This study was designed to investigate the protective effect of ferulic acid (FA) on nerve injury induced by cerebral ischemia. Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery and reperfusion 90 min later in male Sprague-Dawley rats. Daily treatment of the rats with FA was initiated 30 min after the surgery, and was continued for 7 days. The efficacy of FA against nerve injury was assessed by neurological deficit scores as well as pathohistological observation. The expression levels in the brain and level in the peripheral blood of erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF) were analyzed by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), respectively. The results showed that FA attenuated nerve injury of the hippocampus, significantly ameliorated neurological deficits, and increased EPO but not G-CSF expression in the hippocampus and the peripheral blood of ischemic rats. The findings indicate that FA has certain protective effects on the nerve injury of cerebral ischemia, and suggest that promoting EPO in the brain and peripheral blood may be one of the neuroprotective mechanisms of FA. Stroke is a serious threat to human health, and is recognized as the most important factor resulting in nerve injury ,2. EvideAngelicae sinensis is a traditional Chinese medicinal herb that has long been used to treat ischemic stroke and anemia  and granulocyte colony-stimulating factor (G-CSF) expression . They were kept in air-conditioned rooms  on a 12 h light-dark cycle, with free access to food and water. Animal experimental procedures were carried out in strict accordance with the guidelines published by the United States National Institutes of Health  and approved by the ethics committee of Yantai University . Surgical procedures, stroke induction and animal sacrifice (at the end of the observation period) were performed under general anesthesia with intraperitoneal (i.p.) injection of chloral hydrate (350 mg/kg).The sodium salt of FA  was obtained from Haikou Qili Pharmaceutical Co., Ltd.  and edaravone injection was provided by Nanjing Simcere Dongyuan Pharmaceutical Co., Ltd. .The middle cerebral artery occlusion (MCAO) procedure was carried out according to a previously described method with minor modifications . BrieflyThe rats were randomly divided into six groups of 10 rats each. Three groups received different doses of FA , and one group received edaravone (6 mg/kg) by intravenous injection 30 min after ischemia, and once a day thereafter for seven consecutive days. The rats in the sham and vehicle-treated group were injected with saline.Neurological deficits were evaluated using a modified six-point scoring method , by an iSix rats from each group were selected for H&E staining. The rats were deeply anesthetized with choral hydrate and pericardially perfused with 0.9% saline and then with 4% formaldehyde. The entire brain was embedded in paraffin. Nerve injury in the hippocampus was determined by analysis under a microscope . The damage was evaluated by counting the number of surviving neurons per millimeter length of the hippocampus examined under light microscopy.EPO and G-CSF levels in the plasma were measured by ELISA kits . The rats in each group were sacrificed 24 h after the final administration of treatment and plasma was collected. Monoclonal antibodies specific for EPO and G-CSF were pre-coated onto microplates. Standards and samples were added to the wells and were incubated for 30 min at 37°C. After washing away the unbound substances, enzyme-linked polyclonal antibodies specific for EPO and G-CSF were added to the wells and were incubated for 60 min at 37°C. After removing any unbound antibodies, substrates were added to the wells and were incubated for 15 min at 37°C. The intensities of the colors developed were in proportion to the amount of EPO and G-CSF bound to the wells. The optical density of each well was measured with a scanning multi-well spectrophotometer  at a wavelength of 450 nm.After being deeply anesthetized, the rats were transcardially perfused with saline solution, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) 24 h after ischemia. Brains were removed and post-fixed in 4% paraformaldehyde for 4 h, then transferred into 30% sucrose solution until the brains sank to the bottom of the container. Coronal sections (10 μm) were made using a Leica CM1950S cryostat . Sections were blocked with 3% normal goat serum (diluted in PBS containing 0.3% Triton X-100) for 1 h and incubated with rabbit anti-rat polyclonal primary antibodies  overnight at 4°C. After rinsing with PBS, sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit polyclonal IgG  as secondary antibodies (1:200) for 2 h at room temperature. The images from five fields of each ischemic region from six rats in each group were examined using the same brightness and exposure settings. Image-Pro Plus software  was used to analyze positive expression of EPO or G-CSF in each photograph. The artificial unit of mean optical density (MOD) × total per area (TPA) was employed for measurement in the stereological analysis, which indicated the integrated optical density (IOD) of the positive signal in the stained tissue.Neurological deficit scores between groups were analyzed using a non-parametric test. Quantitative data from the experiments were expressed as mean ± standard deviation (SD), and significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s test. In all cases, differences were considered significant if P<0.05.On the seventh day after the surgery, the results showed that FA induced a significant reduction in the neurological deficit score compared with that of the rats treated with vehicle. Treatment with FA at doses of 100 and 200 mg/kg but not 50 mg/kg significantly reduced the neurological deficit score in a dose-dependent manner in the MCAO model rats. Edaravone treatment, as a positive control, also significantly decreased the neurological deficit score .Extensively damaged neurons in the hippocampus were observed and the number of surviving neurons was significantly reduced in the vehicle-treated ischemic rats compared with the sham-treated rats. Neuronal shrinkage and chromatin condensation of nuclei were also observed in the ischemic rats. However, the number of surviving neurons in the FA-treated rats was higher compared with that in the vehicle-treated ischemic rats and numerous surviving neurons were observed in the hippocampus .FA induced significant enhancements in the EPO level in peripheral blood compared with that in the vehicle-treated rats (P<0.05). With regard to G-CSF levels, the rats treated with FA showed no significant difference compared with the vehicle-treated rats .The results showed that EPO expression within the infarct region in the vehicle-treated group was significantly increased compared with that of the sham-operated group. Administration of FA induced a significant enhancement in EPO expression levels in a dose-dependent manner (P<0.01). With regard to G-CSF, cerebral ischemia induced a significant increase in its expression, whereas treatment with FA was not observed to induce a significant difference in the expression of the protein in the infarct region compared with that in the vehicle-treated rats .Nerve injury resulting from focal or global cerebral ischemia is a major cause of mortality and disability in the adult population. Neuronal cell death has been observed to occur several days after ischemic insult and predominantly affects sensitive areas of the brain, such as the hippocampus and the cortex ,19.The results of the present study demonstrated that neuronal cell death occurred in the ischemic brain primarily in the hippocampal area seven days after ischemia. Treatment with FA significantly reduced the neurological deficit score and hippocampal neuron damage in a dose-dependent manner in the rats following MCAO. The results indicated that treatment of the rats with FA for seven days following MCAO ameliorated the neuronal damage.EPO stimulates erythroid cell production, which supports the survival, proliferation and differentiation of erythroid progenitor cells. The sites of expression of the EPO receptor (EPO-R) and the EPO response are in hematopoietic cells and other cell types, including endothelial and neural cells . The tarIn the present study, it was observed that FA induced a significant enhancement of the level of EPO expression in the ischemic brain. The results also demonstrated that FA increased the EPO level in peripheral blood; EPO nay be transferred from the blood into the brain through the blood-brain barrier (BBB) . TherefoAngelicae sinensis is commonly used to promote erythropoiesis for the treatment of anemia, and FA has previously been demonstrated to increase the levels of EPO and G-CSF (The hematopoietic factor G-CSF was recently discovered to act as a protective and neurotrophic factor in the brain. Several studies have described the infarct-reducing and recovery-enhancing effects of G-CSF following ischemic stroke –27. The nd G-CSF ,9, the rThe findings indicate that FA has certain protective effects against the nerve injury induced by cerebral ischemia, and suggest that the promotion of EPO expression in the ischemic brain and peripheral blood may be one of the neuroprotective mechanisms of FA.


Jsc) and fill factor (FF), compared to the layer consisting of only nanoparticles or nanoporous spheres.A scattering layer is utilized by mixing nanoporous spheres and nanoparticles in ZnO-based dye-sensitized solar cells. Hundred-nanometer-sized ZnO spheres consisting of approximately 35-nm-sized nanoparticles provide not only effective light scattering but also a large surface area. Furthermore, ZnO nanoparticles are added to the scattering layer to facilitate charge transport and increase the surface area as filling up large voids. The mixed scattering layer of nanoparticles and nanoporous spheres on top of the nanoparticle-based electrode (bilayer geometry) improves solar cell efficiency by enhancing both the short-circuit current ( In particular, the optimum power conversion efficiency (η) of 2.91% is obtained at the ratio of NP/NS = 3:7, and the trend of η is generally consistent with that of Jsc. The open-circuit voltage (Voc) values are not notably changed among the cells except for the NP/NS = 3:7. From the general trend of parameters, we cautiously consider that the value for the open-circuit voltage in NP/NS = 3:7 is out of the tendency. We consider different nanomorphologies of porous spheres synthesized from the limited number of samples. Open-circuit voltage is represented as Jsc  is not enough to impact Voc. Also, the change of Voc may result from the difference of reverse saturation current J0. We have synthesized nanoporous ZnO spheres by hydrothermal method [The solar cell performance of the DSSCs fabricated with the various ZnO bilayer electrodes was investigated Figure a, and thde model , and betl method , and thel method ,33.Jsc) by the mixed scattering layer is attributed to efficient light scattering and increased surface area.If charge collection probabilities are similar among the cells, quantum efficiency depends on the light trapping inside the solar cell -37. The 0 to 103 Hz) are related to the charge transport/accumulation at dye-attached ZnO/electrolyte interfaces, and the smaller semicircles in high frequency (approximately 103 to 105 Hz) are ascribed to the charge transfer at the interfaces of electrolyte/Pt counter electrode [Rct) in Table Rct. The low resistance favors the transport of the electrons injected within ZnO, thus eventually leading to an effective collection of electrons [Impedance analyses were performed to understand the electrical properties of the synthesized solar cells -41. The lectrode . The implectrons . The betJsc) and fill factor (FF).To improve the utilization of scattering layer in ZnO-based DSSCs, nanoparticles and nanoporous spheres are mixed with various ratios. The nanoporous spheres play an important role in the scattering effect with the large surface area but possess disadvantages of large voids and point contacts between spheres. Nanoparticles clearly advance facile carrier transport with the additional surface area, thereby improving the solar cell efficiency by the enhanced short-circuit current (The authors declare that they have no competing interests.CK carried out the overall scientific experiment and drafted the manuscript. HC and JIK performed the FE-SEM measurements. SL carried out the analysis of electrochemical impedance spectra. JK and SK participated in the manuscript revision. WL and TH helped to check typing errors. BP and TM gave valuable advices about the whole experiments and manuscript as supervisors. All authors read and approved the final manuscript.


