European Community Council Research Articles

Differences in mitochondrial efficiency and heart function after different modes of exercise training in hypertension

Abstract Physical exercise is often used for the cardioprotective purpose in hypertension. However, it's not clear whether the mitochondrial efficacy will depend on the intensity and mode of physical exercise. The aim of the work is to investigate the effect of different modes of exercise training on mitochondria function and cardyohemodynamic in hypertension. All experimental procedures were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609 /EEC). Six-month-old spontaneously hypertensive rats (SHR) and normotensive control–Wistar rats were randomly assigned to 2 different training modes: moderate (20 min) and enhanced (45 min). Swimming training was done five times per week for 5–6 weeks. The functional cardiohemodynamic indicators were registered via microcatheter and Millar Pressure-Volume System. Mitochondrial oxygen consumption registered by Oxygraph. Moderate exercise training mode in SHR had a positive effect on the respiratory chain of mitochondria, respiratory control increased by 25,3%, the mitochondrial membrane potential increased by 47,8%. Moderate exercise training mode protected mitochondrial permeability transition pore (MPTP) formation. The threshold of MPTP opening increased by 1.4 times and the sensitivity of MPTP to inductor Ca2+ decreased. It was shown that end-systolic and end-diastolic pressure of SHR was decreased by 23,8% and 32,7% respectively. Moderate exercise improves end-diastolic heart function in SHR by increasing dP/dtmin and reducing the end-diastolic myocardial stiffness in 8,3 times. Stroke volume increased in 3,3 times, stroke work decreased by 17.2%, which may indicate the heart work efficiency increased after moderate exercise training. The Frank-Starling mechanism efficiency after moderate exercise was increased by 3,7 times. After enhanced exercise mode we didn't observe a protected effect on heart mitochondria and heart function in SHR. The mitochondrial membrane potential didn't increase, mitochondrial respiration in states V2, V3, V4 were significantly decreased. In conclusion, we showed that a moderate mode of exercise in SHR rats increased mitochondrial efficiency and improved heart function. The enhanced mode of exercise was excessive for SHR which was confirmed by suppressing the mitochondrial respiration and end-diastolic function of the heart. Funding Acknowledgement Type of funding sources: None.

Teriflunomide provides protective properties after oxygen-glucose-deprivation in hippocampal and cerebellar slice cultures.

One of the major challenges in emergency medicine is out-of-hospital cardiac arrest (OHCA). Every year, about 53-62/100 000 people worldwide suffer an out-of-hospital cardiac arrest with serious consequences, whereas persistent brain injury is a major cause of morbidity and mortality of those surviving a cardiac arrest. Today, only few and insufficient strategies are known to limit neurological damage of ischemia and reperfusion injury. The aim of the present study was to investigate whether teriflunomide, an approved drug for treatment of relapsing-remitting-multiple-sclerosis, exerts a protective effect on brain cells in an in vitro model of ischemia. Therefore, organotypic slice cultures from rat hippocampus and cerebellum were exposed to oxygen-glucose-deprivation and subsequently treated with teriflunomide. The administration of teriflunomide in the reperfusion time on both hippocampal and cerebellar slice cultures significantly decreased the amount of detectable propidium iodide signal compared with an untreated culture, indicating that more cells survive after oxygen-glucose-deprivation. However, hippocampal slice cultures showed a higher vulnerability to ischemic conditions and a more sensitive response to teriflunomide compared with cerebellar slice cultures. Our study suggests that teriflunomide, applied as a post-treatment after an oxygen-glucose-deprivation, has a protective effect on hippocampal and cerebellar cells in organotypic slice cultures of rats. All procedures were conducted under established standards of the German federal state of North Rhine Westphalia, in accordance with the European Communities Council Directive 2010/63/EU on the protection of animals used for scientific purposes.

Moulds on cementitious building materials-problems, prevention and future perspectives.

Materials rich in organic and inorganic compounds, such as building materials or paints, represent an excellent substrate for the development of moulds. Several conditions affect mould's growth on cementitious materials, such as nutrient and water availability, temperature, pH and moisture. Microorganisms, and especially moulds, attack these surfaces and contribute to their erosion, thereby reducing the life of the structure itself and negatively affecting human health through inhalation, ingestion and dermal contact with spores. Interventions are based on The European Communities Council Directive 89/106/EEC, that obliges the use of materials, products and building elements that are resistant to fungi and other forms of degradation, and that do not constitute a health risk for users and the environment. This mini-review summarises the current state of problems related to mould growth on cementitious building materials, emphasising new innovative approaches for limiting or contrasting their growth. In particular, the use of nanoparticles and the related nanomaterials as well as the potential use of new "biocides" from natural sources is discussed.

