Specific Plasmid Research Articles

Abstract 3442: Functional and molecular analysis of the somatic mutations in the NF-κB pathway inhibitor, CYLD, in nasopharyngeal carcinoma

Abstract Background: NF-κB is an important signaling pathway for regulating multiple signaling cascades involved in cancer development such as inflammation, immune response, cell survival, and cell proliferation. Constitutive activation of the NF-κB pathway plays a key role in the development of NPC. Our previous whole-exome sequencing (WES) study in NPC biopsies has identified five mutations in CYLD, including R758Q. As R758X is a mutational hotspot in CYLD, it has been reported in multiple familial trichoepithelioma type 1, Brooke-Spiegler syndrome and familial cylindromatosis. Thus, we attempted to evaluate the functional effect of CYLD WT and R758Q in regulating NF-κB activity in NPC, which is crucial in NPC progression and development. Methods: C17, C666-1 and NPC43 EBV-positive cell lines and HK1 were co-infected by CYLD wild-type (WT) and mutant constructs with a NF-κB specific dual luciferase promoter plasmid. CRISPR-CAS9 system was used to knock out CYLD. The functional role of CYLD was determined by MTT, CFA and wound healing assays. NF-κB downstream targets expression was evaluated by the method of RT-QPCR. Co-immunoprecipitation (Co-IP) experiments were performed with the Sigma Anti-FLAG M2 magnetic beads and the results were confirmed by Western blot analysis. Female athymic BALB/cAnN (nude) mice were utilized to investigate tumorigenicity. NPC cell lines were treated with cisplatin to check cell sensitivity. Results: The reporter assay showed that CYLD can downregulate NF-κB binding activities in comparison to the vector-alone (VA), but R758Q failed to inhibit the NF-κB binding activities. Over-expression of CYLD can suppress NF-κB downstream target genes expression. Knocking out of CYLD results in higher NF-κB downstream genes expression compared with VA, which confirmed that CYLD can suppress NF-κB activity. R758Q increases these genes expression compared with WT. CYLD can suppress cell growth rate, while R758Q promoted cell proliferation and migration compared to WT. Furthermore, WT CYLD can significantly suppress tumor growth rate, whereas R758Q has the same growth rate with VA, consistent with R758Q loss of WT function, causing tumor suppression. Moreover, CYLD can enhance cell sensitivity to cisplatin treatment compared to the VA. Conclusions: These results suggest that CYLD can suppress NF-κB activity compared to VA, while R758Q lost WT function in NPC. CYLD can inhibit tumorigenesis compared to VA, whereas R758Q induces tumor growth compared to WT. The NPC cell line with CYLD WT is more sensitive to cisplatin treatment. Acknowledgement: Research Grants Council Area of Excellence grant AoE-M06/08 to MLL. Citation Format: Mingdan Deng, Wei Dai, Maria Lung. Functional and molecular analysis of the somatic mutations in the NF-κB pathway inhibitor, CYLD, in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3442.

Inflammation induced by different plasmid types of russian <i>Yersinia pseudotuberculosis</i> strains

In the 2000s, a scientific interest to the Far Eastern scarlet-like fever (FESLF) mainly recorded In Russia and Japanwas remarkably increased. Such clinical and epidemic manifestation of human pseudotuberculosis is related to a certain bacterial clonal lineage characterized by a specific plasmid profile (pVM82, pYV48), sequence type (2ST) as well as the yadA gene allele (1st allele). In our study we examined features of inflammatory changes characterizing plasmidassociated pathogenicity of the FESLF pathogen. In addition, organ histopathology in experimental animals infected intraperitoneally with Y. pseudotuberculosis strains of the four plasmid types 48+:82+; 48+:82-; 48-:82+; 48-:82-; and 48-:82- was investigated. It was found that the mortality rate in animals infected with Y. pseudotuberculosis H-5015 strain (82+:48+) bearing two plasmids with a molecular weight of 82 and 48 MDa was 40%. A picture of diffuse pneumonia with moderate inflammatory infiltration in pulmonary tissue and changes in lymphoid organs characterizing immunodeficiency we observed as early as 3 days postinfection (p.i.). On the contrary, animals infected with Y. pseudotuberculosis H-5015 strain (82+:48) bearing a single plasmid 82 MDa pVM, mortality rate was 30%. A vascular reaction in the lungs and liver as well as deteriorated vascular destructive changes were revealed starting from day 3 and on day 57 days p.i., respectively, which was paralleled with perivascular infiltration mainly by mononuclear cells and focal pneumonia as well as a reaction of bronchialassociated lymphoid tissue and minimal organ destructive changes. On day 7 p.i., signs of granulomatous inflammation in the liver in a form of small mononuclear cell clusters and perivascular compact infiltrates were found. At all time points, lymphoid organ hyperplasia was noted. In case the infection caused by H-5013 strain (48+), the mortality rate was 80%. Inflammatory changes with dominant mononuclear cells were detected as early as 1 day p.i. associated with a picture of large focal bronchopneumonia, more pronounced in the non-survivor animals, also demonstrating signs of severe immunosuppression in the lymphoid organs. Starting from day 3 p.i., lymphoid hypoplasia in the spleen and lymph nodes was detected in all infected animals paralleled with pathogen-associated tissue damage in various organs. Animals infected with the plasmid-free H-5013 strain 48-) resulted in 25% mortality rate. Moreover, starting from day 3 p.i., a slight mononuclear inflammatory infiltration in the lungs and liver, a moderate giant cell reaction in the splenic pulp, and loose perinodal inflammatory infiltration in the lymph nodes were observed. Thus, while modeling infection caused by different plasmid types of Y. pseudotuberculosis, the data on differences in histopathology of changes in diverse organs regarding dynamics and generalization of the inflammatory response, as well as the severity of pathogen-associated damage in the lymphoid tissue were obtained. In case Y. pseudotuberculosis strains contained pVM82 plasmid with or without virulence plasmid pYV vs. single pYV-positive strains, an area of the inflammatory response as well as severity of immunosuppression manifested by splenic and lymph node delymphatization were decreased. It allowed to suggest that pVM82 plasmid found In Russia-originating Y. pseudotuberculosis strains might be implicated in limiting intensity of inflammatory response, bacterial dissemination and severity of lymphoid organ damage.

