Rescue of impaired fear extinction : role of dopaminergic signaling and histone acetylation

submitted by Verena Felizitas Maurer

DOCUMENTARY RESCUE OF POPULAR TRADITIONS OF THE CENTRAL AREA CANTONS HEREDIA PROVINCE

This paper presents the results of documentary rescue demonstrations performed in different counties present in the central area of the province of Heredia.This idea was enhanced thanks to the Organization of the United Nations Educational, Scientific and Cultural Organization (UNESCO) has provided the rescue and preservation of folk traditions, both tangible and intangible, throughout the world.Coupled with this, the Ministry of Education under the direction of Professor Manuel Antonio Bolaños, launched in the administration of La Espriella Pacheco (2002-2006) the development of an educational project aimed at rescuing the popular traditions of the province of Heredia, which under the title"Draft Heredianidad" became one of the main inspiring to be translated into a multimedia all information collected in the process of this research.The final graduation consisted of three major axes: the memory, the media containing all the information collected and a database in which (the) user (s) and to describe the bibliographic production on the subject of popular traditions Heredia has occurred, would help to physically locate these publications.To achieve the objectives we proceeded to conduct visits to public libraries and municipalities in the province as well as interviews with different artistic personalities of the cantons heredianos, thereby gained access to various legends, anecdotes, stories, songs, music and history of the province of Heredia.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+ T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Original Research Article Humoral and cellular immune responses to modified hepatitis B plasmid DNA vaccine in mice

Purpose: To evaluate the immunogenicity and types of immune response of a quality-controlled modified recombinant hepatitis B surface antigen (HBsAg) plasmid encoding HBsAg in mice. Methods: The characterized plasmid DNA was used in the immunization of Balb/c mice. Three groups of mice were intramuscularly injected with three different concentrations (50, 25 and 10 μg/100 μL) of the modified plasmid. Humoral immune response was monitored by enzyme-linked immunosorbent assay (ELISA), while cellular immune response was investigated by analysis of spleen cytokine profile (TNFα, IFN γ and IL2) as well as CD69 expression level in CD4 and CD8 positive cells. Results: In general, the activated CD4 cells showing intracellular cytokines were higher than CD8 positive population of cells (p < 0.05). These findings indicate that the vaccine induced both a humoral and cellular immunity. Cytokine profile also showed high levels of TNFα, IFN γ and IL2 and CD69 expression in the group of animals immunized at a dose of 10 μg when compared to control group (p < 0.05). Conclusion: A 10 μg dose intramuscular injection of the modified DNA-based vaccine encoding HBsAg in mice induces both high humoral and cellular immune responses.

Small-plasmid-mediated antibiotic resistance is enhanced by increases in plasmid copy number and bacterial fitness

Plasmids play a key role in the horizontal spread of antibiotic resistance determinants among bacterial pathogens. When an antibiotic resistance plasmid arrives in a new bacterial host, it produces a fitness cost, causing a competitive disadvantage for the plasmid-bearing bacterium in the absence of antibiotics. On the other hand, in the presence of antibiotics, the plasmid promotes the survival of the clone. The adaptations experienced by plasmid and bacterium in the presence of antibiotics during the first generations of coexistence will be crucial for the progress of the infection and the maintenance of plasmid-mediated resistance once the treatment is over. Here we developed a model system using the human pathogen Haemophilus influenzae carrying the small plasmid pB1000 conferring resistance to β-lactam antibiotics to investigate host and plasmid adaptations in the course of a simulated ampicillin therapy. Our results proved that plasmid-bearing clones compensated for the fitness disadvantage during the first 100 generations of plasmid-host adaptation. In addition, ampicillin treatment was associated with an increase in pB1000 copy number. The augmentation in both bacterial fitness and plasmid copy number gave rise to H. influenzae populations with higher ampicillin resistance levels. In conclusion, we show here that the modulations in bacterial fitness and plasmid copy number help a plasmid-bearing bacterium to adapt during antibiotic therapy, promoting both the survival of the host and the spread of the plasmid.