Down-Regulation of Cell Membrane Localized NTCP Expression in Proliferating Hepatocytes Prevents Hepatitis B Virus Infection

Background: Hepatocyte proliferation could result in the loss of covalently closed circular DNA (cccDNA) and the emergence of cccDNA-cleared nascent hepatocytes, which appear refractory to hepatitis B virus (HBV) reinfection with unknown mechanism(s). This study aims to uncover the underlying molecular mechanism(s) and its related clinical significance on HBV reinfection resistance of proliferating hepatocytes. Methods: The transcriptional inhibition of Sodium taurocholate cotransporting polypeptide (NTCP) expression in proliferating hepatocytes and impact of NTCP protein levels on HBV infection were assayed both in vivo and in vitro. The clinical significance of NTCP down-regulation was explored in a cohort of 68 HBeAg-positive chronic hepatitis B (CHB) patients. Findings: Arresting HepG2-NTCP-tet cells in G0/G1 phase obviously up-regulated NTCP cell membrane expression and thus facilitated HBV infection in vitro. In liver-humanized mice, HBV reinfection occurred only after most hepatocytes had stopped proliferating and cell membrane expression of NTCP returned to normal. In patients, lower NTCP protein expression was correlated well with higher levels of hepatocyte proliferation and less HBsAg expression in HBV-related focal nodular hyperplasia (FNH) tissues. Clinically, significantly lower NTCP protein expression was correlated with more active hepatocyte proliferation in CHB patients with severe active necroinflammation and better antiviral treatment outcome. Mechanistically, p53, cyclin D1, S-phase kinase associated protein 2 and interleukin-6 could transcriptionally down-regulate NTCP expression. Interpretation: Within the milieu of hepatocyte proliferation, lower NTCP expression level renders nascent hepatocytes resistant to HBV reinfection. This may accelerate virus clearance during immune-mediated cell death and compensatory proliferation of survival hepatocytes. Funding Statement: This study was supported by grants from National Science and Technology Major Project of the Ministry of Science and Technology of China (2017ZX10202203, 2017ZX10302201, 2017ZX10202202), the National Natural Science Foundation of China (81672013) and the German Research Foundation (DFG, Collaborative Research Centre SFB-841: A5). Among them, the German Research Foundation (DFG, Collaborative Research Centre SFB-841: A5) supported the studies involved liver-humanized mice. The National Science and Technology Major Project of the Ministry of Science and Technology of China (2017ZX10202203, 2017ZX10302201, 2017ZX10202202), and the National Natural Science Foundation of China (81672013) supported the evolving of the other experiments. Declaration of Interests: All authors have no conflict of interest to disclose. Ethics Approval Statement: The animal experiments involving humanized mice were approved by the City of Hamburg, Germany (G118/16), conducted in accordance with the European Communities Council Directive (86/EEC). The other animals involved in the study were approved by the Bioethics Committees of Peking University, the reference number is LA2018181. The clinical data involved in the study was approved by the Bioethics Committees of Peking University at February 27th, 2018.

The Origin of Microglia Determines the Content of Exosomes and Biological Function