Potential Application of TALENs against Murine Cytomegalovirus Latent Infections.

Cytomegalovirus (CMV) infections are still a global health problem, because the latent viruses persist in humans and cause recurring diseases. Currently, there are no therapies for CMV latent infections and the therapies for active infections are limited by side effects and other problems. It is impossible to eradicate latent viruses in animals. HCMV (human CMV) is specific to human diseases; however, it is difficult to study HCMV due to its host specificity and long life cycle. Fortunately, MCMV (murine CMV) provides an excellent animal model. Here, three specific pairs of transcription activator-like effector nuclease (TALEN) plasmids (MCMV1–2, 3–4, and 5–6) were constructed to target the MCMV M80/80.5 sequence in order to test their efficacy in blocking MCMV lytic replication in NIH3T3 cell culture. The preliminary data showed that TALEN plasmids demonstrate specific targeting and cleavage in the MCMV M80/80.5 sequence and effectively inhibit MCMV growth in cell culture when the plasmid transfection is prior to the viral infection. The most specific pairs of TALEN plasmids (MCMV3–4) were further used to confirm the negative regulation of latent MCMV replication and gene expression in Balb/c mice. The injection of specific TALEN plasmids caused significant inhibition in the copy number level of immediately early gene (ie-1) DNA in five organs of mice, when compared with the controls. The result demonstrated that TALENs potentially provide an effective strategy to remove latent MCMV in animals.

Quinolone resistance mechanisms among third-generation cephalosporin resistant isolates of Enterobacter spp. in a Bulgarian university hospital.

Background: There have been no reports in Bulgaria about quinolone resistance determinants among Enterobacter spp.Aims: To investigate plasmid and chromosomal quinolone resistance rates among 175 third-generation cephalosporin resistant Enterobacter spp. isolates (167 Enterobacter cloacae complex and eight Enterobacter aerogenes isolates) collected at a university hospital in Varna, Bulgaria, as well as to reveal their association with ESBL/AmpC production and a carriage of specific plasmid replicon types.Methods: PCR, isoelectric focusing, replicon typing, sequencing, and epidemiology typing were carried out.Results: A high level of combined third-generation cephalosporin and quinolone resistant Enterobacter spp. was found − 79.4%. The ESBL production rate was 87%, consisting mainly of CTX-M-15 among E. cloacae complex (in 76%) and CTX-M-3 among E. aerogenes (in 88%). Plasmid mediated quinolone resistance (PMQR) determinants were identified in 57% of the isolates. The most commonly detected PMQR determinants were qnrB (90%), consisting mainly of qnrB1 (in 61%), and qnrB9 (in 27%) of the isolates. Both alleles were transferred with CTX-M-15 genes; transconjugants showed HI2 replicons (for qnrB1 positive transconjugants) and were non-typeable (for qnrB9). One Enterobacter spp. isolate produced qnrB4. QnrA1, qnrS1, and aac(6ʹ)-Ib-cr were detected in single isolates only. QnrC, qnrD, qepA, and oqxAB genes were not found. QnrB was associated with CTX-M-15 production, and qnrS1 was linked to CTX-M-3. Alterations in 83 and 87 positions of gyrB in quinolone-resistance determining regions, and 80 position of parC were detected in high level quinolone resistant isolates. Among all the Enterobacter spp. isolates tested, one predominant clone A was identified (53%).Conclusion: Our data showed the necessity of more prudent use of quinolones and third-generation cephalosporins, because of the risk of promoting dissemination, and selection of multiple resistance determinants (ESBL, PMQR) among Enterobacter spp. isolates in Bulgaria.

