Presence of cytochrome P-450-dependent monooxygenase in intimal cells of the hog aorta

Cytochrome P-450-dependent mixed function oxidase activity is present in vascular tissue; however, as far as we could determine, the distribution of monooxygenase activity across the blood vessel wall has not previously been assessed. The aryl-hydrocarbon hydroxylase activity was examined by metabolism of benzo[a]pyrene in microsomes prepared from intimal and smooth muscle cell scrapings of the hog thoracic aorta. Microsomes of intimal cells comprising 95% endothelial cells showed an approximately 2.5-fold increase in aryl-hydrocarbon hydroxylase activity compared with that in microsomes prepared from medial smooth muscle cells. Michaelis-Mentin kinetics for the intimal enzyme yielded an apparent Km value of 11.11 microM and an apparent Vmax of 3-OH benzo[a]pyrene of 40 pmol/mg protein/10 min. Aryl-hydrocarbon hydroxylase activity was dependent on nicotinamide adenine dinucleotide phosphate and was inhibited by 7,8 benzoflavone, SKF 525A, and carbon monoxide. The localization of cytochrome P-450-dependent mixed function oxidase primarily to the intimal surface of the aorta may indicate a role for this enzyme system in vasoregulation and the pathogenesis of atherosclerosis

AP2γ: a new player on adult hippocampal neurogenesis regulation

Since the recognition that the mammalian brain retains the ability to generate newborn neurons with functional relevance throughout life, the matrix of molecular regulators that govern adult neurogenesis has been the focus of much interest. In a recent study published in Molecular Psychiatry, we demonstrate Activating Protein 2γ (AP2γ), a transcription factor previously implicated in cell fate determination in the developing cortex, as a novel player in the regulation of glutamatergic neurogenesis in the adult hippocampus. Using distinct experimental approaches, we showed that AP2γ is specifically present in a subpopulation of transient amplifying progenitors, where it acts as a crucial promoter of proliferation and differentiation of adult-born glutamatergic granule neurons. Strikingly, deficiency of AP2γ in the adult brain compromises the generation of new glutamatergic neurons, with impact on the function of cortico-limbic circuits. Here, we share our view on how AP2γ integrates the transcriptional orchestration of glutamatergic neurogenesis in the adult hippocampus, and consequently, how it emerges as a novel molecular candidate to study the translation of environmental pressures into alterations of brain neuroplasticity in homeostatic, but also in neuropathological contexts.Bial Foundation (427/14); Northern Portugal Regional Operational Programme (NORTE 2020); European Regional Development Fund (FEDER) (projects NORTE-01-0145-FEDER-000013 e NORTE-01-0145-FEDER-000023); Competitiveness Factors Operational Programme (COMPETE)info:eu-repo/semantics/publishedVersio

The application of Bonelike® Poro as a synthetic bone substitute for the management of critical-sized bone defects - A comparative approach to the autograft technique - A preliminary study