Background: It is interesting to know whether microglia cells migrating in different central brain system components, i.e. cortex or in the spinal cord, are subjected to the same maturation processes and share similar functional phenotypes which could account with peculiar pathologies. Methods: We used a combination of pan proteomic platform associated with systemic biology analyses, in order to investigate that microglial cells derived from cortex and spinal cord expressed different phenotypes upon the physiological or pathological conditions Findings: This difference was found in cells and in their secreted exosomes. Cortical microglia showed neurogenesis/tumorigenesis characteristics, the spinal cord microglia revealed mostly involvement in the modulation of the inflammatory response to injury. We confirmed these results by performing functional testing including neurite outgrowth assays, and 3D spheroid cultures, which was used for glioma proliferation analysis. Results indicated that microglia located at different CNS areas reveal not only diverse content but also differential biological functions. Thus, the origin of microglia affected the physio-pathological function. These results also indicate differences in which the glioma is more prevalent in the brain than in spinal cord. Conclusion: Taken together, we demonstrated that microglia cells produce exosomes which can be used as therapeutic nanoparticles in translational medicine either in cancer treatment or in regenerative medicine. In fact, exosomes issued from spinal cord microglia can be addressed for glioma treatment and the ones from cortex microglia for spinal cord or brain traumatic injuries. Microglia exosomes are clearly a new source of therapeutic agents. Funding Statement: This research was supported by a collaboration between the Fundamental and Applied Biology Mass Spectrometry Laboratory (MS) and grants from Ministere de L’Education Nationale, L’Enseignement Superieur et de la Recherche, INSERM, SIRIC ONCOLille Grant INCD) a-DGOS-Inserm 6041aa (IF) and Universite de Lille (AM), APVV 15-0613 (DC), Stefanik SK-FR-2015-0018 (DC, MS), ERANET Axon Repair (DC). Declaration of Interests: The authors declare no competing financial interests in this work. Ethics Approval Statement: The study was performed with the approval and according to the guidelines of the Institutional Animal Care and Use Committee of the Slovak Academy of Sciences and with the European Communities Council Directive (2010/63/EU) regarding the use of animals in Research, Slovak Law for Animal Protection No. 377/2012 and 436/2012.

Effects of treadmill training and EVOO enriched diet on cartilage degeneration in rats with ACLT.

The beneficial effects of Extra Virgin Olive Oil (EVOO), the main fat source in Mediterranean diet, are widely studied thanks to its anti-inflammatory and antioxidant properties. Lubricin is a chondroprotective glycoprotein, with lubricant properties. A joint injury causes an increased cytokine expression, associated with decreased lubricin synthesis and predisposition to cartilage degeneration. The aim of this study was to evaluate the beneficial role of EVOO-enriched diet and physical activity, on osteoarthritic cartilage of rats. These effects were assessed through lubricin and interleukin-6 expression in rat joint tissues. Osteoarthritis was induced mechanically by anterior cruciate ligament transection (ACLT). Forty-eight 3-month-old male Wistar rats were used for this study. Experiments and surgical procedures for ACLT were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (I.A.C.U.C.) of the University of Catania, European Community Council Directive and the Italian Animal Protection Law (Protocol n. 2112015-PR, Italian Ministry of Health). The 48 animals were divided into 6 groups: 1 – control rats (no ACLT, common die); 2 – rats with ACLT and common diet (experimental control); 3 – rats with ACLT, common diet and treadmill training; 4 – rats with ACLT, Sicilian EVOO and treadmill training; 5 – rats with ACLT, Tunisian EVOO and treadmill training; 6 – rats with ACLT, Tunisian EVOO and leaves extract and treadmill training. To this purpose, we performed histomorphometric, histological, immunocytochemical, immunohistochemical and biochemical analysis for lubricin and interleukin-6 evaluations in articular cartilage and synovial fluid of rats. Furthermore, skeletal muscle (anterior tibial) and liver tissue were used to perform histological analyses to exclude respectively hepatic steatosis and sarcopenia. The results showed the beneficial effect of physical activity and EVOO supplementation on the rat articular cartilage. ACLT determined an increase in interleukin-6 expression and a significant decrease in the lubricin expression, while physical activity and EVOO supplemented diet, determined that the values returned to a normal level when compared to the control group. Moreover, EVOO does not cause hepatic steatosis and muscle fibers of all groups did not show damaged histological structure. Our findings suggest that mechanical stimulation is able to increase the release of lubricin in articular cartilage, while the anti-inflammatory properties of EVOO reduces the expression of interleukin-6. In conclusion, the results showed a beneficial effect of the conjunction of EVOO-based diet, corresponding to the Mediterranean diet, and physical activity on the preservation of articular cartilage tissue. Support or Funding Information This study was supported by the University Research Project Grant (Triennial Research Plan 2016–2018), University of Catania, Italy. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Experimental Evaluation of Biocompatibility in Case of Acrylic Materials by Bio-Testing on Lab Animals

