: Verrucous hemangioma (VH) presents clinically as a vascular malformation but has similar histopathologic features to infantile hemangioma. This study characterized the cell population within VH. MATERIAL AND METHODS: Paraffin-embedded sections from two male patients with VH were processed for immunohistochemistry. The expression of SMA, CD34, glucose transporter-1 (Glut-1), D2-40, brachyury, angiotensin converting enzyme (ACE), Oct-4, hemoglobin ζ chain (HBZ), Wilms tumor protein (WT-1) and CD45 was examined. RESULTS: The lymphatic marker, D2-40, was not expressed in VH, whereas Glut-1 was widely expressed in infantile hemangioma, it was only focally expressed by the endothelium of VH. The endothelium of VH expressed the primitive markers, Oct-4, brachyury and ACE. The primitive marker, WT-1, was expressed predominantly on the pericyte layer of both VH and infantile hemangioma. However, HBZ was only expressed in infantile hemangioma. CD45, a mature hematopoetic marker, was expressed by cells within the interstitium, away from the endothelium of VH and infantile hemangioma. DISCUSSION: The expression of the primitive markers, Oct-4, brachyury and ACE on the endothelium, and WT-1 predominantly on the pericyte layer of VH shows a primitive microvascular phenotype similar to infantile hemangioma. However, the absence of the embryonic marker, HBZ, expressed only in first trimester placenta and in proliferating infantile hemangioma, suggests a different cellular origin. HBZ could be used to distinguish between the two conditions.

Sex and age are determinants of the clinical phenotype of primary biliary cirrhosis and response to ursodeoxycholic acid.

UNLABELLED: BACKGROUND, & AIMS: Studies of primary biliary cirrhosis (PBC) phenotypes largely have been performed using small and selected populations. Study size has precluded investigation of important disease subgroups, such as men and young patients. We used a national patient cohort to obtain a better picture of PBC phenotypes. METHODS: We performed a cross-sectional study using the United Kingdom-PBC, patient cohort. Comprehensive data were collected for 2353 patients on diagnosis reports, response to therapy with ursodeoxycholic acid (UDCA), laboratory results, and symptom impact (assessed using the PBC-40 and other related measures). RESULTS: Seventy-nine percent of the patients reported current UDCA, therapy, with 80% meeting Paris response criteria. Men were significantly less likely to have responded to UDCA than women (72% vs 80% response rate; P < .05); male sex was an independent predictor of nonresponse on multivariate analysis. Age at diagnosis was associated strongly and independently with response to UDCA; response rates ranged from 90% among patients who presented with PBC when they were older than age 70, to less than 50% for those younger than age 30 (P < .0001). Patients who presented at younger ages also were significantly more likely not to respond to UDCA therapy, based on alanine aminotransferase and aspartate aminotransferase response criteria, and more likely to report fatigue and pruritus. Women had mean fatigue scores 32% higher than men's (P < .0001). The increase in fatigue severity in women was related strongly (r = 0.58; P < .0001) to higher levels of autonomic symptoms (P < .0001). CONCLUSIONS: Among patients with PBC, response to UDCA, treatment and symptoms are related to sex and age at presentation, with the lowest response rates and highest levels of symptoms in women presenting at younger than age 50. Increased severity of fatigue in women is related to increased autonomic symptoms, making dysautonomia a plausible therapeutic target.

Dense fine-mapping study identifies new susceptibility loci for primary biliary cirrhosis.

We genotyped 2,861 cases of primary biliary cirrhosis (PBC) from the UK PBC Consortium and 8,514 UK population controls across 196,524 variants within 186 known autoimmune risk loci. We identified 3 loci newly associated with PBC (at P<5×10(-8)), increasing the number of known susceptibility loci to 25. The most associated variant at 19p12 is a low-frequency nonsynonymous SNP in TYK2, further implicating JAK-STAT and cytokine signaling in disease pathogenesis. An additional five loci contained nonsynonymous variants in high linkage disequilibrium (LD; r2>0.8) with the most associated variant at the locus. We found multiple independent common, low-frequency and rare variant association signals at five loci. Of the 26 independent non-human leukocyte antigen (HLA) signals tagged on the Immunochip, 15 have SNPs in B-lymphoblastoid open chromatin regions in high LD (r2>0.8) with the most associated variant. This study shows how data from dense fine-mapping arrays coupled with functional genomic data can be used to identify candidate causal variants for functional follow-up.

