Transfer Of Genetic Material Research Articles

Use of static overpressure to assess the role of acoustic cavitation in ultrasound-mediated gene transfection

Ultrasound-mediated DNA delivery (UDD) is a technique that utilizes ultrasound to aid the transfer of genetic materials into bacterial cells of interest (either in their planktonic or biofilm modes), resulting in new functionalities being displayed by the target microorganisms in-situ. The physical mechanism of UDD is poorly understood, which hampers its widespread adoption and scaling. To investigate the role of cavitation in UDD, we utilized a static pressure vessel designed to suppress cavitation while leaving other acoustic effects unaltered. Pseudomonas putida UWC1, a soil bacterium that is not naturally competent, was suspended in water and exposed to 40 kHz burst-mode ultrasound (400 cycle burst, 10 Hz repetition frequency, 60 s total) at an ambient pressure of either 1 bar or 9.5 bars. Noise diagnostics yielded inertial cavitation levels and transformed cells were quantified using a doubly selective reporter plasmid DNA. Samples exposed at 9.5 bars overpressure exhibited a drastic reduction in both cavitation activity and the number of transformed cells compared to those exposed at 1 bar. (Cell viability was not affected by pressure change.) The fact that transformation rates are reduced significantly when cavitation is suppressed suggests that acoustic cavitation is the major contributing factor in UDD.

Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction and protecting transductants from phage lysis.

Phage-inducible chromosomal islands (PICIs) are a widespread family of highly mobile genetic elements that disseminate virulence and toxin genes among bacterial populations. Since their life cycle involves induction by helper phages, they are important players in phage evolution and ecology. PICIs can interfere with the lifecycle of their helper phages at different stages resulting frequently in reduced phage production after infection of a PICI-containing strain. Since phage defense systems have been recently shown to be beneficial for the acquisition of exogenous DNA via horizontal gene transfer, we hypothesized that PICIs could provide a similar benefit to their hosts and tested the impact of PICIs in recipient strains on host cell viability, phage propagation and transfer of genetic material. Here we report an important role for PICIs in bacterial evolution by promoting the survival of phage-mediated transductants of chromosomal or plasmid DNA. The presence of PICIs generates favorable conditions for population diversification and the inheritance of genetic material being transferred, such as antibiotic resistance and virulence genes. Our results show that by interfering with phage reproduction, PICIs can protect the bacterial population from phage attack, increasing the overall survival of the bacterial population as well as the transduced cells. Moreover, our results also demonstrate that PICIs reduce the frequency of lysogenization after temperate phage infection, creating a more genetically diverse bacterial population with increased bet-hedging opportunities to adapt to new niches. In summary, our results identify a new role for the PICIs and highlight them as important drivers of bacterial evolution.

Benefits and Shortcomings of Laboratory Model Systems in the Development of Genetic Therapies.

Gene therapeutic approaches promise treatment or even a cure of diseases that were previously untreatable. Retinal gene therapies tested in clinical trials comprise a wide range of different strategies, including gene supplementation therapies, in vivo gene editing, modulation of splicing mechanisms, or the suppression of gene expression. To guarantee efficient transfer of genetic material into the respective target cells while avoiding major adverse effects, the development of genetic therapies requires appropriate in vitro model systems that allow tests of efficacy and safety of the gene therapeutic approach. In this review, we introduce various in vitro models of different levels of complexity used in the development of genetic therapies and discuss their respective benefits and shortcomings using the example of adeno-associated virus-based retinal gene therapy.

Cryptic Prophages Contribution for Campylobacter jejuni and Campylobacter coli Introgression.