A bio material is a inert and non-viable material with a natural or synthetic origin that comes in to contact with tissues, blood or biological fluids, being used in prosthetic, therapeutic and storage applications, without affecting in any way living organism or their components.Biological systems may produce the deterioration of materials, process known as bio-degradation. In case of oral environment this also includes the dissolution in saliva, chemical and physical degradation, abrasion and erosion caused by the diet components, mastication process and bacteria actions. Most of the materials used in dental therapy may be toxic or may produce allergies until they bind.We evaluated during our experiment the biological consequences (bio-compatibility) that subcutaneous implanting of small acrylic dental fragments have upon tissues in case of white lab mice, keeping an eye on the local tissues reactions. The evaluation of dental materials is made by testing the bio-compatibility. Biologically tests are performed in vivo and in vitro strictly in the lab and on animals.For our experiment we used 20 white mice Wistar line, 8-9 weeks old males, 40 grams, divided in 3 groups; all of them received an acrylic implant. The experiment was centered on the observation of some physiological or pathological effects, local or systemic, that implants may have produced effects that show a toxic answer or the rejection of the material.All experimens was performed in compliance with (European Communities Council Directive 1986 (86/609/EEC) and Ordinance No. 37 of Romanian Government from 2nd February 2002.Materials bio-compatibility was highlighted by anatomic-pathological exams during which we noticed all the modifications at dermis level during the entire period. We observed the phenomenon that occurred around the implants at different time periods, than we collected samples.The data obtained at the end of our experiment allow us to sustain that acrylic materials used in dental implants have all the properties required to be considered as bio-compatible.

Feasibility of In-Situ Aeration of Old Dumping Ground for Land Reclamation

Dumping grounds are characterized by the absence of engineering controls such as base liners and cover layer. Consequently, these dumping grounds present risks for surrounding resources such as soil, groundwater and air. The concern for groundwater contamination by leachate from tropical dumping grounds is heightened due to the greater amounts of rainfall and subsequent infiltration and percolation through the waste mass. The emergent demand for old dumping grounds reclamation drives the need to employ remediation technologies. Generally, in-situ aeration is a remediation method that promotes aerobic conditions in the later stage of dumping ground. It accelerates carbon transfer, reduces remaining organic load, and generally shortens the post closure period. However, high rainfall in tropical areas straitens this technique. For example, pollutants could be easily flushed out and more energy should be required to overcome hydrostatic pressure. Although heavy rainfall could supply sufficient water to the substrate and accelerate degradation of organic matter, it may inhibit aerobic activities due to limited air transfer. The waste characterization from Lorong Halus Dumping Ground (closed dumping ground in Singapore) showed that the waste materials were stabilized after 22 years closure. According to the Waste Acceptance Criteria set by European Communities Council, the waste materials could be classified as inert wastes. One interesting finding was that leachate layer detected was about of 5 - 8 meter depth, which entirely soaked the waste materials. Hence, the reclamation design and operation should be carefully adjusted according to these characters. Lorong Halus Dumping Ground case study can provide a guideline for other tropical closed landfills or dumping grounds.

Are we sufficiently aware of safety in rehabilitation?