Overexpression of a Novel E3 Ligase leads to a Skeletal Muscle Myopathy through Alterations in Cytoskeleton Proteins and Enhanced Protein Degradation

Multiple E3 ubiquitin ligases have been identified to be transcriptionally increased in skeletal muscle under atrophy‐inducing conditions. We have recently observed a novel E3 ligase, F‐box and Leucine‐rich protein 22 (FbxL22), to be elevated during denervation‐induced muscle atrophy. Little is known about the role of FbxL22 in skeletal muscle, with recent research highlighting its impact on cardiac muscle function and sarcomeric protein turnover (Spaich et al., 2012).Subsequently, male C57BL/6 mice (12‐week old) were subjected to in vivo electroporation with a mouse specific FbxL22 plasmid (20ug) in the tibialis anterior (TA) muscle with the contralateral leg serving as an empty vector control (pcDNA3.1). After 14 days, TA muscles were sectioned for H&E stain and GFP visualisation to confirm plasmid incorporation. Muscle mass and cross‐sectional area (CSA) were determined. Western blotting was performed to determine intermediate filament, autophagy and poly‐ubiquitin protein levels in the transfected TA muscles.Interestingly, we observed no difference in muscle mass after 14 days, but we did observe increased numbers of large and small fibers in the FbxL22 transfected muscle. At the protein level, there was a 75% reduction in alpha‐actinin‐2, which was accompanied by 4‐fold increase in desmin and vimentin. We additionally observed a 2‐fold increase in polyubquitination, and 1.5‐fold increase in autophagy markers (p62 and LCB II).Our data highlights the powerful impact of FbxL22 on skeletal muscle structure. Future studies will seek to investigate the chronic effect of FbxL22 on skeletal muscle and characterize its role during the process of muscle atrophy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The origin of plague. Future perspectives of ecological and molecular-genetic synthesis

Plague, infamous due to three devastating pandemics, remains one of the most dangerous human diseases. Its causative agent, the microbe Yersinia pestis, is a priority in the arsenal of possible biological weapons, which requires increased attention to the development of a system of biological (bacteriological) security. A deep knowledge of the natural processes that facilitate the development of the causative agent of plague may be useful for this. There are currently two alternative approaches to determining the origin of Y. pestis – molecular genetics (MG) and ecological. MG-data has led to the innovative idea of the saltation conversion of the psichrophilic saprozoonotic microbe Yersinia pseudotuberculosis O:1b into a population of the pathogenic plague microbe Y. pestis, by horizontal transfer of two specific plasmids pFra and pPst from the external environment, or from other bacteria and inactivated/deletions of genes that have lost their functions in a new habitat, probably in populations of voles (Microtinae) in Asia. The ecological scenario is based on the idea of Darwinian adaptations by way of a quick "quantum" formation of its properties in a parasitic system; the "marmot–flea" (Marmota sibirica – Oropsylla silantiewi), during the transition between the Pleistocene and Holocene periods. Three important factors of quantum speciation were found: heterothermia of marmot bodies during hibernation, the oxidative "burst" of macrophages in hibernating marmots, and stress-induced mutagenesis initiated by the oxidative "burst" of macrophages. This article asserts the complementarity of the ecological and MG approaches. The prospect of a solution to the problem of quantum speciation of the causative agent of the plague, and the development of methods for the treatment and prevention of disease, can be observed in the synthesis of ecological and MG approaches.

Resveratrol Abrogates Hypoxia-Induced Up-Regulation of Exosomal Amyloid-β Partially by Inhibiting CD147.

Hypoxia promotes both total extracellular and exosomal amyloid-β (Aβ) production and aggravates Alzheimer's disease (AD). Resveratrol (RSV) has been proved to be neuroprotective in AD models, and down-regulated the expression of CD147, an additional subunit of γ-secretase. In this study, we aimed to explore the role and mechanisms of RSV in hypoxia-induced upregulation of Aβ, especially exosomal Aβ. SH-SY5Y cells and HEK293 cells overexpressing amyloid precursor protein (APP) as well as C57BL/6 mice were treated with RSV and exposed to hypoxic conditions. The expression of SIRT1 or CD147 was modulated by transfection of specific siRNAs or plasmid. Aβ1-40 and Aβ1-42 levels were determined by ELISA. Hypoxia increased the levels of both Aβ1-40 and Aβ1-42 in the hippocampal lysates and serum-derived exosomes of mice. Hypoxia also increased both Aβ1-40 and Aβ1-42 levels in the total culture medium (CM), cell-derived exosomal lysates, and exosome-free CM of both cell lines. Treatment with RSV abrogated these changes in Aβ expression, inhibited the hypoxia-induced down-regulation of SIRT1 and up-regulation of CD147. Knockdown of SIRT1 promote total Aβ level but has no effect on exosomal Aβs expression. Knockdown of CD147 inhibits both total and exosomal Aβs expression. Furthermore, overexpressing CD147 in cells exposed to hypoxia facilitated the production of Aβ1-40 and Aβ1-42, while application of RSV reduced the CD147 expression as well as Aβ levels in both exosomes and exosome-free CM. These results suggested that RSV abrogated hypoxia-induced up-regulation of total and exosomal Aβ partially by inhibiting CD147.