The effective treatment of non-unions and critical-sized defects remains a challenge in the orthopedic field. From a tissue engineering perspective, this issue can be addressed through the application bioactive matrixes to support bone regeneration, such as Bonelike®, as opposed to the widespread autologous grafting technique. An improved formulation of Bonelike® Poro, was assessed as a synthetic bone substitute in an ovine model for critical-sized bone defects. Bone regeneration was assessed after 5 months of recovery through macro and microscopic analysis of the healing features of the defect sites. Both the application of natural bone graft or Bonelike® Poro resulted in bridging of the defects margins. Untreated defect remained as fibrous non-unions at the end of the study period. The characteristics of the newly formed bone and its integration with the host tissue were assessed through histomorphometric and histological analysis, which demonstrated Bonelike® Poro to result in improved healing of the defects. The group treated with synthetic biomaterial presented bone bridges of increased thickness and bone features that more closely resembled the native spongeous and cortical bone. The application of Bonelike® Poro enabled the regeneration of critical-sized lesions and performed comparably to the autograph technique, validating its octeoconductive and osteointegrative potential for clinical application as a therapeutic strategy in human and veterinary orthopedics.This research was supported by Projects PEst-OE/AGR/UI0211/2011 from FCT , and COMPETE 2020 , from ANI – Projetos ID&T Empresas em Copromoção , by the project “insitu.Biomas – Reinvent biomanufacturing systems by using an usability approach for in situ clinic temporary implants fabrication” with the reference POCI-01-0247-FEDER-017771 , by the project “Print-on-Organs – Engineering bioinks and processes for direct printing on organs” with the reference POCI-01-0247-FEDER-033877 , and by the project “Bone2Move - Development of ‘in vivo’ experimental techniques and modelling methodologies for the evaluation of 4D scaffolds for bone defect in sheep model: an integrative research approach” with the reference POCI-01-0145-FEDER-031146 . Mariana Vieira Branquinho ( SFRH/BD/146172/2019 ), Ana Catarina Sousa ( SFRH/BD/146689/2019 ), and Rui Damásio Alvites ( SFRH/BD/116118/2016 ), acknowledge FCT , for financial support. This research was supported by Projects PEst-OE/AGR/UI0211/2011 from FCT, and COMPETE 2020, from ANI ? Projetos ID&T Empresas em Copromo??o, by the project ?insitu.Biomas ? Reinvent biomanufacturing systems by using an usability approach for in situ clinic temporary implants fabrication? with the reference POCI-01-0247-FEDER-017771, by the project ?Print-on-Organs ? Engineering bioinks and processes for direct printing on organs? with the reference POCI-01-0247-FEDER-033877, and by the project ?Bone2Move - Development of ?in vivo? experimental techniques and modelling methodologies for the evaluation of 4D scaffolds for bone defect in sheep model: an integrative research approach? with the reference POCI-01-0145-FEDER-031146. Mariana Vieira Branquinho (SFRH/BD/146172/2019), Ana Catarina Sousa (SFRH/BD/146689/2019), and Rui Dam?sio Alvites (SFRH/BD/116118/2016), acknowledge FCT, for financial support

Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56

Bacterial AB toxins are secreted key virulence factors that are internalized by target cells through receptor-mediated endocytosis, translocating their enzymatic domain to the cytosol from endosomes (short-trip) or the endoplasmic reticulum (long-trip). To accomplish this, bacterial AB toxins evolved a multidomain structure organized into either a single polypeptide chain or non-covalently associated polypeptide chains. The prototypical short-trip single-chain toxin is characterized by a receptor-binding domain that confers cellular specificity and a translocation domain responsible for pore formation whereby the catalytic domain translocates to the cytosol in an endosomal acidification-dependent way. In this work, the determination of the three-dimensional structure of AIP56 shows that, instead of a two-domain organization suggested by previous studies, AIP56 has three-domains: a non-LEE encoded effector C (NleC)-like catalytic domain associated with a small middle domain that contains the linker-peptide, followed by the receptor-binding domain. In contrast to prototypical single-chain AB toxins, AIP56 does not comprise a typical structurally complex translocation domain; instead, the elements involved in translocation are scattered across its domains. Thus, the catalytic domain contains a helical hairpin that serves as a molecular switch for triggering the conformational changes necessary for membrane insertion only upon endosomal acidification, whereas the middle and receptor-binding domains are required for pore formation. © 2023, The Author(s).This work was supported by National funds through FCT under the project UIDB/04293/2020 and by FEDER funds through Programa Operacional Factores de Competitividade – COMPETE and by national funds through FCT – Fundação para a Ciência e a Tecnologia under the project PTDC/BIA-MIC/29910/2017 to N.M.S.S. A.d.V. was funded by Portuguese national funds through the FCT and, when eligible, by COMPETE 2020 FEDER funds, under the Scientific Employment Stimulus–Individual Call 2021.02251.CEECIND/CP1663/CT0016. We acknowledge access to the HTX crystallization facility (Proposal ID: BIOSTRUCTX_8167) and SOLEIL, ESRF and ALBA synchrotrons for provision of measurement time and thank their staff for help with data collection. The authors acknowledge the support of i3S Scientific Platforms (https://www.i3s.up.pt/scientific-platforms.php) Advanced Light Microscopy, member of the national infrastructure PPBI-Portuguese Platform of BioImaging (supported by POCI-01-0145-FEDER-022122), Animal Facility, Biochemical and Biophysical Technologies and X-ray Crystallography. A special thanks to Dr. Marc Graille and Dr. João Morais Cabral for constructive discussions in structural biology and Dr. Dimitri Panagiotis Papatheodorou for providing plasmid p327