International Journal of Therapy and RehabilitationVol. 20, No. 9 OpinionAre we sufficiently aware of safety in rehabilitation?Massimiliano PolastriMassimiliano PolastriSearch for more papers by this authorMassimiliano PolastriPublished Online:29 Sep 2013https://doi.org/10.12968/ijtr.2013.20.9.426AboutSectionsView articleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareShare onFacebookTwitterLinked InEmail View article References BBC News Health (2013) Physiotherapists given prescribing powers. www.bbc.co.uk/news/health-23752418 (accessed 23 August 2013) Google ScholarConnor M, Ponte PR, Conway J (2002) Multidisciplinary approaches to reducing error and risk in a patient care setting. Crit Care Nurs Clin North Am 14(4): 359–67 Crossref, Google ScholarCouncil of Europe (1996) European Social Charter (revised). http://tinyurl.com/mywy3ol (accessed 3 April 2013) Google ScholarDayton E, Henriksen K (2007) Communication failure: basic components, contributing factors, and the call for structure. Jt Comm J Qual Patient Saf 33(1): 34–47 Google ScholarFerroli P, Caldiroli D, Acerbi F, Scholtze M, Piro A, Schiariti M et al. (2012) Application of an aviation model of incident reporting and investigation to the neurosurgical scenario: method and preliminary data. Neurosurg Focus 33(5): E7 Crossref, Google ScholarFrancis R (2013) The Mid Staffordshire NHS Foundation Trust Public Inquiry. http://tinyurl.com/ksmqu37 (accessed 26 April 2013) Google ScholarKeogh B (2013) Review into the quality of care and treatment provided by 14 hospital trusts in England: overview report. http://tinyurl.com/ph9e267 (accessed 20 July 2013) Google ScholarOfficial Journal of the European Communities (1993) Council Directive 93/104/EC of 23 November 1993 concerning certain aspects of the organization of the working time. http://tinyurl.com/mxxktwy (accessed 3 April 2013) Google ScholarOfficial Journal of the European Communities (2000) Directive 2000/34/EC of the European Parliament and of the Council of 22 June 2000. http://tinyurl.com/m8e8s6z (accessed 3 April 2013) Google ScholarPietrobon R, Lima R, Shah A et al. (2007) Duke Surgery Patient Safety: an open-source application for anonymous reporting of adverse and near-miss surgical events. Ann Surg Innov Res 1: 5 Crossref, Google ScholarPolastri M, Di Eusanio M (2013) Aortic aneurysms repair: perspectives from a subintensive care setting. Int J Ther Rehabil 20(2): 70–1 Link, Google ScholarSingh R, Saleemi A, Walsh K, Popert R, O’Brien T (2003) Near misses in bladder cancer – an airline safety approach to urology. Ann R Coll Surg Engl 85(6): 378–81 Crossref, Google ScholarSperoni KG, Fisher J, Dennis M, Daniel M (2013) What causes near-misses and how are they mitigated? Nursing 43(4): 19–24 Crossref, Google ScholarThompson L (2003) The National Patient Safety Agency allied health professionals project. Int J Ther Rehabil 10(11): 490 Link, Google ScholarWeingart SN, Pagovich O, Sands DZ et al. (2005) What can hospitalized patients tell us about adverse events? Learning from patient-reported incidents. J Gen Intern Med 20(9): 830–6 Crossref, Google Scholar FiguresReferencesRelatedDetails 1 September 2013Volume 20Issue 9ISSN (online): 1759-779X Metrics History Published online 29 September 2013 Published in print 1 September 2013 Information© MA Healthcare LimitedPDF download

Mineral content of different bottled water available in Bangladesh: Assessment of their compliance with current regulations

Nine bottled water brands collected from retail shops and food shops beside the University of Dhaka were analyzed for their major and trace element constituents to ascertain their suitability for human consumption. The results obtained were compared with parametric values (PVs) set by European Community Council Directive 98/83/EC and the guideline values (GVs) recommended by WHO. It was found that the majority of the brands tested were low in mineral contents. Moreover, lower values of hardness, TDS and conductivity than the prescribed limits of WHO showed that water was deficient in essential minerals. Minerals like magnesium, potassium, calcium were present in some cases in such a low concentration that water seemed to be as good as distilled water. In case of heavy metals, only lead and cadmium were found to be below the detection limit (0.001 mg/L) which indicates bottled water brands are better for drinking. DOI: http://dx.doi.org/10.3329/jasbs.v38i1.15316 J. Asiat. Soc. Bangladesh, Sci. 38(1): 7-15, June 2012

σ1Receptors Are Involved in the Visceral Pain Induced by Intracolonic Administration of Capsaicin in Mice

Visceral pain is an important and prevalent clinical condition whose treatment is challenging. Sigma-1 (σ1) receptors modulate somatic pain, but their involvement in pure visceral pain is unexplored. The authors evaluated the role of σ1 receptors in intracolonic capsaicin-induced visceral pain (pain-related behaviors and referred mechanical hyperalgesia to the abdominal wall) using wild-type (WT) (n = 12 per group) and σ1 receptor knockout (σ1-KO) (n = 10 per group) mice, selective σ1 receptor antagonists (BD-1063, S1RA, and NE-100), and control drugs (morphine and ketoprofen). The intracolonic administration of capsaicin (0.01-1%) induced concentration-dependent visceral pain-related behaviors and referred hyperalgesia in both WT and σ1-KO mice. However, the maximum number of pain-related behaviors induced by 1% capsaicin in σ1-KO mice (mean ± SEM, 22 ± 2.9) was 48% of that observed in WT animals (46 ± 4.2). Subcutaneous administration of the σ1 receptor antagonists BD-1063 (16-64 mg/kg), S1RA (32-128 mg/kg), and NE-100 (8-64 mg/kg) dose-dependently reduced the number of behavioral responses (by 53, 62, and 58%, respectively) and reversed the referred hyperalgesia to mechanical control threshold (0.53 ± 0.05 g) in WT mice. In contrast, these drugs produced no change in σ1-KO mice. Thus, the effects of these drugs are specifically mediated by σ1 receptors. Morphine produced an inhibition of capsaicin-induced visceral pain in WT and σ1-KO mice, whereas ketoprofen had no effect in either mouse type. These results suggest that σ1 receptors play a role in the mechanisms underlying capsaicin-induced visceral pain and raise novel perspectives for their potential therapeutic value.