Hepacivirus phenotypes of acute viral clearance and chronic pathologic disease. HCV and GBV-B are also closely phylogenetically related and advances in characterization of the immune systems of New World primates have already led to the use of this model for drug testing and vaccine trials. Herein, we discuss the benefits and caveats of the GBV-B infection model and discuss potential avenues for future development of novel vaccines and immunotherapies.Hepatitis C virus (HCV) infection has become a global public health burden costing billions of dollars in health care annually. Even with rapidly advancing scientific technologies this disease still poses a significant threat due to a lack of vaccines and affordable treatment options. The immune correlates of protection and predisposing factors toward chronicity remain major obstacles to development of HCV vaccines and immunotherapeutics due, at least in part, to lack of a tangible infection animal model. This review discusses the currently available animal models for HCV disease with a primary focus on GB virus B (GBV-B) infection of New World primates that recapitulates the dual  Hepatitis C virus (HCV) is a major chronic disease that has infected greater than 150 million people worldwide. Given the breadth and duration of infection HCV disease could result in an economic crisis reaching into the 100s of billions within the next decade. Although acute infection is usually asymptomatic with self-resolution, 55–85% of infected persons develop chronic disease that can lead to progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma HCC;  which isHepacivirus genus within the Flaviviridae family. The genome is 9.6 kb with a single open reading frame (ORF) that encodes a polyprotein which is further cleaved to yield 10 mature viral proteins namely the structural proteins  and non-structural proteins  for vaccine development is the lack of small animal models that would allow the study of HCV infection, genetic variability of host defenses, and immune modulatory mechanisms induced by the virus. GBV-B is phylogenetically a close relative of HCV and infects only New World monkeys such as tamarins and marmosets  responsible for non-A, non-B hepatitis, Flaviviridae family of viruses. GBV-B in particular was found to be closely related to HCV based on amino acid sequence alignments and phylogenetic analyses  and human pegivirus (HPgV) respectively , the sera of multiple non-primate species were screened for the presence of hepaciviruses  were the first animal model for HCV infection and have contributed significantly to understanding the natural history of HCV (Table 3). Chimpanzees also played a critical role in discovery of HCV  is a non-rodent small mammal indigenous to Southeast Asia, and other than chimpanzees and humans, tree shrews are the only other species known to be susceptible to HCV infection (Table 3).The northern tree shrew .Although mice are naturally resistant to HCV infection, humanized chimeric mice with normal human hepatocytes exhibit prolonged infection following inoculation with HCV . This mo-3 nucleotide substitutions/site/year. By comparison, the error rate for HCV RdRp has been estimated at 1.4 to 1.9 × 10-3 nucleotide substitutions/genome/site/year  and the non-structural  proteins . The nonite/year . These v7–1010 genomic equivalents (GEs)/ml by weeks 2–3 and then plateaus before clearance . Generally GBV-B causes an acute infection, although a few cases of chronicity have been reported. Viral persistence for 2 years has been reported in a tamarin infected by intrahepatic inoculation with a synthetic viral RNA before spontaneous resolution  directed against GBV-B virus controlled virus replication in manner dependent on CL-siRNA dosing, reaching levels of complete inhibition at 5 mg/kg (The GBV-B infection model has also been used for the screening of candidate vaccines and other therapeutic agents such as interferon and ribavarin. Ribavarin treatment reduced viral RNA levels by four logs by inducing error prone replication in culture, but  viremia . Bright  5 mg/kg .During the last decade various reports have shown the utility of GBV-B infection as a surrogate model for HCV. Although the GBV-B/marmoset model has a lower chronicity rate, it has a number of advantages over other animal models including lower cost, availability of chimeric viruses, effectiveness of HCV drugs against GBV-B, and a tractable disease course. While the development of new antivirals has changed the landscape of the HCV field, the cost-prohibitive nature of these treatments underscores the ongoing need for a successful vaccine. Thus, we propose that the GBV-B/marmoset model warrants revisiting as a thoughtful research tool for development of immunotherapeutics, new antivirals, and vaccine modalities for eventual translation into human studies.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.


Furthermore, ∆9-tetrahydrocannabinolic acid A (THCA-A), the biogenetic precursor of THC, is found in the hair of persons who solely handled cannabis material. In the light of the serious consequences of positive test results the mechanisms of drug incorporation into hair urgently need scientific evaluation. Here we show that neither THC nor THCA-A are incorporated into human hair in relevant amounts after systemic uptake. THC-COOH, which is considered an incontestable proof of THC uptake according to the current scientific doctrine, was found in hair, but was also present in older hair segments, which already grew before the oral THC intake and in sebum/sweat samples. Our studies show that all three cannabinoids can be present in hair of non-consuming individuals because of transfer through cannabis consumers, via their hands, their sebum/sweat, or cannabis smoke. This is of concern for e.g. child-custody cases as cannabinoid findings in a child’s hair may be caused by close contact to cannabis consumers rather than by inhalation of side-stream smoke.Hair analysis for cannabinoids is extensively applied in workplace drug testing and in child protection cases, although valid data on incorporation of the main analytical targets, ∆9-tetrahydrocannabinol (THC) and 11- Maximum serum concentrations of THC-COOH were 18 ng/mL (participant 1) and 40 ng/mL (participant 2), respectively  by two volunteers over a 30 day period , the extent of THC incorporation via the bloodstream into hair was evaluated. No THC was detected at any time of sampling in all the head hair, beard hair or body hair samples (limit of detection: 1 pg/mg). From multiple serum samples taken within 8 hours  the estimated AUCvely see  online. vely see  online.  per day . Analysi per day  online.  per day .The results strongly suggest that THCA-A is not incorporated into hair through the bloodstream or via sebum/sweat to a relevant extent. Although this was tested only in one individual, the daily dose of THCA-A was at least an order of magnitude higher than expected in excessive cannabis smokers. Therefore, the THCA-A detected in forensic hair samples 19The incorporation rate of THC via the bloodstream into the hair seems to be negligible low as no THC could be detected in the hair samples of the participants after systemic dronabinol uptake. It follows from Fick’s law that the amount of analyte incorporated into hair should be proportional to the area under the analyte serum concentration over time curve (AUC). Given that the THC AUCFurthermore, the detection of THC-COOH in hair segments did not correlate to the period of THC intake and the presence of THC-COOH in sebum/sweat implicates a relevant contribution to the THC-COOH findings in hair samples by diffusion of the analyte from sebum into the hair matrix. The marked variations in the THC-COOH concentrations between body regions may be explained by differences in the physiology (e.g. presence of apocrine sweat glands in the axillary and pubic region), sampling particularities (e.g. regular shaving of beard hair vs. sampling of hair strands) and a possible transfer of the analyte due to contamination of hair with urine (pubic region). The fact, that THC-COOH was detectable up to 11 weeks past the intake period in beard hair further underlines a relevant incorporation via secretion of sebum which shows a physiological time shift, or by diffusion from surrounding tissuesAt first glance, differentiation of the route of THC-COOH incorporation into hair seems irrelevant as long as positive THC-COOH findings in hair require THC uptake by the individual under investigation. However, considering the presence of THC-COOH in sebum/sweat, a transfer to other persons’ hair is possible. This is particularly true for young children or partners of cannabis consumers . Comparing the maximum serum THC-COOH concentrations detected in persons massively exposed to cannabis smoke in a ‘coffee shop’ (0.5–1.7 ng/mL)For the oral intake of THCA-A only one individual was tested. Although the extraordinary high serum concentrations reached should compensate for this, physiological characteristics of the individual may have led to THCA-A not being incorporated into hair to a measurable extent. In the study with oral intake of dronabinol a relatively low dose of THC was used, which may reflect THC uptake of moderate cannabis smokers, but not of heavy users. Therefore, measurable incorporation of THC from the blood stream cannot be excluded in the case of heavy cannabis smoking. Due to oral administration  the maximum THC serum concentrations were lower than the maximum concentrations generally reached after smoking. Although – following from Fick’s law – incorporation should be proportional to the AUC, the diffusion coefficient may vary with the gradient. High concentration gradients as observed directly after smoking might therefore lead to a more efficient incorporation of THC. Furthermore, the number of individuals tested in this study was low (n = 2) and pharmacokinetic particularities may affect the generalizability of the findings.Knowing the main routes of cannabinoid incorporation into human hair, any interpretation of varying concentrations along the hair shaft in terms of time-resolved patterns of use may lead to false conclusions. Cases with high THC or THC-COOH concentrations in proximal hair segments are in particular critical as they may be interpreted as a recent increase of cannabis consumption. Not over-interpreting THC or THC-COOH findings in hair is of utmost importance in child protection cases, but also in the context of work place drug testing and any forensic application. Practitioners who work with results of hair analysis should be aware of these limitations and the severe consequences false conclusions could entail.Although the results of our study cannot be transferred directly to other cannabinoids or other types of illicit drugs (in particular to less lipophilic and non-acidic compounds) the proportion of drugs incorporated into hair via the bloodstream is largely unknown and should be the focus of further research.th May 2014), and was conducted at the Institute of Forensic Medicine Freiburg, Germany, in accordance with the Declaration of Helsinki Principles and subsequent amendments. Written informed consent was obtained from each participant. Volunteers were recruited from the personal environment of the authors and affirmed that they neither consumed cannabis nor were exposed to cannabis via peers or family members prior to and during the study.The study protocol was approved by the Ethics Committee of the University of Freiburg, Germany (EK-Freiburg 98/14), and the Federal Opium Agency  granted a permit for the intake of dronabinol. The study was registered in a World Health Organization primary register  were collected prior to the intake period, and then on a weekly basis until three weeks after the last intake. The segmented hair samples (1 cm segments) were analysed for THCA-A applying a fully validated LC-MS/MS methodTwo male participants orally ingested 2.5 mg dronabinol (THC) three times daily over a 30 day period. Hair samples  were collected prior to the intake period, and then on a regular basis until several weeks after the last intake. Apart from hair samples, combined sebum/sweat samples were collected using Sebutapes®. All hair samples were analysed for THC and THC-COOH after alkaline hydrolysis applying a fully validated LC-MSHow to cite this article: Moosmann, B. et al. Finding cannabinoids in hair does not prove cannabis consumption. Sci. Rep.5, 14906; doi: 10.1038/srep14906 (2015).