Campylobacter coli and C. jejuni, the causing agents of campylobacteriosis, are described to be undergoing introgression events, i.e., the transference of genetic material between different species, with some isolates sharing almost a quarter of its genome. The participation of phages in introgression events and consequent impact on host ecology and evolution remain elusive. Three distinct prophages, named C. jejuni integrated elements 1, 2, and 4 (CJIE1, CJIE2, and CJIE4), are described in C. jejuni. Here, we identified two unreported prophages, Campylobacter coli integrated elements 1 and 2 (CCIE1 and CCIE2 prophages), which are C. coli homologues of CJIE1 and CJIE2, respectively. No induction was achieved for both prophages. Conversely, induction assays on CJIE1 and CJIE2 point towards the inducibility of these prophages. CCIE2-, CJIE1-, and CJIE4-like prophages were identified in a Campylobacter spp. population of 840 genomes, and phylogenetic analysis revealed clustering in three major groups: CJIE1-CCIE1, CJIE2-CCIE2, and CJIE4, clearly segregating prophages from C. jejuni and C. coli, but not from human- and nonhuman-derived isolates, corroborating the flowing between animals and humans in the agricultural context. Punctual bacteriophage host-jumps were observed in the context of C. jejuni and C. coli, and although random chance cannot be fully discarded, these observations seem to implicate prophages in evolutionary introgression events that are modulating the hybridization of C. jejuni and C. coli species.

Stabilizing bacterial conjugation via conjugation junction proteins

Bacterial conjugation mediates the transfer of genetic material among bacterial communities. The genetic material transferred will contain accessory genes encoding for virulence and antibiotic resistance. The mechanism in which two bacterial cells undergoing conjugation stabilize is not well understood. Previous studies have shown that two proteins, OmpK36 and TraN, found on the outer membrane of recipient and donor cells, respectively, of Klebsiella pneumoniae interact with each other to support efficient conjugation.

A hybrid multifactorial evolutionary algorithm and firefly algorithm for the clustered minimum routing cost tree problem

The clustered minimum routing cost tree (CluMRCT) problem is a recent problem with a wide range of real-life applications, especially in designing computer networks with peer-to-peer architecture. Many multifactorial evolutionary algorithms have been proposed to solve multiple CluMRCT problems simultaneously. However, these algorithms only function effectively on complete graphs with small-and-medium sizes. Moreover, the blindness and randomness in the transfer of genetic materials cause a reduction in the exploitation ability and make these algorithms ineffective to solve low-similarity tasks. This paper proposes a hybrid multitasking algorithm named multifactorial firefly algorithm, which integrates the firefly algorithm’s strong exploitation ability to enhance the self-evolution of each task when facing low-similarity tasks while improving inter-task knowledge transfers by delivering higher-quality solutions. Also, the proposed algorithm is equipped with new encoding and decoding to focus more on potential search areas on both complete and sparse graphs. The experiments and Wilcoxon signed-rank tests were conducted on various instances to verify our proposal with several state-of-the-art methods. The results portrayed that the proposed encoding scheme helped multitasking algorithms improve solution quality by 32% on average. Besides, the statistical test values proved the superiority of the proposed hybrid algorithm in terms of solution quality and convergence trend.

Desvendando mecanismos relacionados a anormalidades meióticas em Crotalaria spectabilis Roth

ABSTRACT The identification of epigenetic marks associated with problems in the meiotic process can enlighten the mechanisms underlying the irregularities and the impacts in the genetic constitution of gametes. Therefore, this study aimed to verify the relationship between the pattern of phosphorylation in serine 10 of histone H3 (H3S10ph), a (peri) centromeric epigenetic mark, with meiotic abnormalities in a wild population of Crotalaria spectabilis Roth. The main abnormalities observed were transfer of genetic material through cytoplasmatic connections, DNA elimination and abnormal spindle array. Different forms of elimination (chromatin fragmentation, ring formation, lagging chromosomes and micronuclei) were observed from the early phases until tetrad formation. The eliminated chromatin was either positive or negative for the immunosignal of H3S10ph, so it may be occurring elimination of acentric fragments, as well as of chromosomes with active or inactive centromeres. Therefore, dysfunctional centromere is not the only candidate cause for elimination. The transfer of genetic material and the abnormal spindle arrays are evidence that this population can produce aneuploid gametes and 2n pollen grains.