Chloride currents in cones modify feedback from horizontal cells to cones in goldfish retina

In neuronal systems, excitation and inhibition must be well balanced to ensure reliable information transfer. The cone/horizontal cell (HC) interaction in the retina is an example of this. Because natural scenes encompass an enormous intensity range both in temporal and spatial domains, the balance between excitation and inhibition in the outer retina needs to be adaptable. How this is achieved is unknown. Using electrophysiological techniques in the isolated retina of the goldfish, it was found that opening Ca(2+)-dependent Cl(-) channels in recorded cones reduced the size of feedback responses measured in both cones and HCs. Furthermore, we show that cones express Cl(-) channels that are gated by GABA released from HCs. Similar to activation of I(Cl(Ca)), opening of these GABA-gated Cl(-) channels reduced the size of light-induced feedback responses both in cones and HCs. Conversely, application of picrotoxin, a blocker of GABA(A) and GABA(C) receptors, had the opposite effect. In addition, reducing GABA release from HCs by blocking GABA transporters also led to an increase in the size of feedback. Because the independent manipulation of Ca(2+)-dependent Cl(-) currents in individual cones yielded results comparable to bath-applied GABA, it was concluded that activation of either Cl(-) current by itself is sufficient to reduce the size of HC feedback. However, additional effects of GABA on outer retinal processing cannot be excluded. These results can be accounted for by an ephaptic feedback model in which a cone Cl(-) current shunts the current flow in the synaptic cleft. The Ca(2+)-dependent Cl(-) current might be essential to set the initial balance between the feedforward and the feedback signals active in the cone HC synapse. It prevents that strong feedback from HCs to cones flood the cone with Ca(2)(+). Modulation of the feedback strength by GABA might play a role during light/dark adaptation, adjusting the amount of negative feedback to the signal to noise ratio of the cone output.

Prevention of Age-Related Macular Degeneration–Like Retinopathy by Rapamycin in Rats

Age-related macular degeneration, a neurodegenerative and vascular retinal disease, is the most common cause of blindness in the Western countries. Evidence accumulates that target of rapamycin is involved in aging and age-related diseases, including neurodegeneration. The target of rapamycin inhibitor, rapamycin, suppresses the senescent cell phenotype and extends life span in diverse species, including mice. Rapamycin decreases senescence-associated phenotypes in retinal pigment epithelial cells in culture. Herein, we investigated the effect of rapamycin on spontaneous retinopathy in senescence-accelerated OXYS rats, an animal model of age-related macular degeneration. Rats were treated with either 0.1 or 0.5 mg/kg rapamycin, which was given orally as a food mixture. In a dose-dependent manner, rapamycin decreased the incidence and severity of retinopathy. Rapamycin improved some (but not all) histological abnormalities associated with retinopathy. Thus, in retinal pigment epithelial cell layers, rapamycin decreased nuclei heterogeneity and normalized intervals between nuclei. In photoreceptor cells, associated neurons, and radial glial cells, rapamycin prevented nuclear and cellular pyknosis. More important, rapamycin prevented destruction of ganglionar neurons in the retina. Rapamycin did not exert any adverse effects on the retina in control disease-free Wistar rats. Taken together, our data suggest the therapeutic potential of rapamycin for treatment and prevention of retinopathy.

Integrity of Helix 2-Helix 3 Domain of the PrP Protein Is Not Mandatory for Prion Replication

The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrP(Sc). We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrP(Sc) and elucidate the conformational changes underlying prions generation.