The aetiology of chronic post-traumatic headache (CPTH) after mild head injury is unclear, but psychological stress and personality factors have been suggested as both causing and maintaining factors. Research is scarce and it is uncertain whether patients with CPTH share a specific personality profile and if the profile differ from other chronic headache patients.To investigate whether CPTH is distinguished from other chronic headache patients in terms of mental distress, general personality characteristics and traits specifically as measured by the The Revised NEO Personality Inventory (NEO-PI-R).Ninety patients with CPTH and 45 control patients with chronic primary headaches were enrolled from the Danish Headache Center. They completed NEO-PI-R personality assessment, The Symptom Checklist (SCL-90-R) and a headache diary for 4 weeks.The scale scores and the levels of psychological distress did not differ between the CPTH and the headache control group. CPTH patients with a headache duration over two years had a higher score on the domain Neuroticism (p=0.002), but lower Extraversion (p=0.001) and lower Openness (p=0.038) on the NEO-PI-R.In contrast to prior hypothesis patients with CPTH were not characterised by a specific personality profile although long lasting CPTH was associated to higher neuroticism and a lower degree of extraversion and openness. No differences were found in the reported level of psychological distress in patients with CPTH compared to a headache control group.No conflict of interest.


Ureaplasma and Mycoplasma species, as well as Gram-positive and Gram-negative bacteria, have been associated with IUI-induced PTB. Bacterial activation of Toll-like receptors and other pattern recognition receptors initiates a cascade of inflammatory signaling via the NF-κB and p38 mitogen-activated protein kinase (MAPK) signaling pathways, prematurely activating parturition. Antenatal antibiotic treatment has had limited success in preventing PTB or fetal inflammation. Administration of anti-inflammatory drugs with antibiotics could be a viable therapeutic option to prevent PTB and fetal complications in women at risk of IUI and inflammation. In this mini-review, we will discuss the potential for anti-inflammatory drugs in obstetric care, focusing on the class of drugs termed “cytokine suppressive anti-inflammatory drugs” or CSAIDs. These inhibitors work by specifically targeting the NF-κB and p38 MAPK inflammatory signaling pathways. Several CSAIDs are discussed, together with clinical and toxicological considerations associated with the administration of anti-inflammatory agents in pregnancy.Preterm birth (PTB) at less than 37 weeks of gestation is the leading cause of neonatal morbidity and mortality. Intrauterine infection (IUI) due to microbial invasion of the amniotic cavity is the leading cause of early PTB (<32 weeks). Commensal genital tract  Preterm birth (PTB), delivery prior to 37 weeks of gestation, is estimated to affect 5–15% of pregnancies worldwide  and the There is growing interest in therapeutic interventions that target the inflammatory labor cascade by blocking the production of pro-inflammatory mediators or up-regulating anti-inflammatory mediators and/or the exogenous administration of anti-inflammatory or pro-resolution mediators. In their review of anti-inflammatory agents for the prevention of labor, Rinaldi et al.  concludeOne in four preterm infants is born to a mother with IUI . These iMycoplasma and Ureaplasma species are some of the most commonly isolated organisms from amniotic fluid in cases of infection-induced PTB  is a non-specific free radical scavenger and NF-κB inhibitor  production in amniotic fluid  and 5-lipoxygenase activity . This le E. coli , 60.Ureaplasma parvum-induced PGE2 production in ovine gestational membranes  peptide that spans the IKKβ NEMO-binding domain disrupting interaction between NEMO and IKKβ  Figure  and coliembranes  but not embranes ; differeE. coli-induced TNF-α and PGE2 production in the fetal compartment, and to a lesser extent in the maternal compartment , prevent the onset of PTL, and minimize risk of FIRS. While prophylactic antibiotic trials have not been overly encouraging, there have been some successes  and exci−/−) resulted in embryonically lethal uncontrolled apoptosis in the liver of mice  and OxZnl (TAK1 inhibitor) in an ovine model of LPS-induced chorioamnionitis showed that the CSAIDs were well tolerated by the fetus, at least in the short-term, with no obvious changes in birth weight or fetal liver function observed , and were more likely to deliver spontaneously before 35 weeks of gestation . These studies suggest that assessing sonographic cervical length may be a useful predictor of risk of microbial invasion of the amniotic cavity and intra-amniotic inflammation (Intrauterine infection is often chronic and usually asymptomatic until the presentation of PTL, at which time it is often too late to treat and the fetus has been irreversibly exposed . The earammation .in vitro and animal models of IUI. These compounds have the potential to be safer and more effective than less selective inhibitors as they target key molecules involved in the pro-inflammatory signaling cascades that prematurely trigger labor. Issues regarding maternal and fetal toxicity, mode of drug delivery, off-target side effects, and appropriate identification of women requiring treatment remain to be addressed. Based on our current appreciation of the importance of IUI and inflammation in the etiology of PTB, the identification and treatment of pregnant women at risk of IUI with effective cytokine signaling inhibitors holds great promise for the prevention of PTB and improvement of neonatal outcomes.Infection and inflammation is the leading cause of PTB, but antenatal antibiotic treatment has had limited success at preventing PTB or improving neonatal outcome , 91. NewThe 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.http://journal.frontiersin.org/article/10.3389/fimmu.2015.00166The Supplementary Material for this article can be found online at Click here for additional data file.


The prognosis of patients with metastatic melanomas is extremely heterogeneous. Therefore, identifying high-risk subgroups by using innovative prediction models would help to improve selection of appropriate management options.In this study, two datasets (GSE7929 and GSE7956) of mRNA expression microarray in an animal melanoma model were normalized by frozen Robust Multi-Array Analysis and then combined by the distance-weighted discrimination method to identify time course-dependent metastasis-related gene signatures by Biometric Research Branch-ArrayTools (BRB)-ArrayTools. Then two datasets (GSE8401 and GSE19234) of clinical melanoma samples with relevant clinical and survival data were used to validate the prognosis signature.A novel 192-gene set that varies significantly in parallel with the increasing of metastatic potentials was identified in the animal melanoma model. Further, this gene signature was validated to correlate with poor prognosis of human metastatic melanomas but not of primary melanomas in two independent datasets. Furthermore, multivariate Cox proportional hazards regression analyses demonstrated that the prognostic value of the 192-gene set is independent of the TNM stage and has higher areas under the receiver operating characteristic curve than stage information in both validation datasets.Our findings suggest that a time course-dependent metastasis-related gene expression signature is useful in predicting survival of malignant melanomas and might be useful in informing treatment decisions for these patients.The online version of this article (doi:10.1186/s13000-014-0155-2) contains supplementary material, which is available to authorized users. The incidence of malignant melanoma, an aggressive form of skin cancer, is increasing rapidly globally, including in China . It has Gene expression profiling has been used to establish molecular signatures for classifying the subtypes of primary tumors and predicting the clinical outcome of multiple cancers including malignant melanoma ,6. PreviThe gene expression profiles of four independent datasets deposited in the Gene Expression Omnibus (GEO)  were analyzed [The GSE7929 comprises the gene expression microarray data of 32 human melanoma cells samples including 11 replicas of parental melanoma cell line A375 (LeiATCC) and 21 derived cell lines with different metastatic potentials selected based on a xenotransplant metastasis model [The GSE8401 dataset was generated from 83 melanoma samples (31 primary and 52 metastatic tumors) that were collected from patients undergoing surgery were collected from 1992 to 2001 at the Massachusetts General Hospital and Harvard Medical School as a part of the diagnostic workup or planning of therapy . Fifty-tThe GSE19234 dataset comprises 44 melanoma tumors with metastasis were collected at the New York University Medical Center from 1988 to 1997 [As claimed in the original publications, all the studies on these datasets had been approved by the local ethical committees and samples were collected with written informed consent from the patients.Two human melanoma cell line datasets GSE7929 and GSE7956 were used to identifying time course-dependent metastasis-related gene signatures. All gene expression profiles were normalized with frozen Robust Multi-Array Analysis (fRMA), a procedure in which allows arrays are not longer clustered per study but per platform . DWD is A time course analysis of the combined datasets of GSE7929 and GSE7956 was performed with the expression profiles of different generations of metastatic cell lines set as time-course data. A BRB-ArrayTool plug-in was used to perform regression analysis of time course expression data to identify the genes whose expression varies over time. The round numbers of different generations of selected cells were set as different time points. The tests were performed at a false discovery rate (FDR) threshold of 0.05. A list of genes that satisfied this threshold was produced and used as gene sets to assess correlations with poor survival of patients with melanomas in the validation datasets.Two clinical melanoma sample datasets (GSE8401 and GSE19234) were used to validate the prognostic significance of the time course-dependent metastasis-related gene signature. For the validated datasets, series matrix files containing normalized data were obtained from the GEO. Survival risk prediction of the gene expression data by the time course-dependent metastatic gene signature was performed based on principal components with BRB-ArrayTools software. Survival risk prediction was based on 10-fold cross-validation and classified patients into low- and high-risk groups according to the prognostic indexes.Distributions of overall survival were assessed using the Kaplan-Meier curve method and log-rank statistics based on 100 permutations. Multivariate analyses of prognostic factors were based on the Cox proportional hazards model. Area under the receiver operating characteristic (ROC) curves were used as a performance measure for prognostic factors. Permutated Kaplan-Meier curve analysis was performed using BRB-ArrayTools. All other statistical analyses were performed with Medcalc Software. P values less than 0.05 were considered statistically significant.In this study, a gene set that correlated with the metastatic potential of melanoma cell lines was first identified from the gene expression profiling of two datasets (GSE7929 and GSE7956). To increase power, these two data sets were merged by the DWD method after fRMA normalization. As seen in Figure The merged dataset comprised of 71 samples among which the parental lines samples were assigned as “time course 0” n = 21); metastasis-derived cell lines established after one round of selection as “time course 1” (n = 24); derived cell lines after two or three rounds of selection as “time course 2” (n = 26). Cells at different metastatic stages had distinct expression patterns. As seen in Figure 1; metastNext, the robustness with which the 192 metastasis-related gene expression signature in predicts survival in patients with melanomas was further tested with whole-genome mRNA expression profiling data obtained from two completely independent datasets (GSE8401 and GSE19234). Using the prognostic indexes based on our gene signature, patients were partitioned into high- and low-risk groups. As seen in Figure Multivariate Cox proportional hazards regression analyses showed that the prognostic value of our 192-gene signature was independent of TNM stage in both validation datasets  method and could enhance prediction of survival of metastatic melanoma patients by clinical staging . A more In this study, we re-analyzed the same dataset of animal melanoma models as that assessed by Xu et al. . Unlike Interpretation of gene function showed that the 192 genes are enriched significantly in extracellular matrix receptor interaction, focal adhesion and the β-transforming growth factor signaling pathway. Many of these genes have previously been implicated in melanoma metastasis and prognosis prediction, including MYC, GPNMB, GAB2, and SATB1 -15. TherIn conclusion, the time course-dependent gene signature we have developed is useful for predicting survival of patients with metastatic melanomas and might be useful for informing treatment decisions.