Computational Methods for the Discovery and Annotation of Viral Integrations.

The transfer of genetic material between viruses and eukaryotic cells is pervasive. Somatic integrations of DNA viruses and retroviruses have been linked to persistent viral infection and genotoxic effects. Integrations into germline cells, referred to as Endogenous Viral Elements (EVEs), can be co-opted for host functions. Besides DNA viruses and retroviruses, EVEs can also derive from nonretroviral RNA viruses, which have often been observed in piRNA clusters. Here, we describe a bioinformatic framework to annotate EVEs in a genome assembly, study their widespread occurrence and polymorphism and identify sample-specific viral integrations using whole genome sequencing data.

Homologous Recombination as a Fundamental Genome Surveillance Mechanism during DNA Replication.

Accurate and complete genome replication is a fundamental cellular process for the proper transfer of genetic material to cell progenies, normal cell growth, and genome stability. However, a plethora of extrinsic and intrinsic factors challenge individual DNA replication forks and cause replication stress (RS), a hallmark of cancer. When challenged by RS, cells deploy an extensive range of mechanisms to safeguard replicating genomes and limit the burden of DNA damage. Prominent among those is homologous recombination (HR). Although fundamental to cell division, evidence suggests that cancer cells exploit and manipulate these RS responses to fuel their evolution and gain resistance to therapeutic interventions. In this review, we focused on recent insights into HR-mediated protection of stress-induced DNA replication intermediates, particularly the repair and protection of daughter strand gaps (DSGs) that arise from discontinuous replication across a damaged DNA template. Besides mechanistic underpinnings of this process, which markedly differ depending on the extent and duration of RS, we highlight the pathophysiological scenarios where DSG repair is naturally silenced. Finally, we discuss how such pathophysiological events fuel rampant mutagenesis, promoting cancer evolution, but also manifest in adaptative responses that can be targeted for cancer therapy.

Genomic Analysis of Global Staphylococcus argenteus Strains Reveals Distinct Lineages With Differing Virulence and Antibiotic Resistance Gene Content.

Infections due to Staphylococcus argenteus have been increasingly reported worldwide and the microbe cannot be distinguished from Staphylococcus aureus by standard methods. Its complement of virulence determinants and antibiotic resistance genes remain unclear, and how far these are distinct from those produced by S. aureus remains undetermined. In order to address these uncertainties, we have collected 132 publicly available sequences from fourteen different countries, including the United Kingdom, between 2005 and 2018 to study the global genetic structure of the population. We have compared the genomes for antibiotic resistance genes, virulence determinants and mobile genetic elements such as phages, pathogenicity islands and presence of plasmid groups between different clades. 20% (n = 26) isolates were methicillin resistant harboring a mecA gene and 88% were penicillin resistant, harboring the blaZ gene. ST2250 was identified as the most frequent strain, but ST1223, which was the second largest group, contained a marginally larger number of virulence genes compared to the other STs. Novel S. argenteus pathogenicity islands were identified in our isolates harboring tsst-1, seb, sec3, ear, selk, selq toxin genes, as well as chromosomal clusters of enterotoxin and superantigen-like genes. Strain-specific type I modification systems were widespread which would limit interstrain transfer of genetic material. In addition, ST2250 possessed a CRISPR/Cas system, lacking in most other STs. S. argenteus possesses important genetic differences from S. aureus, as well as between different STs, with the potential to produce distinct clinical manifestations.

2021 Joseph W. Richards Fellowship – Summary Report: Toward On-Chip Electronic Monitoring of Bacterial Conjugation

Intercellular conjugation is a primary communication method by which bacteria transfer genetic material from one cell to another. Because conjugation mediates the transfer of genetic material, it enables the spread of antibiotic resistance and other virulence factors within a population. Current techniques for its study have low throughput and require extensive sample preparation, making real-time monitoring of conjugation dynamics challenging.To address these shortcomings, we are exploring two-dimensional (2D) materials as a strain-sensitive platform for on-chip, electronic monitoring of conjugation events.