Apoe variants in an iberian alzheimer cohort detected through an optimized sanger sequencing protocol

The primary genetic risk factor for late onset Alzheimer’s disease (LOAD) is the APOE4 allele of Apolipoprotein E (APOE) gene. The three most common variants of APOE are determined by single nucleotide polymorphisms (SNPs) rs429358 and rs7412. Our aim was to estimate allele and genotype frequencies of APOE variants in an Iberian cohort, thus helping to understand differences in APOE-related LOAD risk observed across populations. We analyzed saliva or buccal swab samples from 229 LOAD patients and 89 healthy elderly controls (=68 years old) from Northern Portugal and Castile and León region, Spain. The genotyping was performed by Sanger sequencing, optimized to overcome GC content drawbacks. Results obtained in our Iberian LOAD and control cohorts are in line with previous large meta-analyses on APOE frequencies in Caucasian populations; however, we found differences in allele frequencies between our Portuguese and Spanish subgroups of AD patients. Moreover, when comparing studies from Iberian and other Caucasian cohorts, differences in APOE2 and APOE4 frequencies and subsequent different APOE-related LOAD risks must be clarified. These results show the importance of studying genetic variation at the APOE gene in different populations (including analyses at a regional level) to increase our knowledge about its clinical significance.This work was supported by ‘European Commission’ and ‘European Regional Development Fund’ (FEDER) under the project “Análisis y correlación entre el genoma completo y la actividad cerebral para la ayuda en el diagnóstico de la enfermedad de Alzheimer” (Project 0378_AD_EEGWA_2_P), (Cooperation Programme INTERREG V-A Spain-Portugal POCTEP 2014– 2020) and the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020. Portuguese funds are supporting this work through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). IG, AML, SM, and NP are funded by FCT: CEECIND/02609/2017, IF/01262/2014, CEECIND/00684/2017, and through the Decreto-Lei n◦ 57/2016 de 29 de Agosto, respectively. Spanish funds are supporting this work through ‘Ministerio de Ciencia e Innovación–Agencia Estatal de Investigación’ and FEDER under project PGC2018-098214-A-I00 and by “CIBER en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN)” through “Instituto de Salud Carlos III” co-funded with FEDER funds

Friedreich ataxia patient tissues exhibit increased 5-hydroxymethylcytosine modification and decreased CTCF binding at the FXN locus

© 2013 Al-Mahdawi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,distribution, and reproduction in any medium, provided the original author and source are credited.This article has been made available through the Brunel Open Access Publishing Fund.Friedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene, which induces epigenetic changes and FXN gene silencing. Bisulfite sequencing studies have identified 5-methylcytosine (5 mC) DNA methylation as one of the epigenetic changes that may be involved in this process. However, analysis of samples by bisulfite sequencing is a time-consuming procedure. In addition, it has recently been shown that 5-hydroxymethylcytosine (5 hmC) is also present in mammalian DNA, and bisulfite sequencing cannot distinguish between 5 hmC and 5 mC.The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 242193/EFACTS (CS), the Wellcome Trust [089757] (SA) and Ataxia UK (RMP) to MAP

Dynamic clamp with StdpC software

Dynamic clamp is a powerful method that allows the introduction of artificial electrical components into target cells to simulate ionic conductances and synaptic inputs. This method is based on a fast cycle of measuring the membrane potential of a cell, calculating the current of a desired simulated component using an appropriate model and injecting this current into the cell. Here we present a dynamic clamp protocol using free, fully integrated, open-source software (StdpC, for spike timing-dependent plasticity clamp). Use of this protocol does not require specialist hardware, costly commercial software, experience in real-time operating systems or a strong programming background. The software enables the configuration and operation of a wide range of complex and fully automated dynamic clamp experiments through an intuitive and powerful interface with a minimal initial lead time of a few hours. After initial configuration, experimental results can be generated within minutes of establishing cell recording

Evidence for distinct coastal and offshore communities of bottlenose dolphins in the north east Atlantic.