The aim of this paper was to conduct a systematic review of the published literatures comparing the use of mechanical chest compression device and manual chest compression during cardiac arrest (CA) with respect to short-term survival outcomes and neurological function.Databases including MEDLINE, EMBASE, Web of Science and the ClinicalTrials.gov registry were systematically searched. Further references were gathered from cross-references from articles by handsearch. The inclusion criteria for this review must be human prospective controlled studies of adult CA. Random effects models were used to assess the risk ratios and 95 % confidence intervals for return of spontaneous circulation (ROSC), survival to admission and discharge, and neurological function.Twelve trials , involving 11,162 participants, were included in the review. The results of this meta-analysis indicated no differences were found in Cerebral Performance Category (CPC) scores, survival to hospital admission and survival to discharge between manual cardiopulmonary resuscitation (CPR) and mechanical CPR for out-of-hospital CA (OHCA) patients. The data on achieving ROSC in both of in-hospital and out-of-hospital setting suggested poor application of the mechanical device . OHCA patients receiving manual resuscitation were more likely to attain ROSC compared with load-distributing bands chest compression device . The in-hospital studies suggested increased relative harm with mechanical compressions for ratio of survival to hospital discharge . However, the results were not statistically significant between different kinds of mechanical chest compression devices and manual resuscitation in survival to admission, discharge and CPC scores for OHCA patients and survival to discharge for in-hospital CA patients.The ability to achieve ROSC with mechanical devise was inferior to manual chest compression during resuscitation. The use of mechanical chest compression cannot be recommended as a replacement for manual CPR, but rather a supplemental treatment in an overall strategy for treating CA patients. Sudden cardiac arrest (CA) occurs when someone’s heart stops beating unexpectedly. Minimally interrupted, regular and appropriate cardiopulmonary resuscitation (CPR) can keep blood flowing to the victim’s vital organs while the heart is not pumping –4. UnforMachines have been developed to take over this chest pumping action using pneumatically driven or load-distributing bands (LDBs) mechanisms, because the machines do not pause or get tired, and deliver uninterrupted chest-compressions with a predefined depth and rate . Some stA meta-analysis was performed to compare any type of mechanical chest compression device with manual chest compression in the management of patients suffered from CA in out-of-hospital and in-hospital settings. Human prospective randomized controlled studies comparing compressions delivered via any type of powered, automatic mechanical compression device versus manual compression were considered for inclusion. Studies explicitly including patients with CA caused by drowning, hypothermia and toxic substances were excluded.The primary outcome for this meta-analysis was return of spontaneous circulation (ROSC) defined as spontaneous palpable pulse. Secondary outcomes included survival to hospital admission for out-of-hospital cardiac arrest (OHCA) patients only, survival to hospital discharge and good neurological outcome after hospital discharge, with Cerebral Performance Category (CPC) scores one or two for both in-hospital and out-of-hospital patients.A search strategy was pursued, using the following search terms: “mechanical”, “manual”, “chest compression” and “cardiopulmonary resuscitation”. Searches were conducted in MEDLINE (1946 to 31 August 2015), EMBASE (1950 to 31 August 2015), Web of Science  and the ClinicalTrials.gov registry (on 31 August 2015). We handsearched bibliographies of included papers. The search was inclusive of studies in any language.Data selection and data extracted were performed among pairs of independent reviewers, and the results were confirmed by a third review author. Discrepancies were discussed and adjudicated by the team consensus. In reporting the results of this systematic review, the authors have followed the recommended guidelines from the Quality of Reporting of Meta-Analysis (QUOROM) Statement.I2 statistics. Funnel plots and Egger’s regression test were used to assess the potential for reporting bias.Data were checked and entered into the Stata 12.0  database for further analysis. Using a random-effects model, we calculated the risk ratios (RR) and 95 % confidence intervals (CI) for ROSC, arrival to hospital with a spontaneous palpable pulse, survival at discharge and CPC score. The presence of heterogeneity between trials was assessed using the In the original review, the comprehensive search identified 678 citations . Two independent review authors reviewed 678 citations by titles; possible citations were selected for review by abstract. After review by abstract, potential relevant were identified and reviewed by full article. After exclusion, 12 studies were found meeting the inclusion criteria, included 9 out-of-hospital studies and 3 in-hospital studies –34. The N = 8886), including 7 out-of-hospital studies (N = 8590) and 3 in-hospital studies (N = 296). These data were entered into a forest plot respectively, resulting in risk ratio of 0.87  for OHCA patients and 0.71  for in-hospital CA patients  was 83.4 % for OHCA patients, which indicated that the treatment effect might not be the same for each device when being compared with manual compression. After analyzed separately according to the device type, a decrease in ratio of ROSC was observed with the use of AutoPulse as compared with manual chest compressions , while non-significant effect was observed between LUCAS and manual chest compression  for OHCA.ROSC was reported in 10 studies . Furthermore, when device type was analyzed separately, the treatment effect was not significant for AutoPulse , for LUCAS  and for Thumper (0/7:1/10) with manual chest compression, respectively.8 studies comprising a total of 9975 OHCA patients met the selection criteria. Although an analysis combining multi-mechanical CPR devices produced an insignificant treatment effect   and 2 in-hospital studies (N = 200). Data reported in the out-of-hospital studies suggested non-significant effect between manual and mechanical compressions , although the in-hospital studies suggested increased relative harm with mechanical compressions  , subgroup analyses were conducted to evaluate the effect of different CPR device on patients with OHCA. The use of any type mechanical chest compression had no significant differences in ratio of survival to discharge with manual chest compression .Several included studies reported data of survival to hospital discharge, including 7 out-of-hospital studies N = 488 and 2 Six trial including 8825 OHCA patients provided data for the CPC score of this review. The pooled meta-analytic results for good neurological function (defined as a CPC score of one or two) at hospital discharge were not significant , which indicated a similar treatment effect for good neurological outcome with the use of mechanical and manual chest compressions Fig. . The staP value from Egger’s regression test showed no significant statistical evidence of publication bias for both out-of-hospital and in hospital CA .Meanwhile, the funnel plot indicated the publication bias of the review was acceptable Figs.  and 9. TThe goal of CPR treatment for CA patients is to achieve ROSC and normal neurological function as early as possible while minimizing end-organ damage and dysfunction in the interim. Several animal and human studies have demonstrated an inverse relationship between poor chest compression quality and short-term survival –37. The Twelve studies  were identified after the search of the literatures, comprising five studies about LUCAS, four about AutoPulse, three about Thumper and one about vest CPR (one paper including both LUCAS and AutoPulse). The publication dates of these included studies span over 40 years. The results of this meta-analysis indicated no difference is found in CPC scores, survival to hospital admission and survival to discharge between manual and mechanical CPR for OHCA patients. The data on achieving ROSC in both of in-hospital and out-of-hospital setting and survival to discharge for in-hospital patients suggested poor application of the mechanical device. Considering the heterogeneity in types of mechanical devices used, we identified studies that reported LUCAS and AutoPulse in ROSC with mechanical chest compressions compared with manual chest compressions respectively, the results shown that OHCA patients receiving manual resuscitation were more likely to attain ROSC compared with AutoPulse; however, the difference was not significant between LUCAS and manual resuscitation. On the whole, available data from clinical trials did not provide sufficient evidence to permit conclusions on the effectiveness of mechanical chest compressions as compared with manual chest compressions, which was similar to the conclusion of another recent review and meta-analysis about OHCA patients [Many factors affect the chances of survival after cardiac arrest, including early recognition of arrest, effective cardiopulmonary resuscitation and defibrillation, and post-resuscitation care. A critical issue is the delay between collapse and the start of the intervention. For OHCA patients, witnessed CA receiving bystander basic life support might improve outcome . One of In this meta-analysis, there were some limitations. The first limitation was that some of the included studies were prospective observational trials or phased controlled trials. It was possible that selection bias could be introduced that the paramedics tended to have a lower threshold for initiation of resuscitation. A second limitation was that none of studies were blinded. Because the rescuers who decided when to initiate CPR were not blinded, introducing the possibility of rescuers preferential application of a device to patients thought to have a very poor prognosis in the hope that use of the device might lead to better outcomes than attained with standard care. Thirdly, these included studies were conducted across nearly 50 years, which demonstrated marked heterogeneity in types of mechanical devices used, timing of device application, CPR quality of manual chest compression and post-resuscitation management. Although we had made a subgroup analysis according to the types of mechanical devices  for OHCA patients, the heterogeneity could not be eliminated and would reduce the statistical power of the approach based on the limited data. Finally, very limited data were available from those studies comparing long-term survival and neurological function score, results from these studies were not pooled.In this systematic review, the combined meta-analysis of mechanical chest compression devices compared with manual chest compressions shown a better result with manual chest compression in ROSC rates for both out-of-hospital and in-hospital CA patients and survival of discharge for in-hospital CA patients. When analyzed separately, only AutoPulse was found to be inferior to manual chest compressions with ratio of ROSC. We believed that the mechanical chest compression should not be seen as a replacement for manual CPR, but rather a supplemental treatment in an overall strategy for treating CA patients.