Antibiotic-resistant Klebsiella pneumoniae and Escherichia coli high-risk clones and an IncFII(k) mosaic plasmid hosting Tn1 (blaTEM-4) in isolates from 1990 to 2004

We describe the genetic background of bla(TEM-4) and the complete sequence of pRYC11::bla(TEM-4), a mosaic plasmid that is highly similar to pKpQIL-like variants, predominant among TEM-4 producers in a Spanish hospital (1990 to 2004), which belong to Klebsiella pneumoniae and Escherichia coli high-risk clones responsible for the current spread of different antibiotic resistance genes. Predominant populations of plasmids and host adapted clonal lineages seem to have greatly contributed to the spread of resistance to extended-spectrum cephalosporins.

Haemophilus influenzae clinical isolates with plasmid pB1000 bearing blaROB-1: fitness cost and interspecies dissemination

Plasmid pB1000 is a mobilizable replicon bearing the bla(ROB-1) beta-lactamase gene that we have recently described in Haemophilus parasuis and Pasteurella multocida animal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000', in four Haemophilus influenzae clinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzae isolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae, giving rise to recipients resistant to ampicillin and cefaclor (MICs, > or =64 microg/ml). Stability experiments showed that pB1000 is stable in H. influenzae without antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzae strains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceae act as an antimicrobial resistance reservoir for H. influenzae. Further, since P. multocida is the only member of this family that can colonize both humans and animals, we propose that P. multocida is the vehicle for the transport of pB1000 between animal- and human-adapted members of the Pasteurellaceae.

Novel blaROB-1-bearing plasmid conferring resistance to β-lactams in Haemophilus parasuis isolates from healthy weaning pigs

Haemophilus parasuis, the causative agent of Glässer's disease, is one of the early colonizers of the nasal mucosa of piglets. It is prevalent in swine herds, and lesions associated with disease are fibrinous polyserositis and bronchopneumonia. Antibiotics are commonly used in disease control, and resistance to several antibiotics has been described in H. parasuis. Prediction of H. parasuis virulence is currently limited by our scarce understanding of its pathogenicity. Some genes have been associated with H. parasuis virulence, such as lsgB and group 1 vtaA, while biofilm growth has been associated with nonvirulent strains. In this study, 86 H. parasuis nasal isolates from farms that had not had a case of disease for more than 10 years were obtained by sampling piglets at weaning. Isolates were studied by enterobacterial repetitive intergenic consensus PCR and determination of the presence of lsgB and group 1 vtaA, biofilm formation, inflammatory cell response, and resistance to antibiotics. As part of the diversity encountered, a novel 2,661-bp plasmid, named pJMA-1, bearing the blaROB-1 β-lactamase was detected in eight colonizing strains. pJMA-1 was shown to share a backbone with other small plasmids described in the Pasteurellaceae, to be 100% stable, and to have a lower biological cost than the previously described plasmid pB1000. pJMA-1 was also found in nine H. parasuis nasal strains from a separate collection, but it was not detected in isolates from the lesions of animals with Glässer's disease or in nontypeable Haemophilus influenzae isolates. Altogether, we show that commensal H. parasuis isolates represent a reservoir of β-lactam resistance genes which can be transferred to pathogens or other bacteria.

Healthcare Computer Reasoning Addressing Chronically Ill Societies Using IoT: Deep Learning AI to the Rescue of Home-Based Healthcare

The authors present a proposal to develop intelligent assisted living environments for home based healthcare. These environments unite the chronical patient clinical history sematic representation with the ability of monitoring the living conditions and events recurring to a fully managed Semantic Web of Things (SWoT). Several levels of acquired knowledge and the case based reasoning that is possible by knowledge representation of the health-disease history and acquisition of the scientific evidence will deliver, through various voice based natural interfaces, the adequate support systems for disease auto management but prominently by activating the less differentiated caregiver for any specific need. With these capabilities at hand, home based healthcare providing becomes a viable possibility reducing the institutionalization needs. The resulting integrated healthcare framework will provide significant savings while improving the generality of health and satisfaction indicators.

