Somatic Genome Editing: Technical Challenges and Ethical Appraisal

The ability to modify the DNA sequences with molecular tools that employ nucleases has made it possible to edit genomes. Somatic genome editing is the procedure to alter the genome of somatic cells, making the changes introduced into the nucleotide sequence not inheritable. Powerful tools have been developed for therapeutic purposes to correct monogenic inherited disorders that often cause premature death and for which effective treatment options are not available. To ensure positive impact and minimise potential harms of these techniques, require to understand their limits in addition to apply values and principles that guide their use. This study reviews technical challenges of genome editing and appraises the Ethics of its application

Effects of human and porcine bile on the proteome of Helicobacter hepaticus

<p>Abstract</p> <p>Background</p> <p><it>Helicobacter hepaticus </it>colonizes the intestine and liver of mice causing hepatobiliary disorders such as hepatitis and hepatocellular carcinoma, and has also been associated with inflammatory bowel disease in children. In its habitat, <it>H. hepaticus </it>must encounter bile which has potent antibacterial properties. To elucidate virulence and host-specific adaptation mechanisms of <it>H. hepaticus </it>modulated by human or porcine bile, a proteomic study of its response to the two types of bile was performed employing two-dimensional gel electrophoresis (2-DE) and mass spectrometry.</p> <p>Results</p> <p>The 2-DE and mass spectrometry analyses of the proteome revealed that 46 proteins of <it>H. hepaticus </it>were differentially expressed in human bile, 18 up-regulated and 28 down-regulated. In the case of porcine bile, 32 proteins were differentially expressed of which 19 were up-regulated, and 13 were down-regulated. Functional classifications revealed that identified proteins participated in various biological functions including stress response, energy metabolism, membrane stability, motility, virulence and colonization. Selected genes were analyzed by RT-PCR to provide internal validation for the proteomic data as well as provide insight into specific expressions of motility, colonization and virulence genes of <it>H. hepaticus </it>in response to human or porcine bile.</p> <p>Conclusions</p> <p>Overall, the data suggested that bile is an important factor that determines virulence, host adaptation, localization and colonization of specific niches within host environment.</p

Characterisation of Campylobacter jejuni genes potentially involved in phosphonate degradation

Potential biological roles of the Campylobacter jejuni genes cj0641, cj0774c and cj1663 were investigated. The proteins encoded by these genes showed sequence similarities to the phosphonate utilisation PhnH, K and L gene products of Escherichia coli. The genes cj0641, cj0774c and cj1663 were amplified from the pathogenic C. jejuni strain 81116, sequenced, and cloned into pGEM-T Easy vectors. Recombinant plasmids were used to disrupt each one of the genes by inserting a kanamycin resistance (KmR) cassette employing site-directed mutagenesis or inverse PCR. Campylobacter jejuni 81116 isogenic mutants were generated by integration of the mutated genes into the genome of the wild-type strain. The C. jejuni mutants grew on primary isolation plates, but they could not be purified by subsequent passages owing to cell death. The mutant C. jejuni strains survived and proliferated in co-cultures with wild-type bacteria or in media in which wild-type C. jejuni had been previously grown. PCR analyses of mixed wild-type/mutant cultures served to verify the presence of the mutated gene in the genome of a fraction of the total bacterial population. The data suggested that each mutation inactivated a gene essential for survival. Rates of phosphonate catabolism in lysates of E. coli strain DH5α were determined using proton nuclear magnetic resonance spectroscopy. Whole-cell lysates of the wild-type degraded phosphonoacetate, phenylphosphonate and aminomethylphosphonate. Significant differences in the rates of phosphonate degradation were observed between lysates of wild-type E. coli, and of bacteria transformed with each one of the vectors carrying one of the C. jejuni genes, suggesting that these genes were involved in phosphonate catabolism

\u3cem\u3eHelicobacter pylori\u3c/em\u3e infection in Havana, Cuba. Prevalence and \u3cem\u3ecagA\u3c/em\u3e status of the strains