Vascular complications are major causes of morbidity and mortality in type 2 diabetes patients. Mitochondrial reactive oxygen species (ROS) generation and a lack of efficient antioxidant machinery, a result of hyperglycaemia, mainly contribute to this problem. Although advances in therapy have significantly reduced both morbidity and mortality in diabetic individuals, diabetes-associated vascular complications are still one of the most challenging health problems worldwide. New healing options are urgently needed as current therapeutics are failing to improve long-term outcomes. Particular effort has recently been devoted to understanding the functional relationship between chromatin structure regulation and the persistent change in gene expression which is driven by hyperglycaemia and which accounts for long-lasting diabetic complications. A detailed investigation into epigenetic chromatin modifications in type 2 diabetes is underway. This will be particularly useful in the design of mechanism-based therapeutics which interfere with long-lasting activating epigenetics and improve patient outcomes. We herein provide an overview of the most relevant mechanisms that account for hyperglycaemia-induced changes in chromatin structure; the most relevant mechanism is called “metabolic memory.” Diabetes is a leading cause of morbidity and mortality across the world , 2. PopuFree radical accumulation in the vasculature of diabetic patients is considered to be the most significant mechanism in long-lasting vascular complications , 18. TheκB-p65 gene, resulting in the increased expression of its product in the diabetic setting . Ov. Ov28].


AHCY gene. Main characteristics are psychomotor delay including delayed myelination and myopathy  from birth, mostly associated with hypermethioninaemia, elevated serum creatine kinase levels and increased genome wide DNA methylation. The prime function of AHCY is to hydrolyse and efficiently remove S-adenosylhomocysteine, the by-product of transmethylation reactions and one of the most potent methyltransferase inhibitors. In this study, we set out to more specifically characterize DNA methylation changes in blood samples from patients with AHCY deficiency. Global DNA methylation was increased in two of three analysed patients. In addition, we analysed the DNA methylation levels at differentially methylated regions (DMRs) of six imprinted genes  as well as Alu and LINE1 repetitive elements in seven patients. Three patients showed a hypermethylation in up to five imprinted gene DMRs. Abnormal methylation in Alu and LINE1 repetitive elements was not observed. We conclude that DNA hypermethylation seems to be a frequent but not a constant feature associated with AHCY deficiency that affects different genomic regions to different degrees. Thus AHCY deficiency may represent an ideal model disease for studying the molecular origins and biological consequences of DNA hypermethylation due to impaired cellular methylation status.S-adenosylhomocysteine hydrolase (AHCY) deficiency is a rare autosomal recessive disorder in methionine metabolism caused by mutations in the  AHCY  removes SAH (S-adenosyl-L-homocysteine) formed in each S-adenosylmethionine (SAM)-dependent methyltransferase reaction by catalysing its hydrolysis to adenosine and homocysteine . In eukaAHCY gene resulting in a reduced activity of AHCY have been reported in eight patients [3H]dCTP into DNA of patients compared to that of control individuals  obtained in one measurement of patient B and the mean 5-methyl-cytosine methylation and standard deviation [%] detected in one measurement of 5 female and 5 male control samples, respectively. (EPS)Click here for additional data file.