Design and synthesis of E3 ligase RNF5 inhibitors as innovative strategy to trigger mutant CFTR rescue in Cystic Fibrosis

In Cystic Fibrosis (CF) the deletion of phenylalanine 508 (F508del) in the CFTR anion channel is associated to misfolding and defective gating of the mutant protein. Among the known proteins involved in CFTR processing, one of the most promising drug target is the ubiquitin ligase RNF5, which normally promotes F508del-CFTR degradation. In this context, a small molecule RNF5 inhibitor is expected to chemically mimic a condition of RNF5 silencing, thus preventing mutant CFTR degradation and causing its stabilization and plasma membrane trafficking. Hence, by exploiting a virtual screening (VS) campaign, the hit compound inh-2 was discovered as the first-in-class inhibitor of RNF5. Evaluation of inh-2 efficacy on CFTR rescue showed that it efficiently decreases ubiquitination of mutant CFTR and increases chloride current in human primary bronchial epithelia. Based on the promising biological results obtained with inh-2, this thesis reports the structure-based design of potential RNF5 inhibitors having improved potency and efficacy. The optimization of general synthetic strategies gave access to a library of analogues of the 1,2,4-thiadiazol-5-ylidene inh-2 for SAR investigation. The new analogues were tested for their corrector activity in CFBE41o- cells by using the microfluorimetric HS-YFP assay as a primary screen. Then, the effect of putative RNF5 inhibitors on proliferation, apoptosis and the formation of autophagic vacuoles was evaluated. Some of the new analogs significantly increased the basal level of autophagy, reproducing RNF5 silencing effect in cell. Among them, one compound also displayed a greater rescue of the F508del-CFTR trafficking defect than inh-2. Our preliminary results suggest that the 1,2,4-thiadiazolylidene could be a suitable scaffold for the discovery of potential RNF5 inhibitors able to rescue mutant CFTRs. Biological tests are still ongoing to acquire in-depth knowledge about the mechanism of action and therapeutic relevance of this unprecedented pharmacological strategy.

Fleet Management System for Search and Rescue Operations

Over one million people lost their lives in the last twenty years from natural disasters like wildfires, earthquakes and man-made disasters. In such scenarios the usage of a fleet of robots aims at the parallelization of the workload and thus increasing speed and capabilities to complete time sensitive missions. This work focuses on the development of a dynamic fleet management system, which consists in the management of multiple agents cooperating in order to accomplish tasks. We presented a Mixed Integer Programming problem for the management and planning of mission’s tasks. The problem was solved using both an exact and a heuristic approach. The latter is based on the idea of solving iteratively smaller instances of the complete problem. Alongside, a fast and efficient algorithm for estimation of travel times between tasks is proposed. Experimental results demonstrate that the proposed heuristic approach is able to generate quality solutions, within specific time limits, compared to the exact one.

Graphene And Carbon Nanotube Nanocomposite For Gene Transfection.

Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5m(2)/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency.

Carbon Nanoparticles For Gene Transfection In Eukaryotic Cell Lines.

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

Overlapped Sequence Types (sts) And Serogroups Of Avian Pathogenic (apec) And Human Extra-intestinal Pathogenic (expec) Escherichia Coli Isolated In Brazil.

Avian pathogenic Escherichia coli (APEC) strains belong to a category that is associated with colibacillosis, a serious illness in the poultry industry worldwide. Additionally, some APEC groups have recently been described as potential zoonotic agents. In this work, we compared APEC strains with extraintestinal pathogenic E. coli (ExPEC) strains isolated from clinical cases of humans with extra-intestinal diseases such as urinary tract infections (UTI) and bacteremia. PCR results showed that genes usually found in the ColV plasmid (tsh, iucA, iss, and hlyF) were associated with APEC strains while fyuA, irp-2, fepC sitDchrom, fimH, crl, csgA, afa, iha, sat, hlyA, hra, cnf1, kpsMTII, clpVSakai and malX were associated with human ExPEC. Both categories shared nine serogroups (O2, O6, O7, O8, O11, O19, O25, O73 and O153) and seven sequence types (ST10, ST88, ST93, ST117, ST131, ST155, ST359, ST648 and ST1011). Interestingly, ST95, which is associated with the zoonotic potential of APEC and is spread in avian E. coli of North America and Europe, was not detected among 76 APEC strains. When the strains were clustered based on the presence of virulence genes, most ExPEC strains (71.7%) were contained in one cluster while most APEC strains (63.2%) segregated to another. In general, the strains showed distinct genetic and fingerprint patterns, but avian and human strains of ST359, or ST23 clonal complex (CC), presented more than 70% of similarity by PFGE. The results demonstrate that some zoonotic-related STs (ST117, ST131, ST10CC, ST23CC) are present in Brazil. Also, the presence of moderate fingerprint similarities between ST359 E. coli of avian and human origin indicates that strains of this ST are candidates for having zoonotic potential.