Phenotypic Prediction: Linking in vitro Virulence to the Genomics of 59 Salmonella enterica Strains.

The increased availability of whole-genome-sequencing techniques generates a wealth of DNA data on numerous organisms, including foodborne pathogens such as Salmonella. However, how these data can be used to improve microbial risk assessment and understanding of Salmonella epidemiology remains a challenge. The aim of this study was to assess variability in in vitro virulence and genetic characteristics between and within different serovars. The phenotypic behavior of 59 strains of 32 different Salmonella enterica serovars from animal, human and food origin was assessed in an in vitro gastro-intestinal tract (GIT) system and they were analyzed for the presence of 233 putative virulence genes as markers for phenotypic prediction. The probability of in vitro infection, P(inf), defined as the fraction of infectious cells passing from inoculation to host cell invasion at the last stage of the GIT system, was interpreted as the in vitro virulence. Results showed that the (average) P(inf) of Salmonella serovars ranged from 5.3E-05 (S. Kedougou) to 5.2E-01 (S. Typhimurium). In general, a higher P(inf) on serovar level corresponded to higher reported human incidence from epidemiological reporting data. Of the 233 virulence genes investigated, only 101 showed variability in presence/absence among the strains. In vitro P(inf) was found to be positively associated with the presence of specific plasmid related virulence genes (mig-5, pef, rck, and spv). However, not all serovars with a relatively high P(inf), > 1E-02, could be linked with these specific genes. Moreover, some outbreak related strains (S. Heidelberg and S. Thompson) did not reveal this association with P(inf). No clear association with in vitro virulence P(inf) was identified when grouping serovars with the same virulence gene profile (virulence plasmid, Typhoid toxin, peg operon and stk operon). This study shows that the in vitro P(inf) variation among individual strains from the same serovar is larger than that found between serovars. Therefore, ranking P(inf) of S. enterica on serovar level alone, or in combination with a serovar specific virulence gene profile, cannot be recommended. The attribution of single biological phenomena to individual strains or serovars is not sufficient to improve the hazard characterization for S. enterica. Future microbial risk assessments, including virulence gene profiles, require a systematic approach linked to epidemiological studies rather than revealing differences in characteristics on serovar level alone.

Genetic Manipulation of Chlamydia trachomatis: Chromosomal Deletions.

Progress in understanding molecular mechanisms contributing to chlamydial pathogenesis has been greatly facilitated by recent advances in genetic manipulation of C. trachomatis. Valuable approaches such as random, chemically induced mutagenesis or targeted, insertion-based gene disruption have led to significant discoveries. We describe herein a technique for generating definitive null strains via complete deletion of chromosomal genes in C. trachomatis. Fluorescence-reported allelic exchange mutagenesis (FRAEM), using the suicide vector pSUmC, enables targeted deletion of desired chromosomal DNA. The protocol provided here describes steps required to produce transformation competent chlamydiae, generate a specific allelic exchange plasmid construct, carry out mutagenesis, and isolate clonal populations of resulting mutant strains.