The lack of a genetic transformation system that allows integration of a foreign gene into the genome is a serious hindrance to progress in biological research on seaweeds. In contrast, physiological research in seaweeds has a long history of focus on the establishment of cell polarity and multicellularity  35S RNA promoter that has been used for foreign gene expression in seaweeds prior to establishment of the transient gene expression has rather low activity in seaweed cells  creating expression constructs particularly suited to seaweed cells; (2) transferring the expression constructs into cells; (3) ensuring integration of expression constructs into the genome; and (4) selecting genetically transformed cells. It has already been confirmed that combining codon-optimized coding regions and endogenous strong promoters results in high-efficiency expression in seaweed cells -resistant and uracil-auxotrophic mutants were used as recipients for genetic transformation , it is possible that if 5-FOA-resistant uracil auxotrophic strains are obtained in a variety of other seaweeds, a genetic transformation system such as that in C. merolae could be established for each. However, preliminary data clearly indicate that there is substantial intrinsic 5-FOA-resistance in Bangia fuscopurpurea and it would therefore be impossible to develop a UMPS-dependent transformation system, at least in this seaweed. Thus, it will be necessary to assess the degree of 5-FOA-sensitivity of other seaweeds.Isolation of genetically transformed cells is quite important for the production of transgenic lines. For the red unicellular alga Porphyridium sp. using SMM sensitivity , transcription activator-like effector nuclease, and the clustered regulatory interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9), and subsequent repair of the breaks via homologous recombination or non-homologous end joining (NHEJ) in the genome (Gaj et al., Chlamydomonas reinhardtii (Sizova et al., Agrobacterium-mediated transformation in plants. Thus, genome sequences of seaweeds are being carefully surveyed to identify the U6 small RNA genes and also strong promoters, but the prospects for using Agrobacterium for transformation of seaweeds would need to be tested.At present, the ZFN genome editing method has been successfully used for targeted mutagenesis in algae only in A number of paths that could be taken to establish a gene-targeting system for seaweeds have been described above. The two most critical technological issues to address include the stable integration of targeted foreign genes into the genome, and effective antibiotic-based selection of genetically transformed cells. Gene targeting may also be possible via genome editing with gene-specific artificial endonucleases; however, much would be necessary to optimize this type of system for seaweeds. Currently, the most feasible way to establish a system for genetic transformation is to insert foreign genes into the genome by site-specific homologous recombination after introduction of constructs using particle bombardment or microinjection methods and subsequent antibiotic-dependent selection of genetically transformed cells.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although microRNAs are highly conserved among species, the functional implications of existing lineage-specific changes and their role in determining differences between humans and other great apes have not been specifically addressed. We analyzed the recent evolutionary history of 1,595 human microRNAs by looking at their intra- and inter-species variation in great apes using high-coverage sequenced genomes of 82 individuals including gorillas, orangutans, bonobos, chimpanzees and humans. We explored the strength of purifying selection among microRNA regions and found that the seed and mature regions are under similar and stronger constraint than the precursor region. We further constructed a comprehensive catalogue of microRNA species-specific nucleotide substitutions among great apes and, for the first time, investigated the biological relevance that human-specific changes in microRNAs may have had in great ape evolution. Expression and functional analyses of four microRNAs  revealed that lineage-specific nucleotide substitutions and changes in the length of these microRNAs alter their expression as well as the repertoires of target genes and regulatory networks. We suggest that the studied molecular changes could have modified crucial microRNA functions shaping phenotypes that, ultimately, became human-specific. Our work provides a frame to study the impact that regulatory changes may have in the recent evolution of our species. Over the last years, attention has been focused on the role that regulatory elements may have played in shaping diversification among species and individuals that share extended genomic similarities. Certain specific changes in transcription factors and non-coding RNAs have been shown to be under positive selection and to contribute to determine phenotypic differences between species such as humans and chimpanzees ,2. AmongmiRNAs are initially transcribed by RNA polymerase II as primary miRNAs, from which Drosha-mediate cleavage gives a ~70 nt hairpin precursor miRNA (pre-miRNA), that is processed by Dicer in the cytoplasm into a miRNA duplex formed by two ~22 nt mature miRNA strands, 5p and 3p . One of miRNAs can be located either in intergenic regions or within genes, and they can be organized as single miRNAs or as clusters containing several miRNAs, which are usually transcribed together in a polycistronic primary miRNA ,12. MostPrevious studies have shown that the miRNA repertoire has constantly increased during metazoan evolution. It is estimated that, in mammals, half of the new-born miRNAs would be deleterious and eliminated by purifying selection; however, some of them become integrated into gene regulatory networks having low chances to be subsequently lost ,14–16. TSo far, the study of primate miRNAs has been mostly focused on the identification of orthologous miRNAs in different species. Firstly by comparative genomic approaches taking advantage of the high level of sequence conservation of miRNA genes across species –21; and It has been shown that selective pressures have differentially affected miRNA regions in animal evolution. The seed region has been commonly considered as being the most conserved region of miRNAs as well as the main region for target gene recognition and consequently for gene regulation. Nevertheless, increasingly number of studies has shown that other positions along the mature region could be under similar selective constraint as demonstrated by a research in metazoans showing that the seed region and nucleotides 13 to 16 of the mature molecule are the most conserved miRNA nucleotides . This agGenomic coordinates of 1,595 human pre-miRNAs containing 2,233 mature miRNAs were obtained from miRBase (release 19). Nucleotides 2 to 8 of the 5’ end of mature miRNAs were defined as the seed region. We considered flanking 5’ and 3’ regions of 200 bp upstream and downstream of the pre-miRNA, respectively. We defined miRNAs as clustered when they were located less than 8 Kb from another miRNA and as isolated otherwise. We classified miRNAs as intragenic when they overlapped an annotated RefSeq gene and as intergenic otherwise. Intragenic miRNAs were further classified as intronic, exonic or non-coding. We also grouped miRNAs regarding their molecular age ,30 and tGorilla gorilla dielhi from [Data was taken from Prado-Martinez et al.  where 82lhi from  data sinlhi from  excludinlhi from .For the inter-species variation analyses, we used the density of single nucleotide variants (SNV density) fixed in at least one of the populations along the phylogeny as a surrogate measure of purifying selection. Given that the time of divergence between the great ape populations is relatively small, we assumed the number of fixed substitutions along the great ape phylogeny to be proportional to the substitution rate. We excluded all positions that were polymorphic in at least one great ape population (1000 Genomes database) . We usedThe PCA was performed using the prcomp function in R. The variables used in the PCA were SNV density for the pre-miRNA and other miRNA features such as miRNA clustering, expression levels, involvement in disease, molecular age and localization regarding other transcriptional units. The 576 miRNAs for which these features were available were included in the PCA. The correlation study between the expression data and the SNV density included miRNA gene expression from five human tissues  . We usedThis work was conducted according to relevant Spanish and International guidelines. Tissue samples were collected after natural death and thus it was done in a non-invasive way without disturbing, threatening or harming the animals. Blood samples were explicitly not taken for this study.® Tissue DNA Kit from Omega BioTek . Mouse brain RNA and human RNA samples were purchased at Ambion .Gorilla brain tissue was provided by the Barcelona Zoo (Spain). Chimpanzee and macaque brain and testis tissues were provided by the Biomedical Primate Research Centre (Netherlands). Total RNA was extracted with the RNeasy Mini Kit . DNA was extracted using the EZNABamH1 and XhoI restriction sites and transfected in HeLa cells grown as previously described [5 cells/well in 12-well plates. After 24h, cells were double-transfected with Lipofectamine 2000 (Invitrogen) and 0.75 μg of DNA with the plasmid constructions carrying either the human or non-human pre-miRNAs plus a control reference miRNA. Transfections were optimized to an efficiency of over 70% by monitoring the expression of the fluorescent ZsGreen1 protein. Three independent experiments each including three technical replicates were performed. For transcriptome analyses SH-SY5Y cells were transfected with 100 μM of miRNA mimic variants  as previously described [™ miRNA Mimic Transfection Control with Dy547, Dharmacon) was used to optimize the transfection to an efficiency of over 70%. Four independent experiments were performed. In both approaches non-human miRNA sequences were taken from chimpanzee, except for mir-541, for which we took the macaque sequence since no chimpanzee mir-541 has been described so far.For expression level analyses, pre-miRNA genomic sequences of the selected miRNAs  and ~100 bp flanking regions at each side were PCR amplified  following manufacturer’s protocol. Standard curves were calculated separately for each miRNA variant by pooling all samples from transfection experiments. Expression level comparisons between human and non-human miRNA variants were calculated from RT templates 20x diluted using the method published by Pfaffl [miRNA expression analyses were performed by RT-qPCR starting from 100–300 ng of total RNA from either different mammalian tissues (for measuring tissue expression) or transfected HeLa cells (for measuring expression level differences) using specific primers for each species variant . Primersy Pfaffl  by quantwww.ebi.ac.uk/arrayexpress) under accession number E-MTAB-3617.RNA samples from four independent transfection experiments of each miRNA variant in SH-SY5Y cells were used for microarray expression experiments in Agilent SurePrint G3 Human Gene Expression microarrays (8x60k) starting from 300ng of total RNA. Data were analyzed using the Array File Maker (AFM) 4.0 software package . We considered genes differently expressed when comparing human and non-human miRNA mimic variants  and when comparing each variant with the negative mimic control . Analyses on the biological functions associated with deregulated genes were performed using the Ingenuity Pathway Analysis software (IPA). Microarray data is publically available at the ArrayExpress database  . In ordeMinimum free energy (MFE) of the secondary miRNA structures were based on RNAfold predictions. miRNA sequences were taken from miRBase (release 21). Non-human miRNA sequences were taken from chimpanzee, except mir-541 that was taken from macaque.To systematically study the recent evolutionary history of miRNAs we used high-coverage sequencing data from 82 individuals from ten great ape populations including humans . We firsTo rule out the possibility that we did not observe differences between the seed and the rest of the mature miRNA due to short evolutionary distances in the analysis that do not provide enough information, we calculated average PhastCons conservation scores in each of the miRNA regions using pre-computed values for all primates . In accoTo assess whether any feature of the miRNAs, such as their levels of expression, their molecular age, their role in disease or their location respect to other transcriptional units, was correlated with their levels of constraint we ran a principal component analysis. The first component was mostly explained by the number of diseases each miRNA is involved in (data according to Human miRNA Disease Database) , whereasIdentifying miRNA species-specific substitutions along the great ape phylogeny could provide new insights into the impact these changes may have had in the recent evolutionary history of great apes. We calculated the number of fixed nucleotide substitutions in miRNAs along the different great ape populations  and founSince mutations in the mature miRNA sequence are predicted to have a stronger impact on miRNA function than mutations in the precursor sequence we focused on the first as potential candidates for functional studies. We considered the 82 miRNAs with human-specific nucleotide substitutions in the mature region and selected the miRNAs with the highest average expression levels in brain, a tissue with high levels of miRNA regulation activity , based oTo test if human-specific substitutions in miRNAs could implicate differences in miRNA expression, we measured the expression of the four selected miRNAs in HeLa cells transfected with either the human or the non-human pre-miRNAs. Expression levels of the three miRNAs with nucleotide substitutions located in the mature out of the seed region were significantly different between the human and non-human miRNAs  . SpecifiDifferences in expression levels between versions of miRNAs could be also determined by differences in their molecular stabilities. To address this, we calculated the minimum free energy (MFE) of the human and non-human predicted pre-miRNA secondary structures using RNAfold . InteresApart from differences in expression levels, nucleotide substitutions in the mature region could change the spectrum of miRNA target genes, thus we investigated which genes could be regulated by each miRNA variant thorough miRNA overexpression experiments. SH-SY5Y neuroblastoma cells were singled transfected with miRNA mimics of either the human, the non-human primate version of each mature miRNA or a negative control. All four miRNAs modified the expression of a variable number of genes  . The humKCTD11, SLC12A6, and PMP22  were deregulated by the non-human miR-503-3p in the transcriptome experiments and predicted as candidate targets for the same variant . These two miRNAs are located within a large cluster in the human chromosome 14 that was originated before the radiation of modern placental mammals . miRNAs ntiation ; SLC12A6isorders  and PMP2s system . Our ress system , in a cls system . Althougs system , in acco variant . As expede novo or they can undergo a neo-functionalization process from existing miRNAs by mutation. We speculate that other putative mechanisms could give rise to changes in miRNA function such as differential biogenesis giving precursor molecules of different lengths among species. We suggest that this could be the case for mir-299 and mir-508 for which the hairpin length between humans and the other great apes has been described to be different. Gene duplication is another common mechanism responsible of the birth of most of the miRNAs [In general, miRNAs can appear e miRNAs  as it hae miRNAs . RemarkaWe engaged in a deep study of the selective pressures acting on miRNAs molecules to elucidate the functional relevance that certain species-specific modifications may have had in shaping phenotypic differences among species. We find that the whole mature region and not particularly the seed region is under selective constrain, highlighting the importance of the entire mature domain in the miRNA function. We have thoroughly studied the possible functional consequences of human-specific substitutions in the mature region of four miRNAs and show that miR-299-3p and miR-541-3p are conserved miRNAs with neuronal functions and thus evolutionary changes in these miRNAs may have had an impact in gene regulatory networks ultimately related to the evolution of the nervous system. Conversely, miR-503-3p and miR-508-3p regulate a low number of genes, have a restrictive pattern of expression and, although they seem to be involved in development, their functional role is still very vague. We show that specific nucleotide substitutions in the mature region and/or changes in the length of existing miRNAs may affect miRNA expression and hypothesize that both could be mechanisms by which miRNAs acquire new regulatory functions. Our study addresses for the first time the role that human-specific substitutions in miRNAs could have had in our recent evolutionary history and enlarges our understanding of how the non-coding genome may have contributed to shape human-specific traits.S1 Fig(PDF)Click here for additional data file.S1 TableInformation based on Prado-Martinez et al. (2013).(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableTop right in grey: p-values for the generalized linear model used to compare conservation among different miRNA regions. The model takes into account GC content: SNV density ~ GC content + miRNA region. Bottom left in white: p-values for the paired t-test comparing concatenated nucleotide diversity values of each miRNA region. Asterisks indicate p-value < 0.05.(PDF)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 TableMFE in kcal/mol of the secondary structures predicted for the four studied miRNAs according to RNAfold.(PDF)Click here for additional data file.S6 TableData based on the Ingenuity Pathway Analysis software.(PDF)Click here for additional data file.S7 Table(XLS)Click here for additional data file.S8 Table(XLS)Click here for additional data file.