Susceptibility to infection and pathogenicity of White Spot Disease (WSD) in non-model crustacean host taxa from temperate regions

Despite almost two decades since its discovery, White Spot Disease (WSD) caused by White Spot Syndrome Virus (WSSV) is still considered the most significant known pathogen impacting the sustainability and growth of the global penaeid shrimp farming industry. Although most commonly associated with penaeid shrimp farmed in tropical regions, the virus is also able to infect, cause disease and kill a wide range of other decapod crustacean hosts from temperate regions, including lobsters, crabs, crayfish and shrimp. For this reason, WSSV has recently been listed in European Community Council Directive 2006/88. Using principles laid down by the European Food Safety Authority (EFSA) we applied an array of diagnostic approaches to provide a definitive statement on the susceptibility to White Spot Syndrome Virus (WSSV) infection in seven ecologically or economically important crustacean species from Europe. We chose four marine species: Cancer pagurus, Homarus gammarus, Nephrops norvegicus and Carcinus maenas; one estuarine species, Eriocheir sinensis and two freshwater species, Austropotamobius pallipes and Pacifastacus leniusculus. Exposure trials based upon natural (feeding) and artificial (intra-muscular injection) routes of exposure to WSSV revealed universal susceptibility to WSSV infection in these hosts. However, the relative degree of susceptibility (measured by progression of infection to disease, and mortality) varied significantly between host species. In some instances (Type 1 hosts), pathogenesis mimicked that observed in penaeid shrimp hosts whereas in other examples (Types 2 and 3 hosts), infection did not readily progress to disease, even though hosts were considered as infected and susceptible according to accepted principles. Results arising from challenge studies are discussed in relation to the potential risk posed to non-target hosts by the inadvertent introduction of WSSV to European waters via trade. Furthermore, we highlight the potential for susceptible but relatively resistant hosts to serve as models to investigate natural mitigation strategies against WSSV in these hosts. We speculate that these non-model hosts may offer a unique insight into viral handling in crustaceans.

Growth Defects and Impaired Cognitive–Behavioral Abilities in Mice with Knockout for Eif4h, a Gene Located in the Mouse Homolog of the Williams-Beuren Syndrome Critical Region

Protein synthesis is a tightly regulated, energy-consuming process. The control of mRNA translation into protein is fundamentally important for the fine-tuning of gene expression; additionally, precise translational control plays a critical role in many cellular processes, including development, cellular growth, proliferation, differentiation, synaptic plasticity, memory, and learning. Eukaryotic translation initiation factor 4h (Eif4h) encodes a protein involved in the process of protein synthesis, at the level of initiation phase. Its human homolog, WBSCR1, maps on 7q11.23, inside the 1.6 Mb region that is commonly deleted in patients affected by the Williams-Beuren syndrome, which is a complex neurodevelopmental disorder characterized by cardiovascular defects, cerebral dysplasias and a peculiar cognitive-behavioral profile. In this study, we generated knockout mice deficient in Eif4h. These mice displayed growth retardation with a significant reduction of body weight that began from the first week of postnatal development. Neuroanatomical profiling results generated by magnetic resonance imaging analysis revealed a smaller brain volume in null mice compared with controls as well as altered brain morphology, where anterior and posterior brain regions were differentially affected. The inactivation of Eif4h also led to a reduction in both the number and complexity of neurons. Behavioral studies revealed severe impairments of fear-related associative learning and memory formation. These alterations suggest that Eif4h might contribute to certain deficits associated with Williams-Beuren syndrome.

MiRNA Expression Profile after Status Epilepticus and Hippocampal Neuroprotection by Targeting miR-132

When an otherwise harmful insult to the brain is preceded by a brief, noninjurious stimulus, the brain becomes tolerant, and the resulting damage is reduced. Epileptic tolerance develops when brief seizures precede an episode of prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. We investigated how prior seizure preconditioning affects the miRNA response to status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status epilepticus in animals that had previously received either seizure preconditioning (tolerance) or no preconditioning (injury), and mature miRNA levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs was increased, relative to control, after status epilepticus alone, and expression of 12 miRNAs was decreased. Increased miR-132 levels were matched with increased binding to Argonaute-2, a constituent of the RNA-induced silencing complex. In tolerant animals, expression responses of >40% of the injury-group-detected miRNAs differed, being either unchanged relative to control or down-regulated, and this included miR-132. In vivo microinjection of locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132 depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death. Thus, our data strongly suggest that miRNAs are important regulators of seizure-induced neuronal death.