Recombinant Transcription Factors (TFs) Fused to ZEBRA Minimal Transduction Domain (MD) for Modulation of mRNA Transcripts.

Transcription factors (TFs) are key players in the control of gene expression and consequently all major cellular process, ranging from cell fate determination to cell cycle control and response to the environment.In particular cases, their ectopic expression has shown great promise in cell reprogramming for regenerative medicine, ontogenesis studies, and cell modeling. The current reprogramming methods mainly rely on gene transfer, therefore require technological improvements to limit genetic imprinting and improve safety. Direct protein delivery could represent an attractive alternative. Cell-penetrating peptides (CPPs) fused to recombinant TFs or other proteins involved in the epigenetic definition of cells have great potential in this context. We have thus developed the direct vectorization of Oct4, Sox2, or Nanog TFs and the posttranscriptional regulatory RNA-binding protein Lin28a by using the minimal transduction domain (MD11) of Epstein-Barr virus ZEBRA protein.This section describes the molecular cloning and production of different TFs fused to ZEBRA MD11 domain in the E. coli expression system. We also include the optimized purification conditions for each recombinant protein. The treatment of primary fibroblasts as well as cord blood-derived hematopoietic stem cells is also described. Finally, the transcriptional activation of the target genes following the transfer of TFs analyzed by quantitative PCR is presented.Our work primarily finds applications for advanced medicinal products, an area that requires novel therapy designs and delivery systems devoid of genetic material transfer to improve safety.

Ultrasonic particles: An approach for targeted gene delivery.

Gene therapy has been widely investigated for the treatment of genetic, acquired, and infectious diseases. Pioneering work utilized viral vectors; however, these are suspected of causing serious adverse events, resulting in the termination of several clinical trials. Non-viral vectors, such as lipid nanoparticles, have attracted significant interest, mainly due to their successful use in vaccines in the current COVID-19 pandemic. Although they allow safe delivery, they come with the disadvantage of off-target delivery. The application of ultrasound to ultrasound-sensitive particles allows for a direct, site-specific transfer of genetic materials into the organ/site of interest. This process, termed ultrasound-targeted gene delivery (UTGD), also increases cell membrane permeability and enhances gene uptake. This review focuses on the advances in ultrasound and the development of ultrasonic particles for UTGD across a range of diseases. Furthermore, we discuss the limitations and future perspectives of UTGD.

Genome Analysis of Acinetobacter lwoffii Strains Isolated from Permafrost Soils Aged from 15 Thousand to 1.8 Million Years Revealed Their Close Relationships with Present-Day Environmental and Clinical Isolates.