Bottlenose dolphin stock structure in the northeast Atlantic remains poorly understood. However, fine scale photo-id data have shown that populations can comprise multiple overlapping social communities. These social communities form structural elements of bottlenose dolphin (Tursiops truncatus) [corrected] populations, reflecting specific ecological and behavioural adaptations to local habitats. We investigated the social structure of bottlenose dolphins in the waters of northwest Ireland and present evidence for distinct inshore and offshore social communities. Individuals of the inshore community had a coastal distribution restricted to waters within 3 km from shore. These animals exhibited a cohesive, fission-fusion social organisation, with repeated resightings within the research area, within a larger coastal home range. The offshore community comprised one or more distinct groups, found significantly further offshore (>4 km) than the inshore animals. In addition, dorsal fin scarring patterns differed significantly between inshore and offshore communities with individuals of the offshore community having more distinctly marked dorsal fins. Specifically, almost half of the individuals in the offshore community (48%) had characteristic stereotyped damage to the tip of the dorsal fin, rarely recorded in the inshore community (7%). We propose that this characteristic is likely due to interactions with pelagic fisheries. Social segregation and scarring differences found here indicate that the distinct communities are likely to be spatially and behaviourally segregated. Together with recent genetic evidence of distinct offshore and coastal population structures, this provides evidence for bottlenose dolphin inshore/offshore community differentiation in the northeast Atlantic. We recommend that social communities should be considered as fundamental units for the management and conservation of bottlenose dolphins and their habitat specialisations

Astrobiological Complexity with Probabilistic Cellular Automata

Search for extraterrestrial life and intelligence constitutes one of the major endeavors in science, but has yet been quantitatively modeled only rarely and in a cursory and superficial fashion. We argue that probabilistic cellular automata (PCA) represent the best quantitative framework for modeling astrobiological history of the Milky Way and its Galactic Habitable Zone. The relevant astrobiological parameters are to be modeled as the elements of the input probability matrix for the PCA kernel. With the underlying simplicity of the cellular automata constructs, this approach enables a quick analysis of large and ambiguous input parameters' space. We perform a simple clustering analysis of typical astrobiological histories and discuss the relevant boundary conditions of practical importance for planning and guiding actual empirical astrobiological and SETI projects. In addition to showing how the present framework is adaptable to more complex situations and updated observational databases from current and near-future space missions, we demonstrate how numerical results could offer a cautious rationale for continuation of practical SETI searches.Comment: 37 pages, 11 figures, 2 tables; added journal reference belo

MutLα heterodimers modify the molecular phenotype of Friedreich ataxia

This article has been made available through the Brunel Open Access Publishing Fund.Background: Friedreich ataxia (FRDA), the most common autosomal recessive ataxia disorder, is caused by a dynamic GAA repeat expansion mutation within intron 1 of FXN gene, resulting in down-regulation of frataxin expression. Studies of cell and mouse models have revealed a role for the mismatch repair (MMR) MutS-heterodimer complexes and the PMS2 component of the MutLα complex in the dynamics of intergenerational and somatic GAA repeat expansions: MSH2, MSH3 and MSH6 promote GAA repeat expansions, while PMS2 inhibits GAA repeat expansions. Methodology/Principal Findings: To determine the potential role of the other component of the MutLα complex, MLH1, in GAA repeat instability in FRDA, we have analyzed intergenerational and somatic GAA repeat expansions from FXN transgenic mice that have been crossed with Mlh1 deficient mice. We find that loss of Mlh1 activity reduces both intergenerational and somatic GAA repeat expansions. However, we also find that loss of either Mlh1 or Pms2 reduces FXN transcription, suggesting different mechanisms of action for Mlh1 and Pms2 on GAA repeat expansion dynamics and regulation of FXN transcription. Conclusions/Significance: Both MutLα components, PMS2 and MLH1, have now been shown to modify the molecular phenotype of FRDA. We propose that upregulation of MLH1 or PMS2 could be potential FRDA therapeutic approaches to increase FXN transcription. © 2014 Ezzatizadeh et al.This article has been made available through the Brunel Open Access Publishing Fund