There is a great paucity of information about Helicobacter pylori infection in the countries of the Caribbean basin. Almost no studies have been performed to determine the prevalence, antibiotic resistance or virulence factors of the bacterium. To measure the prevalence of H. pylori infection among patients attending endoscopy in three clinics in Havana, Cuba, to evaluate clarithromycin resistance, and to determine the cagA status of the strains obtained. Endoscopy was performed and biopsies were obtained from 117 successive patients attending the Institute of Oncology, the Institute of Gastroenterology, and the Calixto Garcia Hospital in Havana, Cuba. Biopsies were maintained at –70 ºC before being cultured on three different media (two selective and one non-selective) and incubated for 7 days at 37 °C under a microaerobic atmosphere. The presence of H. pylori was identified by oxidase, catalase and urease activities. DNA was extracted, and PCR was performed with primers H2761676 which amplify a 397 bp fragment of the cagA gene. Clarithromycin susceptibility was measured by the gel diffusion method. The diagnoses of patients were: 1 gastric carcinoma; 19 duodenal ulcers; 8 gastric ulcers; and 89 non-ulcer dyspepsia, including (62) gastritis, (9) hiatal hernia,(2) biliary reflux, (1) gastric polyps, and (15) no abnormality. Among the 117 biopsies tested, 83 were H. pylori positive (70.9%). The cagA status determined for 35 cases gave a positive result in 31 cases (88.5%). Only 3% of the strains were resistant to clarithromycin. The prevalence of Helicobacter pylori infection in the symptomatic population of La Habana is the same as reported for other developing countries. Most strains were cagA positive and are likely harbour the cag pathogenicity island. The low resistance to clarithromycin in the strains studied probably reflects the low degree of use of the antibiotic in this population

Genetic microheterogeneity and phenotypic variation of Helicobacter pylori arginase in clinical isolates

BACKGROUND: Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. RESULTS: The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (>100-fold) in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3' end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. CONCLUSION: These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori

The complete genome sequence and analysis of the Epsilonproteobacterium \u3cem\u3eArcobacter butzleri\u3c/em\u3e

Arcobacter butzleri is a member of the epsilon subdivision of the Proteobacteria and a close taxonomic relative of established pathogens, such as Campylobacter jejuni and Helicobacter pylori. Here we present the complete genome sequence of the human clinical isolate, A. butzleri strain RM4018. Methodology/Principal Findings: Arcobacter butzleri is a member of the Campylobacteraceae, but the majority of its proteome is most similar to those of Sulfuromonas denitrificans and Wolinella succinogenes, both members of the Helicobacteraceae, and those of the deep-sea vent Epsilonproteobacteria Sulfurovum and Nitratiruptor. In addition, many of the genes and pathways described here, e.g. those involved in signal transduction and sulfur metabolism, have been identified previously within the epsilon subdivision only in S. denitrificans, W. succinogenes, Sulfurovum, and/or Nitratiruptor, or are unique to the subdivision. In addition, the analyses indicated also that a substantial proportion of the A. butzleri genome is devoted to growth and survival under diverse environmental conditions, with a large number of respiration-associated proteins, signal transduction and chemotaxis proteins and proteins involved in DNA repair and adaptation. To investigate the genomic diversity of A. butzleri strains, we constructed an A. butzleri DNA microarray comprising 2238 genes from strain RM4018. Comparative genomic indexing analysis of 12 additional A. butzleri strains identified both the core genes of A. butzleri and intraspecies hypervariable regions, where, 70% of the genes were present in at least two strains. Conclusion/Significance: The presence of pathways and loci associated often with non-hostassociated organisms, as well as genes associated with virulence, suggests that A. butzleri is a free-living, water-borne organism that might be classified rightfully as an emerging pathogen. The genome sequence and analyses presented in this study are an important first step in understanding the physiology and genetics of this organism, which constitutes a bridge between the environment and mammalian hosts

The Complete Genome Sequence and Analysis of the Epsilonproteobacterium Arcobacter butzleri