Targeting cholesterol metabolism in neuroblastoma by acting on HDACs

Event Abstract Back to Event Targeting cholesterol metabolism in neuroblastoma by acting on HDACs Maria João Nunes1, 2, Daniela Costa1, Rita Mendes De Almeida1, Margarida Castro-Caldas1, 3, Maria João Gama1, 2 and Elsa Rodrigues1, 2* 1 Research Institute for Medicines (iMed.ULisboa), Portugal 2 Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Portugal 3 Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal A wide range of brain disorders are associated with imbalances in protein acetylation levels and treatment with various histone deacetylase (HDAC) inhibitors (HDACi) has been shown to correct these deficiencies, emerging as a promising new strategy for therapeutic intervention in neurodegenerative diseases. We have demonstrated that in neuroblastoma cells HDACi dramatically increase mRNA levels of genes involved in cholesterol efflux and elimination, while decreasing the expression of genes involved in synthesis and uptake, leading to a decrease in intracellular cholesterol content. These results suggested that this class of compounds could potentially be used to modulate brain cholesterol metabolism in pathological conditions. We used primary cultures of mice neurons and astrocytes treated with the pan-HDACi trichostatin A, and with class-specific HDAC inhibitors, namely, the class I inhibitor Mocetinostat (MGCD0103) and the class II (IIa) inhibitor MC1568, to evaluate the mRNA levels of a panel of genes involved in cholesterol synthesis, up-take, transport and elimination. Although we could only partially recapitulate the results obtained in the neuroblastoma cells, we could identify class I HDACs as major modulators of neuronal cholesterol homeostasis. In addition, when comparing the expression profiles of neurons and astrocytes, we identified Mvk, Cyp46a1 and Apoe as genes that are affected by both TSA and MGCD0103 in both cells. In order to further dissect the role of class I HDACs we specifically silenced class I HDACs by transfecting TALEN specific plasmids in SH-SY5Y neuroblastoma cells. Our results point to a role of class I HDACs as therapeutic targets both in neuroblastoma and neurodegenerative disorders with disturbances in cholesterol homeostasis. Acknowledgements This work was supported by FEDER and national funds from Fundação para a Ciência e Tecnologia (FCT) (UID/DTP/04138/2013 and fellowship SFRH/BPD/95855/2013 to MJN). Keywords: cholesterol metabolism, HDACs, HDAC inhibitors, neurodegeneration, Neuroblastoma Conference: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019), Lisboa, Portugal, 30 May - 1 Jun, 2019. Presentation Type: Poster presentation Topic: Cellular and Molecular Neurosciences Citation: Nunes M, Costa D, Mendes De Almeida R, Castro-Caldas M, Gama M and Rodrigues E (2019). Targeting cholesterol metabolism in neuroblastoma by acting on HDACs. Front. Cell. Neurosci. Conference Abstract: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019). doi: 10.3389/conf.fncel.2019.01.00012 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 16 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Prof. Elsa Rodrigues, Research Institute for Medicines (iMed.ULisboa), Lisboa, Portugal, elsa.rodrigues@ff.ulisboa.pt Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Maria João Nunes Daniela Costa Rita Mendes De Almeida Margarida Castro-Caldas Maria João Gama Elsa Rodrigues Google Maria João Nunes Daniela Costa Rita Mendes De Almeida Margarida Castro-Caldas Maria João Gama Elsa Rodrigues Google Scholar Maria João Nunes Daniela Costa Rita Mendes De Almeida Margarida Castro-Caldas Maria João Gama Elsa Rodrigues PubMed Maria João Nunes Daniela Costa Rita Mendes De Almeida Margarida Castro-Caldas Maria João Gama Elsa Rodrigues Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Effects of polyamines on the ultrafiltration of plasmid DNA.

It is well established that the structure of plasmid DNA is a strong function of solution ionic conditions due to changes in intramolecular electrostatic interactions between the charged phosphate groups along the DNA backbone. Multivalent cations like spermine and spermidine play a critical role in compacting and controlling the structure of supercoiled DNA in living cells. The objective of this work was to investigate the effects of these polyamines on the ultrafiltration of plasmid DNA, including possible opportunities to use these polycations to enhance the purification of specific plasmid isoforms. Data were obtained using a wide range of spermine and spermidine concentrations to evaluate DNA transmission through Biomax polyethersulfone ultrafiltration membranes. Spermine has a very strong effect on DNA transmission, with the sieving coefficient of the supercoiled plasmid decreasing by more than an order of magnitude upon addition of only 15 μM spermine. A comparable change in DNA transmission required >300 μM of the trivalent spermidine. The polyamines were able to significantly increase the selectivity for the separation of DNA from a model protein, but they were unable to provide a significant increase in the selectivity for separating DNA isoforms under the conditions examined in this study. The results do demonstrate that both spermine and spermidine can be used to control the extent of DNA transmission/purification during ultrafiltration. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2765, 2019.

SPECIFIC FEATURES OF MOLECULAR EPIDEMIOLOGY OF IMPORTED MORBIDITY CAUSED BY SALMONELLA ENTERITIDIS INTRODUCED STRAINS WITH SPECIFIC PLASMID TYPES

Aim. To compare the plasmid characteristics of Salmonella Enteritidis populations isolated from human patients in the Primorsky and Khabarovsk Territories to determine the causes and mechanisms of the morbidity gain associated with Salmonella infections caused by specific plasmid types. Materials and methods. The plasmid analysis of S. Enteritidis isolates from 7057 patients, including 6120 patients in the Primorsky Territory and 937 in the Khabarovsk Territory were performed over the period of 2006-2013. The plasmid profile was analyzed by the method of Kado C.I. and Liu S.T. (1981). Results. The concept of the synchronous morbidity among people living in different administrative territories caused by specific plasmid types of S. Enteritidis was established. The need to identify this type of morbidity is predicated on its targeted control and prevention measures. Conclusion. The nonintegrated epidemiological surveillance of salmonellosis covering certain administrative territories is unable to control the whole process of synchronous morbidity; additional infection control of strategies is needed.