The acute event of thoracic aortic dissection carries with it high mortality and morbidity. Despite optimal initial surgical or medical management strategies, the risk of further complications in the long-term, including aneurysmal dilatation and false lumen (FL) expansion, are not insignificant. Adequate follow-up of such conditions requires dedicated imaging where relevant prognostic indicators are accurately assessed. We perform a systematic review of the literature and report the current evidence for the use of magnetic resonance imaging (MRI) in assessment of chronic aortic dissection. We then make a comparison with traditional imaging modalities including computed tomography and echocardiography. We discuss new ways in which MRI may extend existing aortic assessment, including identification of blood-flow dynamics within the TL and FL using phase-contrast imaging. Aortic dissection is a catastrophic complication of aortic wall disease associated with high mortality and morbidity. The underlying process of the aortic wall disruption is most commonly secondary to atherosclerotic disease  or a known connective tissue disease . Using data from the International Registry of Acute Aortic Dissection (IRAD), aortic dissection is more common in men, with mean age of 63 years and an incidence of up to 0.8% at autopsy –4. AccorIt is well recognized that persisting or chronic aortic dissection  is a risk factor for further aortic dilatation and dissection extension –9. In thA key issue regarding the management of chronic aortic dissections is a progressive increase in the size of the false lumen (FL). The reported incidence of partial or complete distal aortic FL patency in type A dissection patients is significant, ranging between 31 and 89% . Hence, The most commonly used modalities are computed tomography (CT) and echocardiography, however magnetic resonance imaging (MRI) has recently emerged as a comprehensive, non-ionizing imaging tool well suited to serial measurements in this group of patients Figure  13). In. In13). This review aims to compare the clinical utility of alternative imaging modalities for the follow-up of chronic aortic dissection by means of systematic review of the current literature, with emphasis upon the advantages and disadvantages of MRI in this setting, and discuss the future role of MRI in assessment of chronic aortic dissection.We performed a systematic review of the current literature using pre-existing guidelines , to asceTwelve studies were included for this review from the literature selection process  MRI studies utilizing low-field strengths (0.5–1.0 T) to assess chronic aortic dissection, results are favorable. Compared with CTA, the assessment of FL partial thrombosis was shown to be comparable , 30. UsiWhile these early studies highlighted the potential of MRI in imaging aortic dissection, current MRI scanners possess far superior technology and the ability to assess additional dynamic aspects of blood-flow and the aortic wall environment. This key potential advantage that MRI affords over other imaging modalities offers the prospect of developing new prognostic indicators that move beyond the simple measurement of vessel dimensions alone .A typical MRI protocol for imaging of the aorta will include GRE and black blood-weighted images for anatomic definition Figure B. The adThe ability to assess and quantify blood-flow movement within the aorta is gained when PC imaging is utilized Figure D. The abAn important application of MRI is in the identification of a number of important post-operative conditions. For example, the use of contrast-enhanced 3D MRA can distinguish aortic pseudoaneurysm from periprosthetic hematoma via identified areas of high signal intensity that are indicative of peri-graft flow . FurtherBlood-flow quantification using PC or velocity mapping can demonstrate bidirectional flow within a FL; such turbulent flow may induce aortic wall shear stress which may be associated with elevated risk of aneurysmal dilatation or tear –26, 42. Echocardiography is generally tolerated well by patients and does not require use of contrast or ionizing radiation. It is also widely available and may be performed at the bedside. It can assess FL flow, FL thrombosis, and FL communication with the true lumen (TL); all significant prognostic markers , 34, 43.In a study in 2000 by Di Cesare et al., 29 patients who had undergone surgery for type A dissection all underwent follow-up imaging with TEE, conventional MRI, and contrast-enhanced 3D breath-hold MRA . ImagingAlthough TEE can provide useful prognostic information in the acute setting, current recommendations suggest MRI or CT to be more useful for long-term follow-up , 43. GivComputed tomography angiography has essentially replaced conventional angiography in the assessment of aortic disease secondary to the reduced associated morbidity, lack of invasiveness, and lower cost associated with this modality. CTA is additionally readily available in the vast majority of clinical centers, and has a high reproducibility and low intra- and inter-operator variability . Its draThe time to complete a CTA examination is relatively short 4–20 s) and the use of electrocardiographic (ECG) gating techniques allows reliable artifact-free imaging of the aortic root and coronary arteries when necessary , 50, 51.–20 s andDespite improvements in modern multi-detector CT technology, limited temporal resolution can prevent reliable resolution of rapidly moving structures such as an intimal flap or native valve leaflets . Ganten As the traditional gold standard , aortogrn = 2)  as an adjunctive assessor of prognosis in chronic aortic dissection has been explored in several studies –58. SuchHemodynamic quantification appears to be the major advantage of MRI over other imaging modalities in chronic aortic dissection. Encoding of all three spatial directions of a volumetric data set utilizing 3D velocity-encoded cine MRI relative to the cardiac cycle, is commonly referred to as 4D flow MRI . The velClough et al. have shown excellent accuracy of 4D PC MRI in velocity assessment (when compared with the MRI gold standard of 2D PC MRI) . In theiIn the setting of chronic aortic dissection, the ideal imaging modality for use in follow-up has high sensitivity and specificity, is non-invasive, and can accurately identify not only aortic dimensions but also progressive changes in relative flow between true and FLs. MRI can achieve these objectives, as well as the assessment of significant prognostic indicators such as FL thrombosis. CT and echocardiography remain the two most widely used alternatives, given their easy access, rapid acquisition, and non-invasiveness. There is a paucity in the current literature comparing these imaging modalities in the context of chronic aortic disease. In patients requiring multiple studies, the risks of repeated contrast/radiation exposure  and inter-observer variability in assessed aortic dimensions remain a concern. New techniques in MRI include more accurate hemodynamic assessment and the use of 4D imaging to demonstrate blood-flow and potential wall shear stress. However, the relevance of such techniques to long-term prognosis warrants further prospective investigation. Given its advantages, and when available, MRI is suggested as a suitable imaging modality in the follow-up of chronic aortic dissection.Cardiovascular Magnetic Resonance Sydney receives sponsorship funding from Siemens Ltd. . There was no involvement from Siemens in the development or preparation of this manuscript.


The immune system is the built-in host defense mechanism against infectious agents as well as cancer. Protective immunity against cancer was convincingly demonstrated in the 1940s with syngeneic animal models . Since then, the last century’s dream has been to effectively prevent and cure cancers by immunological means. This dream has slowly but surely become a reality . The successful examples of immunoprophylaxis and therapy against cancers include: (i) targeted therapy using monoclonal antibodies ; (ii) allogeneic hematopoietic stem cell transplantion to elicit graft-versus-cancer effect against a variety of hematopoietic malignancies ; (iii) vaccination for preventing cancers with clear viral etiology such as hepatocellular carcinoma and cervical cancer ; (iv) T cell checkpoint blockade against inhibitory pathways including targeting CTLA-4 and PD-1 inhibitory molecules for the treatment of melanoma and other solid tumors ; (v) antigen-pulsed autologous dendritic cell vaccination against prostate cancer ; and (vi) the transfer of T cells including those genetically engineered with chimeric antigen receptors allowing targeting of B cell neoplasms .This article provides an overview on the exciting and expanding immunological arsenals against cancer, and discusses critical remaining unanswered questions of cancer immunology. The inherent specificity and memory of the adaptive immune response towards cancer will undoubtedly propel cancer immunotherapy to the forefront of cancer treatment in the immediate near future. Study of the fundamental mechanisms of the immune evasion of cancer shall also advance the field of immunology towards the development of effective immunotherapeutics against a wide spectrum of human diseases. Cancer immunotherapy has come a long way -16. In tCancer immunotherapy has already entered the mainstream of oncology . ExistinThree kinds of cancer immunotherapeutics have emerged as key breakthroughs in clinical cancer care in 2013. These advances include T cell checkpoint blockers  [+ T cells were observed in the cerebrospinal fluid. More importantly, complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment at the time of the report. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Thus, CAR-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. Indeed, Michel Sadelain’s group treated five relapsed acute B cell lymphocytic leukemia subjects with autologous T cells expressing a CD19-reactive CD28/CD3ζ second-generation dual-signaling CAR [Several proof-of-principle studies have demonstrated the huge potential of utilizing synthetic immunology to engineer CAR-expressing or TCR-expressing T cells for the adoptive therapy of select cancers including lymphocytic leukemia -46. Builia (CLL) . In Apriia (CLL) . In bothling CAR . All pat+ breast cancer, denosumab for giant cell tumor of the bone, bevacizumab for both first and second line therapy for metastatic colorectal cancer in combination with chemotherapy. Ever since anti-CD3 antibody was approved in 1986 for the treatment of autoimmune diseases, more than 35 antibodies have been introduced to the market with a significant portion of them indicated for cancer therapy including rituximab (anti-CD20 antibody), Herceptin (anti-Her2 antibody), and Ipilimumab (anti-CTLA-4 antibody).2013 continues to witness the increasing application of monoclonal antibody for cancer immunotherapy . ApproveThe current enthusiasm in cancer immunology raises the question of whether all cancers may be amenable by immune intervention. As a basis for addressing this question and making existing therapies more effective, there are four critical unanswered questions Table . First, Slowly but surely there has been a growing paradigm shift in the understanding of biology and immunology of cancer. This is evident from the essay by Douglas Hanahan and Robert Weinberg on the Hallmarks of Cancer (The Next Generation) in 2011 which added “avoiding immune destruction” and “tumor-promoting inflammation” as another two hallmarks of cancer to their original perspective . Given tImmunotherapy of cancer is no longer a dream. Gone is the time when elimination of cancer by immunological means was anecdotal or achievable only in animal studies. Immune-based therapies have now demonstrated efficacy in a range of clinical studies and types of cancer. Adoptive transfer of tumor-reactive T cells can cure select patients with advanced metastatic disease that have exhausted all other options. Other reagents, such as selective manipulation of T cell checkpoint blockers in cancer microenvironment, offer the possibility of off-the-shelf dosing or novel combinatorial therapies. More than ever before, the field of cancer immunology is permeated with a sense of optimism . The keyThe authors declare that they have no competing interests.All authors wrote, reviewed and approved the manuscript.