BACKGROUND: Arcobacter butzleri is a member of the epsilon subdivision of the Proteobacteria and a close taxonomic relative of established pathogens, such as Campylobacter jejuni and Helicobacter pylori. Here we present the complete genome sequence of the human clinical isolate, A. butzleri strain RM4018. METHODOLOGY/PRINCIPAL FINDINGS: Arcobacter butzleri is a member of the Campylobacteraceae, but the majority of its proteome is most similar to those of Sulfuromonas denitrificans and Wolinella succinogenes, both members of the Helicobacteraceae, and those of the deep-sea vent Epsilonproteobacteria Sulfurovum and Nitratiruptor. In addition, many of the genes and pathways described here, e.g. those involved in signal transduction and sulfur metabolism, have been identified previously within the epsilon subdivision only in S. denitrificans, W. succinogenes, Sulfurovum, and/or Nitratiruptor, or are unique to the subdivision. In addition, the analyses indicated also that a substantial proportion of the A. butzleri genome is devoted to growth and survival under diverse environmental conditions, with a large number of respiration-associated proteins, signal transduction and chemotaxis proteins and proteins involved in DNA repair and adaptation. To investigate the genomic diversity of A. butzleri strains, we constructed an A. butzleri DNA microarray comprising 2238 genes from strain RM4018. Comparative genomic indexing analysis of 12 additional A. butzleri strains identified both the core genes of A. butzleri and intraspecies hypervariable regions, where <70% of the genes were present in at least two strains. CONCLUSION/SIGNIFICANCE: The presence of pathways and loci associated often with non-host-associated organisms, as well as genes associated with virulence, suggests that A. butzleri is a free-living, water-borne organism that might be classified rightfully as an emerging pathogen. The genome sequence and analyses presented in this study are an important first step in understanding the physiology and genetics of this organism, which constitutes a bridge between the environment and mammalian hosts

The Vaginal Microbiome during Pregnancy in Health and Disease

This study appraises the progress in the understanding of the composition of the vaginal microflora with a focus on the microbiome during pregnancy. This knowledge is presented with the background of the global health contribution, along with the importance of these microbial communities to pregnancy. A brief review of current methods employed to investigate the structure of these microbial populations is included. Two types of studies, cross-sectional and longitudinal, have been used to characterise the vaginal microbiota; both types are reviewed since they provide information that serves to piece together a more complete picture of the vaginal microflora and its changes during pregnancy. The identity of microbes present in the vagina are examined in the context of health and disease, and, more specifically, in the setting of pregnancy outcomes. The protective role of lactobacilli in maintaining a healthy vaginal environment is evaluated, with analyses of the different roles of various Lactobacillus spp. Classifications of the vaginal microbiota into vagitypes in non-pregnant and pregnant women are discussed. The associations of specific taxa with three adverse pregnancy results, namely, miscarriage, stillbirth, and preterm birth, are examined in some detail. Longitudinal studies investigating changes in the bacterial community composition and taxa abundance demonstrate that this microbiota decreases in richness and diversity relative to those present in non-pregnant microbiomes. Notwithstanding the significant effort made to characterise the vagina bacterial microbiota, a large number of issues remain to be fully understood

Posthumanism: Creation of \u27New Men\u27 through technological innovation

he posthumanist project proposes directing the evolution of human beings by promoting their improvement through technological means to create a variety of entities that will have few or no common characteristics with current humans. Its agenda is extremely broad and this study mostly addresses enhancement of the human organism through genetic modification techniques. An overview of posthumanist values and a brief discussion of its philosophical background provide a framework to understand its ideals. Genetics and ethics are employed to assess some claims of the posthumanist program of creating evolved humans; in particular, the capabilities and limitations of techniques for somatic and germline genome editing. Consequences of the creation of posthumans are discussed in relation to accepted current human beings and values. It is concluded that the posthumanist program rests on a large number of hypotheses without sufficient evidence and with little or no consideration of the consequences of its implementation

Responses of Four Campylobacterales to Cadmium Stress

Abstract unavailable for this Book Chapter Description of the Book, Metal Ions in Biology and Medicine: The aims of this 10th volume are to present the latest scientific progress in the fields of biochemistry, chemistry and cell biology using examples such as the therapeutic applications of complex metal ligands. Progress in our understanding of the roles played by metals in cells has resulted in the rise of metal-based medicinal products which are currently a major research field. This book provides the latest findings on: - the effects of metals in cancer, infectious disease, endocrine disorders and all other diseases affecting human beings. - toxicological aspects of metal ions - metal-based medicinal products [Retrieved from the publisher\u27s website] ISBN: 978-274200714