Gephyrin Regulates GABAergic and Glutamatergic Synaptic Transmission in Hippocampal Cell Cultures

Gephyrin is a scaffold protein essential for stabilizing glycine and GABA(A) receptors at inhibitory synapses. Here, recombinant intrabodies against gephyrin (scFv-gephyrin) were used to assess whether this protein exerts a transynaptic action on GABA and glutamate release. Pair recordings from interconnected hippocampal cells in culture revealed a reduced probability of GABA release in scFv-gephyrin-transfected neurons compared with controls. This effect was associated with a significant decrease in VGAT, the vesicular GABA transporter, and in neuroligin 2 (NLG2), a protein that, interacting with neurexins, ensures the cross-talk between the post- and presynaptic sites. Interestingly, hampering gephyrin function also produced a significant reduction in VGLUT, the vesicular glutamate transporter, an effect accompanied by a significant decrease in frequency of miniature excitatory postsynaptic currents. Overexpressing NLG2 in gephyrin-deprived neurons rescued GABAergic but not glutamatergic innervation, suggesting that the observed changes in the latter were not due to a homeostatic compensatory mechanism. Pulldown experiments demonstrated that gephyrin interacts not only with NLG2 but also with NLG1, the isoform enriched at excitatory synapses. These results suggest a key role of gephyrin in regulating transynaptic signaling at both inhibitory and excitatory synapses.

Low‐voltage activated Kv1.1 subunits are crucial for the processing of sound source location in the lateral superior olive in mice

Voltage-gated potassium (Kv) channels containing Kv1.1 subunits are strongly expressed in neurons that fire temporally precise action potentials (APs). In the auditory system, AP timing is used to localize sound sources by integrating interaural differences in time (ITD) and intensity (IID) using sound arriving at both cochleae. In mammals, the first nucleus to encode IIDs is the lateral superior olive (LSO), which integrates excitation from the ipsilateral ventral cochlear nucleus and contralateral inhibition mediated via the medial nucleus of the trapezoid body. Previously we reported that neurons in this pathway show reduced firing rates, longer latencies and increased jitter in Kv1.1 knockout (Kcna1−/−) mice. Here, we investigate whether these differences have direct impact on IID processing by LSO neurons. Single-unit recordings were made from LSO neurons of wild-type (Kcna1+/+) and from Kcna1−/− mice. IID functions were measured to evaluate genotype-specific changes in integrating excitatory and inhibitory inputs. In Kcna1+/+ mice, IID sensitivity ranged from +27 dB (excitatory ear more intense) to −20 dB (inhibitory ear more intense), thus covering the physiologically relevant range of IIDs. However, the distribution of IID functions in Kcna1−/− mice was skewed towards positive IIDs, favouring ipsilateral sound positions. Our computational model revealed that the reduced performance of IID encoding in the LSO of Kcna1−/− mice is mainly caused by a decrease in temporal fidelity along the inhibitory pathway. These results imply a fundamental role for Kv1.1 in temporal integration of excitation and inhibition during sound source localization.

Thyroid Hormone Receptor β (TRβ) and Liver X Receptor (LXR) Regulate Carbohydrate-response Element-binding Protein (ChREBP) Expression in a Tissue-selective Manner

Thyroid hormone (TR) and liver X (LXR) receptors are transcription factors involved in lipogenesis. Both receptors recognize the same consensus DNA-response element in vitro. It was previously shown that their signaling pathways interact in the control of cholesterol elimination in the liver. In the present study, carbohydrate-response element-binding protein (ChREBP), a major transcription factor controlling the activation of glucose-induced lipogenesis in liver, is characterized as a direct target of thyroid hormones (TH) in liver and white adipose tissue (WAT), the two main lipogenic tissues in mice. Using genetic and molecular approaches, ChREBP is shown to be specifically regulated by TRbeta but not by TRalpha in vivo, even in WAT where both TR isoforms are expressed. However, this isotype specificity is not found in vitro. This TRbeta specific regulation correlates with the loss of TH-induced lipogenesis in TRbeta(-/-) mice. Fasting/refeeding experiments show that TRbeta is not required for the activation of ChREBP expression particularly marked in WAT following refeeding. However, TH can stimulate ChREBP expression in WAT even under fasting conditions, suggesting completely independent pathways. Because ChREBP has been described as an LXR target, the interaction of LXR and TRbeta in ChREBP regulation was assayed both in vitro and in vivo. Each receptor recognizes a different response element on the ChREBP promoter, located only 8 bp apart. There is a cross-talk between LXR and TRbeta signaling on the ChREBP promoter in liver but not in WAT where LXR does not regulate ChREBP expression. The molecular basis for this cross-talk has been determined in in vitro systems.