Infantile haemangioma expresses embryonic stem cell markers.

BACKGROUND: The origin of infantile haemangioma (IH) remains enigmatic. A primitive mesodermal phenotype origin of IH with the ability to differentiate down erythropoietic and terminal mesenchymal lineages has recently been demonstrated. AIMS: To investigate the expression of human embryonic stem cell (hESC) markers in IH and to determine whether IH-derived cells have the functional capacity to form teratoma in vivo. METHODS: Immunohistochemical staining and quantitative reverse transcription PCR were used to investigate the expression of hESC markers in IH biopsies. The ability of cells derived from proliferating IH to form teratomas in a mouse xenograft model was investigated. RESULTS: The hESC markers, Oct-4, STAT-3 and stage-specific embryonic antigen 4 were collectively expressed on the endothelium of proliferating IH lesions, whereas Nanog was not. Nanog was expressed by cells in the interstitium and these cells did not express Oct-4, stage-specific embryonic antigen 4 or STAT-3. Proliferating IH-derived cells were unable to form teratomas in severely compromised immunodeficient/non-obese diabetic mice. CONCLUSION: The novel expression of hESC on two different populations of cells in proliferating IH and their inability to form teratomas in vivo infer the presence of a primitive cellular origin for IH downstream from hESC.

Learning impairment by Δ(9)-tetrahydrocannabinol in adolescence is attributable to deficits in chunking.

Cannabis is the most popular illicit drug used by adolescents. Yet, there are only a few studies that have examined the effects of cannabis use on learning and memory during this sensitive and important neurodevelopmental stage. Male adolescent Sprague-Dawley rats were treated with Δ(9)-tetrahydrocannabinol (THC, 6 mg/kg) daily for 27 days and concurrently trained in a spatial learning and memory task. The chronic effects of cannabis use were specifically examined by assessing animal behaviour during the 'postacute' period (17 h after drug exposure), when minimal acute drug burden is expected to be present. The postacute period is a good model for cannabis use patterns in human adolescents. In addition, we investigated whether the hierarchical organization of working memory (chunking) was impaired by THC-treatment. We show that THC exposure impairs adolescent learning when tested in the postacute period, and that THC impairs the ability of animals to use a chunking strategy.

Infantile haemangioma expresses tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), TRAIL receptors, osteoprotegerin and receptor activator for nuclear factor кB ligand (RANKL).

AIMS: To investigate the expression of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors and decoy receptors, including osteoprotegerin (OPG) in infantile haemangioma (IH). METHODS AND RESULTS: Immunostaining, Western blotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used on IH biopsies and haemangioma explant-derived cells (HaemEDCs). TRAIL and its receptors and decoy receptors, including OPG, are expressed in proliferating IH tissues and in HaemEDCs. Cells forming the endothelium of immature capillaries of proliferating IHs express abundant OPG and show punctate von Willebrand Factor (vWF) staining. As the cells mature and assume the characteristic of endothelial cells they increase expression of vWF, but lose expression of OPG. The endothelium of IH shows minimal expression of receptor activator for nuclear factor кB ligand (RANKL) compared with a small population of RANKL-positive cells located within the interstitium between microvessels. Proliferating HaemEDCs express significantly higher levels of OPG and decoy receptor 2 than the matched tissue samples. Increased OPG expression is detected in the extracellular matrix and in the conditioned medium of HaemEDCs. CONCLUSIONS: Our data suggest that OPG through the TRAIL pathway, but not the RANKL pathway, plays a role in regulating anti-apoptosis during the development of IH.