Simple SummaryArctic ecosystems are an extreme habitat characterized by a negative average annual temperature and the presence of permafrost that occupies about 25% of the land. Permafrost can retain viable microorganisms for several million years and therefore it is a source of unique “ancient” microbes. Acinetobacter lwoffii are aerobic chemoorganotrophic bacteria widespread in a variety of natural and artificial environments, and have been reported as hospital pathogens associated with nosocomial infections. Here, we carried out a genome-wide analysis of five strains of A. lwoffii isolated from permafrost aged from 15 thousand to 1.8 million years. Surprisingly, we did not reveal genetic determinants that distinguish them from modern clinical and environmental A. lwoffii isolates. On the phylogenetic tree permafrost strains do not form a separate cluster, but are related to various clinical isolates. The genomes of clinical and permafrost strains contain similar mobile elements and prophages, which indicates an intense horizontal gene transfer. Like clinical isolates, permafrost strains harbored antibiotic resistance genes, although plasmids from the modern strains are enriched with antibiotic resistance genes compared to permafrost ones. The obtained results indicate that thawing of permafrost caused by global warming could release new potentially pathogenic strains of Acinetobacter into the modern biosphere.Microbial life can be supported at subzero temperatures in permafrost up to several million years old. Genome analysis of strains isolated from permafrost provides a unique opportunity to study microorganisms that have not previously come into contact with the human population. Acinetobacter lwoffii is a typical soil bacterium that has been increasingly reported as hospital pathogens associated with bacteremia. In order to identify the specific genetic characteristics of ancient permafrost-conserved strains of A. lwoffii and their differences from present-day clinical isolates, we carried out a genome-wide analysis of five strains of A. lwoffii isolated from permafrost aged from 15 thousand to 1.8 million years. Surprisingly, we did not identify chromosomal genetic determinants that distinguish permafrost strains from clinical A. lwoffii isolates and strains from other natural habitats. Phylogenetic analysis based on whole genome sequences showed that permafrost strains do not form a separate cluster and some of them are most closely related to clinical isolates. The genomes of clinical and permafrost strains contain similar mobile elements and prophages, which indicates an intense horizontal transfer of genetic material. Comparison of plasmids of modern and permafrost strains showed that plasmids from the modern strains are enriched with antibiotic resistance genes, while the content of genes for resistance to heavy metals and arsenic is nearly the same. The thawing of permafrost caused by global warming could release new potentially pathogenic strains of Acinetobacter.

Starvation-induced cell fusion and heterokaryosis frequently escape imperfect allorecognition systems in an asexual fungal pathogen

BackgroundAsexual fungi include important pathogens of plants and other organisms, and their effective management requires understanding of their evolutionary dynamics. Genetic recombination is critical for adaptability and could be achieved via heterokaryosis — the co-existence of genetically different nuclei in a cell resulting from fusion of non-self spores or hyphae — and the parasexual cycle in the absence of sexual reproduction. Fusion between different strains and establishment of viable heterokaryons are believed to be rare due to non-self recognition systems. Here, we investigate the extent and mechanisms of cell fusion and heterokaryosis in the important asexual plant pathogen Verticillium dahliae.ResultsWe used live-cell imaging and genetic complementation assays of tagged V. dahliae strains to analyze the extent of non-self vegetative fusion, heterokaryotic cell fate, and nuclear behavior. An efficient CRISPR/Cas9-mediated system was developed to investigate the involvement of autophagy in heterokaryosis. Under starvation, non-self fusion of germinating spores occurs frequently regardless of the previously assessed vegetative compatibility of the partners. Supposedly “incompatible” fusions often establish viable heterokaryotic cells and mosaic mycelia, where nuclei can engage in fusion or transfer of genetic material. The molecular machinery of autophagy has a protective function against the destruction of “incompatible” heterokaryons.ConclusionsWe demonstrate an imperfect function of somatic incompatibility systems in V. dahliae. These systems frequently tolerate the establishment of heterokaryons and potentially the initiation of the parasexual cycle even between strains that were previously regarded as “incompatible.”

Molecular evidence of introgression of Asian germplasm into a natural Castanea sativa forest in Spain

Abstract Evidence has been documented in Europe of introgression, the transfer of genetic material, between Asian chestnut species (Castanea crenata and C. mollissima) and the native C. sativa through spontaneous hybridization and subsequent backcrossing. However, the extent of this introgression has not been monitored in a particular forest stand or in adult and juvenile trees simultaneously. We assessed introgression in a natural C. sativa forest in northwest Spain and developed a reliable method to detect the presence of Asian germplasm of Castanea spp. A total of 34 adult trees and 42 saplings were genotyped at 13 SSRs in a forest where ink-disease-resistant C. crenata and C. mollissima seedlings had been planted in the 1940s. The 13 SSR loci selected were highly polymorphic and 115 different alleles were detected for the individuals sampled. Bayesian clustering identified two groups for C. sativa and one group each for C. mollissima and C. crenata. Within the forest, 70.6 per cent of adults and 28.6 per cent of juveniles were classified as pure C. sativa. Most juveniles were C. sativa × C. crenata (>40 per cent) and ca. 10 per cent were C. crenata × C. mollissima hybrids. Six new alleles private to C. crenata are reported here. The study quantifies, for the first time in Europe, introgression of Asian germplasm into a natural C. sativa forest. It also examines the extent of introgression in offspring and provides a method to detect exotic germplasm in C. sativa forests. We discuss why the forest studied may benefit from transfer of alleles involved in ink-disease resistance and why introgression will be detrimental to drought tolerance in offspring.