Body image disturbance is a core feature of anorexia nervosa (AN). Attitudinal and cognitive biases as well as fundamental perceptual differences have been hypothesized to play a role in this disturbance.This study investigated body image dissatisfaction and distortion, haptic perception and perfectionism in 30 patients with AN and 31 age-matched healthy controls. Participants completed perceptual tasks and self-report measures.As predicted, participants with AN scored significantly higher on body dissatisfaction, perfectionism measures and had greater body distortion (as assessed by a body size estimation task). Cognitive–affective factors and perfectionism were highly correlated with body image distortion in AN. No significant differences were found between groups on the generic perception task.Findings did not confirm the hypothesis of fundamental perceptual inefficiencies in body image disturbance in individuals with AN. Despite renewed interest in fundamental perceptual factors implicated in body image disturbance, these findings suggest that it continues to be important to focus treatment on cognitive affective biases versus fundamental perceptual inefficiencies. Body image disturbance is one of the diagnostic criteria  for AnorA growing consensus exists that two types of body representation may be impaired in AN: the body schema and the body image . The Distortions in either the body schema or the body image could result in body size overestimation. Recent research in eating disorders has focused on attitudinal components of body image disturbance, with individuals with AN showing higher levels of body dissatisfaction than healthy controls , is defined as the recognition of external stimuli, through the combination of somatosensory and proprioceptive perception . Haptic Further haptic-perceptual impairment was noticed in somatosensory tasks  persistiThe parietal lobe is also thought to be responsible for the integration of proprioceptive and visual information regarding one’s own body, with this integration forming the basis of the physical body representation . DisordThis study therefore first explored haptic perceptual task performance in individuals with a current diagnosis of AN as compared to age-matched controls; secondly, the attitudinal and perceptual aspects of body image disturbance, and their relative contribution to body image distortion in AN were investigated.The following hypotheses were tested: 1) individuals with AN would make greater body size estimation (BSE) errors than healthy controls (HC); 2) individuals with AN would have greater body dissatisfaction than HC; 3) individuals with AN would perform worse than HC on a measure of haptic perception; 4) the AN group would score more highly on measures of perfectionism compared to HC individuals; and 5) correlations would exist between attitudes to body image, haptic perception, perfectionism and distorted body image in the AN group.Sixty-one adult females were recruited to the study. Thirty females with a clinical diagnosis of AN or EDNOS-AN were recruited from eating disorder services of hospitals in South London. Diagnosis was made by trained clinicians using a semi-structured interview schedule and DSM-IV criteria. Thirty-one age-matched females were recruited to the HC group. A screening questionnaire confirmed the absence of diagnosed eating disorders and other psychiatric disorders. Study volunteers with an intellectual disability, head injury or non-fluent English were excluded from the study.Participants were asked to read the information sheet, sign the consent form and complete the screening form. The study was approved by a National Health Service ethics committee. The self-report measures were completed followed by the perceptual tasks.The Silhouettes Body Perception Scale.The original authors were able to detect a significant difference between AN (n = 16) and HC groups (n = 16) on right-handed tasks with this measure )Findings did not confirm the presence of fundamental haptic perceptual impairments in body image distortion in individuals with AN. Future work should explore whether or not previous findings in the literature demonstrating sub-optimal visual perceptual performance in AN could contribute to body image distortion. The findings did confirm the strong relationship between body image disturbance and cognitive-affective factors. This highlights the importance of continuing to focus on treatment interventions that target cognitive-affective biases and high levels of perfectionism as opposed to correcting underlying fundamental perceptual inefficiencies.The authors declare that they have no competing interest.AW - Conceived and designed the protocol, conducted experiments, analysed the results, drafted the paper. RL - Conducted the experiments, took part in analysis, revision of the paper. VM – co supervised the project, participated in planning the study and revised the paper. KT – principal investigator, planned the study protocol, helped with the participant recruitment, supervised the project, edited drafts, submitted the paper. All authors read and approved the final manuscript.


Peanut allergic patients may react to small amounts of the allergen with symptoms that can be life-threatening. The management of peanut allergy (PA) relies on allergen avoidance and adrenaline auto-injector for rescue treatment in cases at risk of anaphylaxis. Biomarkers of severity and threshold could improve the management of PA.To assess the utility of the basophil activation test (BAT) to predict the severity and the threshold of reactivity to peanut on double-blind-placebo-controlled-peanut-challenges (DBPCPC).Patients with positive DBPCPC were included in the study. The severity of the allergic reaction on DBPCPC was scored and the threshold dose was determined. Skin prick test, specific IgE to peanut and its components and BAT to peanut extract (PE) were performed on the day of the challenge.44 peanut allergic children (median age 5 years) reacted to peanut on DBPCPC with clinical symptoms than ranged from oral allergy syndrome to anaphylaxis. 61% of patients reacted to 0.1g of peanut protein. The mean %CD63+ basophil at 10 and 100 ng/ml of PE was independently associated with severity (p=0.012) whilst CD-sens (1/EC50x100) was independently associated with threshold (p=0.039) of allergic reactions to peanut. Severity and threshold parameters were correlated both at the clinical  and at the basophil level .Basophil reactivity and sensitivity are associated with severity and threshold of allergic reactions to peanut on DBPCPC. Further studies are needed to define prognostic cut-offs values for BAT to determine severity and thresholds of reactivity in peanut allergic patients.


Partial root-zone drying has been successfully applied under field conditions to woody and non-woody crops; yet the few previous trials with olive trees have produced contrasting results. Thirty year-old olive trees (Olea europaea ‘var. Chetoui’) in a Tunisian grove were exposed to four treatments from May to October for three-years: ‘control’ plants received 100% of the potential evapotranspirative demand (ETc) applied to the whole root-zone; ‘PRD100’ were supplied with an identical volume of water to the control plants alternated between halves of the root-zone every ten-days; ‘PRD50’ were given 50% of ETc to half of the root-system, and; ‘rain-fed’ plants received no supplementary irrigation. Allowing part of the root-zone to dry resulted in reduced vegetative growth and lower yield: PRD100 decreased yield by ~47% during productive years. During the less productive years of the alternate bearing cycle, irrigation had no effect on yield; this suggests that withholding of water during ‘off-years’ may enhance the effectiveness of irrigation over a two-year cycle. The amount and quality of oil within the olive fruit was unaffected by the irrigation treatment. Photosynthesis declined in the PRD50 and rain-fed trees due to greater diffusive limitations and reduced biochemical uptake of CO2. Stomatal conductance and the foliar concentration of abscisic acid (ABA) were not altered by PRD100 irrigation, which may indicate the absence of a hormonal root-to-shoot signal. Rain-fed and PRD50 treatments induced increased stem water potential and increased foliar concentrations of ABA, proline and soluble sugars. The stomata of the olive trees were relatively insensitive to super-ambient increases in [CO2] and higher [ABA]. These characteristics of ‘hydro-passive’ stomatal behaviour indicate that the ‘Chetoui’ variety of olive tree used in this study lacks the physiological responses required for the successful exploitation of PRD techniques to increase yield and water productivity. Alternative irrigation techniques such as partial deficit irrigation may be more suitable for ‘Chetoui’ olive production.The productivity of olive trees in arid and semi-arid environments is closely linked to irrigation. It is necessary to improve the efficiency of irrigation techniques to optimise the amount of olive fruit produced in relation to the volume of water used. Partial root-zone drying (PRD) is a water saving irrigation technique that theoretically allows the production of a root-to-shoot signal that modifies the physiology of the above-ground parts of the plant; specifically reducing stomatal conductance ( Olea europaea L.) is largely constrained by the availability of water during the summer months when the fruit develops  of 380 ppm, leaf temperature of 25°C and relative humidity ranged between 45 and 55%. Instantaneous transpiration efficiency was expressed as the ratio of A to gs. Mesophyll conductance was calculated using the variable J method involving simultaneous measurements of gas-exchange and chlorophyll fluorescence parameters as described by Harley et al.  were conducted in the field using the method of Centritto et al. . Th. Th100 tpiration , as allooduction  (eg.  and increased foliar [ABA] may suggest that the varieties of olive used in this study (var. ‘Chetoui’) and others  , and fu  and bei hybrid) . Not allartments . The absers eg. , 48) lac lacgs vaes) (eg. ). In ess30 irrigation  (The results of this study indicate that PRD irrigation was relatively ineffective in enhancing the yield of olive fruit relative to the volume of water utilised in irrigation . This ma2]  or [ABA]2]  may indi100 treatment relative to the control, despite receiving the same volume of water. Yield was 68 and 95% lower in the PRD50 and rain-fed treatments. The yield of fruit relative to the amount of water used was significantly lower under PRD in comparison to application of water to the whole root-zone. Supplementary irrigation did not enhance olive fruit yield during the less productive ‘off-year’, suggesting that co-ordination of the supply of water with the alternate bearing cycle may enhance water-productivity on a two-year basis. The quality and quantity of oil produced by equal amounts of olive fruit from each irrigation treatment was identical. Lower A was observed in the PRD50 and rain-fed treatments due to higher diffusive (2-uptake. A similar pattern was not observed in the PRD100 treatment, possibly indicating that a root-to-shoot signal inducing stomatal closure was not present. Stomatal conductance was identical in the control and PRD100 treatments, as were Ψs and foliar [ABA]. Stomatal closure occurred in the PRD50 and rain-fed olive trees, with a relatively small reduction in gs of 19–29%, which corresponded to lower Ψs and higher concentrations of the osmotic regulators ABA, proline and soluble sugars (2] (Partial root-zone drying has been utilised to improve the water productivity of numerous crops. The successful application of PRD to olives would permit the optimisation of yield relative to water-use in a crop grown in drought prone areas. However, while the results of laboratory based split-root studies of olive trees have been promising; the efficacy of PRD irrigation in the field has been equivocal. In this study, during productive ‘on-years’, yield was significantly reduced by 47% in the PRDiffusive  and biociffusive  constraie sugars . The lacgars (2]  and [ABAgars (2]  may indi