A Full MALDI-Based Approach to Detect Plasmid-Encoded KPC-Producing Klebsiella pneumoniae.

KPC-producing Klebsiella pneumoniae represents a severe public health concern worldwide. The rapid detection of these isolates is of fundamental importance for the adoption of proper antibiotic treatment and infection control measures, and new applications of MALDI-TOF MS technology fit this purpose. In this study, we present a full MALDI-based approach to detect plasmid-encoded KPC-producing strains, accomplished by the automated detection of a KPC-specific peak (at 11,109 m/z) by a specific algorithm integrated into the MALDI Biotyper system (Bruker Daltonik), and the confirmation of carbapenemase activity by STAR-Carba imipenem hydrolysis assay. A total of 6209 K. pneumoniae isolates from Italy and Germany were investigated for the presence of the KPC-related peak, and a subset of them (n = 243) underwent confirmation of carbapenemase activity by STAR-Carba assay. The novel approach was further applied directly to positive blood culture bottles (n = 204), using the bacterial pellet obtained with Sepsityper kit (Bruker Daltonik). The novel approach enabled a reliable and very fast detection of KPC-producing K. pneumoniae strains, from colonies as well as directly from positive blood cultures. The automated peak detection enabled the instant detection of KPC-producing K. pneumoniae during the routine identification process, with excellent specificity (100%) and a good sensitivity (85.1%). The sensitivity is likely mainly related to the prevalence of the specific plasmid harboring clones among all the KPC-producing circulating strains. STAR-Carba carbapenemase confirmation showed 100% sensitivity and specificity, both from colonies and from positive blood cultures.

Cloning and expression of enterovirus 71 capsid protein 1 in a probiotic Bifidobacterium pseudocatenulatum.

This study investigated cloning and expression of enterovirus 71 viral capsid protein 1 (EV71-VP1) in Bifidobacterium pseudocatenulatum (B.pseudocatenulatum) M115. To achieve this, a codon-optimized gene coding for EV71-VP1 was analysed, designed, synthesized and cloned into a plasmid vector flanked by a transcriptional promoter and terminator sequences. The promoter was based on that of P919, a constitutive promoter of the gene encoding the large ribosomal protein of B.bifidum BGN4, while the terminator was based on that of the peptidase N gene of Lactococcus lactis. The construct was amplified in Escherichia coli XL1-blue and then transferred into B.pseudocatenulatum M115 by electrotransformation. Western blot analysis revealed that the EV71-VP1 was intracellularly expressed in B.pseudocatenulatum M115 under the control of the selected heterologous promoter. In addition, plasmid stability analysis showed the construct was maintained stably for more than 160 generations, enough for most future applications. The results derived from this study open the possibility to utilize the bacterium carrying a specific expression plasmid as cell factory for the production of proteins with high commercial and health-promoting value. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the first successful expression of a codon-optimized gene coding for enterovirus 71 viral capsid protein 1 (EV71-VP1) in Bifidobacterium pseudocatenulatum M115, a novel probiotic strain isolated from human intestines. The EV71-VP1 was constitutively expressed under the control of P919 promoter derived from B.bifidum BGN4 in the cytoplasm of bacterial cells supporting the use of heterologous promoter and terminator sequences for viral gene expression in Bifidobacterium species.

CircRNA of AR-suppressed PABPC1 91 bp enhances the cytotoxicity of natural killer cells against hepatocellular carcinoma via upregulating UL16 binding protein 1.

Hepatocellular carcinoma (HCC) is a heterogeneous malignancy type with limited approaches for treatment. Additionally, inappropriate immune therapy indicates that the understanding the underlying mechanism of HCC is necessary. The aim of the present study was to investigate the influence of a novel circular RNA (circRNA), circRNA of AR-suppressed PABPC1 91 bp (CircARSP91), on immune surveillance induced by natural killer (NK) cells. An in vitro cell cytotoxicity assay was performed to determine the cytotoxicity of NK cells against HCC cells. A specific plasmid for circRNA overexpression was used to establish stable cell lines. Additionally, samples from patients with HCC were analyzed to determine the association between the present in vitro data and those of clinical settings. CircARSP91 could increase the susceptibility of HCC cells to NK cell cytotoxicity. Following screening multiple factors that could influence the activation of NK cells, it was determined that such a phenotype may be caused by upregulating UL16 binding protein 1 (ULBP1) expression in HCC cells at the mRNA and protein levels. Additionally, the data generated from patient samples significantly support a positive association between CircARSP91 and ULBP1. In conclusion, CircARSP91 could enhance innate immune surveillance by strengthening the cytotoxicity of NK cells, implying that circRNA may serve a role in tumor immunity.

DNMT1, a Novel Regulator Mediating mTORC1/mTORC2 Pathway-Induced NGF Expression in Schwann Cells

Schwann cells play an important role in maintaining the normal function of peripheral nerves via the secretion of nerve growth factor (NGF). The mTOR signaling pathway is known as a kind of Ser/Thr protein kinase that regulates various cell functions. DNA methyltransferase 1 (DNMT1) is an epigenetic regulator and downstream target of the mTOR pathway. In the present study, we explored the relationship between NGF expression and the mTOR pathway/DNMT1 in RSC96 cells. The results showed that both rapamycin and Torin 1 downregulated NGF expression via the inhibition of phospho-mTOR (Ser 2448) and phospho-S6K1 (Thr 389). Similarly, the silencing of RAPTOR and RICTOR decreased NGF expression by 56.7% and 52.4%, respectively, in RSC96 cells compared with the control siRNA treatment, which was accompanied by reduced phospho-S6K1 (Thr 389). The mTOR/S6K1 activator MHY1485 increased NGF expression by 28.7% and 17.1% 1day and 2day after stimulation, respectively, compared to the corresponding control group in RSC96 cells. Furthermore, DNMT1 was enhanced by 94.5% and 42.5% with mTOR pathway inhibitor (rapamycin and Torin 1, respectively) treatment for 3day compared with the control group. Additionally, the inhibition of DNMT1 with a chemical inhibitor or a specific shRNA plasmid upregulated NGF in RSC96 cells. In summary, our findings suggest that DNMT1 is the downstream target of the mTOR pathway and mediates the mTOR pathway inhibition-induced reduction in NGF expression in Schwann cells. Activation of the mTOR signaling pathway and/or inhibition of DNMT1 increased NGF expression, which may benefit patients suffering from NGF deficiencies, such as diabetic peripheral neuropathy.

Optimizing DNA transduction by selection of mutations that evade bacterial defense systems

ABSTRACTWe recently developed a platform where phage-transducing particles optimize DNA delivery to a wide range of hosts. Here, we use this platform to optimize DNA transduction into hosts that naturally restrict specific DNA sequences. We first show that a specific plasmid is restricted for transduction into a particular Salmonella strain. Using the platform, we select for a mutated plasmid that overcomes the restriction barrier. Insertion of the non-mutated sequence into a permissive plasmid restricts transduction. We further show that epigenetic modification enables the DNA to evade restriction by the putative defense system. Our results validate this straightforward genetic approach for optimization of DNA transduction into new hosts.

Abstract B008: PMEPA1 isoform specific regulation of androgen and TGF beta signaling in prostate cancer

Abstract Introduction and Objectives: The dysfunctions of androgen receptor (AR) and TGF-β signaling contribute to prostate tumorigenesis and cancer progression. Our laboratory has identified PMEPA1 as an androgen responsive gene with prostate abundance, as well as PMEPA1/NEDD4 and AR negative feedback loop in the regulation of AR levels in prostate cancer cells. In addition, it has also been established by other groups that PMEPA1 inhibits TGF-β signaling via a similar negative feedback loop as a TGF-β responsive gene. Five isoforms are transcribed from distinct promoters within the PMEPA1 locus. PMEPA1 isoforms were shown to have variations at the N-terminus of the protein. This study focuses on understanding of the expression and biologic functions of PMEPA1 isoforms in CaP. Methods: The PMEPA1 isoforms were evaluated for expressions in multiple CaP cell lines, trensfectant derivatives, and prostate tumors. LNCaP cells were treated with R1881 (0, 0.1, 1.0 nM) and DU-145 and PC-3 cells were treated with TGF-β (0, 5 and 25 ng/ml) for 24 hours. PMEPA1 isoform specific plasmids and siRNAs were transfected into LNCaP, DU-145, and PC-3 cells individually. Cell proliferation was analyzed by cell counting, cell plating efficiency assay, and soft agar assay. The protein levels of PMEPA1 isoforms, AR, PSA, TGF-β receptor I, Smad2 were detected by immunoblotting, and the transcript levels of KLK3 (PSA), TGF-β responsive genes including THBS1, NEDD9 and COL1A1 were evaluated by QRT-PCR. The TGF-β signaling activity was measured by SMAD reported dual-luciferase assay. The transcript levels of PMEPA1-252, PMEPA3-287-STAG1, AR, PSA in 120 matched human benign and malignant frozen tissue were evaluated with Q-PCR. Results: The expressions of PMEPA1-252 and PMEPA2-344 isoforms were restricted to androgen-responsive prostate cancer cells in comparison to broader expression pattern of other isoforms (PMEPA3-287/STAG1, PMEPA4-259 and PMEPA5-237). The expression of PMEPA1-2 was androgen regulated, whereas expression of PMEPA 3-5 was regulated by TGF-β. Only PMEPA1-252 inhibited cell growth of LNCaP, DU-145 and PC-3 cells. In contrast, PMEPA2-4 promoted cell growth of DU-145 and PC-3 cells. Only PMEPA1-252 mediated AR protein degradation and inhibited AR signaling. In contrast, PMEPA3-287-STAG1 and PMEPA4-259 inhibited TGF-β signaling luciferase activity and transcript levels of THBS1, NEDD9 and COL1A1. All PMEPA1 isoforms were found to have no effects on protein level of TGF-β receptor I. Moreover, the deletion mutants of N-terminus and transmembrane domains interrupted the isoform specific inhibitory effects on AR/TGF-β signaling. The transcript level of PMEPA1-252 was higher than PMEPA3-287-STAG1 in benign prostate tissue. Different from PMEPA1-252, the expression of PMEPA3-287-STAG1 was not found to decrease in prostate tumor tissue. There was no significant correlation between the expression of PMEPA1-252 and PMEPA3-287-STAG1 in prostate tissue. The transcript of PMEPA1-252 was associated with PSA, but the expression of PMEPA3-287-STAG1 was found not to be associated with PSA or AR in CaP cells. Conclusions: The PMEPA isoforms appear to underscore distinct biologic functions in the context of androgen and TGF-β signaling. Intensively studied PMEPA1-252 was specific for AR degradation in prostate cancer cells and was consistent with previous observations of association of AR upregulation with loss of PMEPA1 in prostate cancer. The roles of PMEPA1 isoforms need to be better defined in prostate cancer and other cancers. Funding: This study was supported by CPDR, USUHS, HU0001-10-2-0002 to I.L.R. Citation Format: Hua Li, Shashwat Sharad, Lakshmi Ravindranath, Gyorgy Petrovics, Yongmei Chen, Alagarsamy Srinivasan, Inger Rosner, Albert Dobi, Shiv Srivastava. PMEPA1 isoform specific regulation of androgen and TGF beta signaling in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B008.

The role of E2F1-topoIIβ signaling in regulation of cell cycle exit and neuronal differentiation of human SH-SY5Y cells

This study aims to test the role of E2F1-topoIIβ signaling in neuronal differentiation of SH-SY5Y cells. With retinoic acid (RA) induction, a high percentage of cells were found to be arrested at the G0/G1 phase, with decreased levels of cyclinD1, CDK4, phosphorylation status of pRb and E2F1, in addition to an elevated level of p27. The cells were shown to differentiate into neuronal phenotypes characterized by highly expressed neuronal markers, MAP2 and enriched topoIIβ, and remarkable neurite outgrowth. Exogenously forced E2F1 expression with a specific E2F1 plasmid led to suppression of topoIIβ expression and disruption of the neuronal differentiation of SH-SY5Y cells. On further examination using the ChIP assay, we found that E2F1 bound directly to the promoter region of topoIIβ, and its binding ability was inversely correlated with topoIIβ expression in response to RA induction. Thus, our findings suggest that E2F1-topoIIβ signaling may play a role in regulation of cell cycle exit and neuronal differentiation.

Is blaCTX-M-1 Riding the Same Plasmid Among Horses in Sweden and France?

A predominance of the blaCTX-M-1/IncHI1 plasmid combination in horses has been reported in Czech-Republic, Denmark, and The Netherlands. To clarify a possible specific plasmid epidemiology of blaCTX-M-1 in horses in a European perspective, a collection of 74 extended-spectrum beta-lactamase-producing Escherichia coli recovered from diseased horses in France and Sweden during the period 2009-2014 was investigated in respect of their genetic relatedness, plasmid content, and molecular features. Overall, 80% of E. coli isolates from diseased horses harbored blaCTX-M-1 on large IncHI1 plasmids with plasmid sequence type (pST) 2 and pST9 more prevalent in Sweden and France, respectively. In French isolates, IncI1/pST3 plasmids were also identified. The CTX-M-1-producing E. coli belonged principally to the clonal complex 10. ST641, together with its single locus variant, and ST1730 constituted two other major groups. The rep-PCR clustering highlighted a clonal dissemination of E. coli CTX-M-1 producers in different regions of the same country and during several years. The STs found in our isolates were also reported in The Netherlands, suggesting a common source of contamination in Europe, although only further cooperative investigation will clarify this issue. On the other hand, the spread of IncI1/pST3 plasmids among horses constitutes another worrisome issue considering their successful spread in other animal hosts such as chicken or bovines. Monitoring evolution and propagation of antimicrobial resistance in equine environment is a priority to avoid further propagation of antimicrobial resistance mechanisms threatening human and animal health.