Encapsulation Strategies for Bacillus thuringiensis: From Now to the Future.

Bacillus thuringiensis (Bt) has been recognized for its high potential in the control of various agricultural pests. Developments in micro/nanotechnology have opened new perspectives for the production of more efficient formulations that can overcome some obstacles associated with its use in the field, such as formulation instability and loss of activity as a result of the degradation of pesticidal protein by its exposure to ultraviolet radiation, among other problems. This review describes current studies and recent discoveries related to Bt and processes for the encapsulation of Bt derivatives, such as Cry pesticidal proteins. Different techniques are described, such as extrusion, emulsion, spray drying, spray cooling, fluidized bed, lyophilization, coacervation, and electrospraying to obtain micro- and nanoparticulate systems. It is noteworthy that products based on microorganisms present less risk to the environment and non-target organisms. However, systematic risk assessment studies of these new Bt biopesticides are necessary, considering issues, such as interactions with other organisms, the formation of toxic secondary metabolites, or the interspecific transfer of genetic material. Given the great potential of these new formulations, a critical assessment is provided for their future use, considering the technological challenges that must be overcome to achieve their large-scale production for efficient agricultural use.

Gene Therapy Approaches

Gene therapy can be described broadly as the transfer of genetic material to control a disease or at least to enhance a patient's clinical status. The transformation of viruses into genetic shuttles is one of the core principles of gene therapy, which will introduce the gene of interest into the target tissue and cells. To do this, safe strategies have been invented, using many viral and non-viral vector delivery. Two major methods have emerged: modification in vivo and modification ex vivo. For gene therapeutic approaches which are focused on lifelong expression of the therapeutic gene, retrovirus, adenovirus, adeno-associated viruses are acceptable. Non-viral vectors are much less successful than viral vectors, but because of their low immune responses and their broad therapeutic DNA ability, they have advantages. The addition of viral functions such as receptor-mediated uptake and nuclear translocation of DNA may eventually lead to the development of an artificial virus in order to improve the role of non-viral vectors. For human use in genetic conditions, cancers and acquired illnesses, gene transfer techniques have been allowed. The ideal delivery vehicle has not been identified, although the accessible vector systems are capable of transporting genes in vivo into cells. Therefore, only with great caution can the present viral vectors be used in human beings and further progress in the production of vectors is required. Current progresses in our understanding of gene therapy approaches and their delivery technology, as well as the victors used to deliver therapeutic genes, are the primary goals of this review. For that reason, a literature search on PubMed and Google Scholar was carried out using different keywords.

The Distinctive Evolution of orfX Clostridium parabotulinum Strains and Their Botulinum Neurotoxin Type A and F Gene Clusters Is Influenced by Environmental Factors and Gene Interactions via Mobile Genetic Elements.

Of the seven currently known botulinum neurotoxin-producing species of Clostridium, C. parabotulinum, or C. botulinum Group I, is the species associated with the majority of human botulism cases worldwide. Phylogenetic analysis of these bacteria reveals a diverse species with multiple genomic clades. The neurotoxins they produce are also diverse, with over 20 subtypes currently represented. The existence of different bont genes within very similar genomes and of the same bont genes/gene clusters within different bacterial variants/species indicates that they have evolved independently. The neurotoxin genes are associated with one of two toxin gene cluster types containing either hemagglutinin (ha) genes or orfX genes. These genes may be located within the chromosome or extrachromosomal elements such as large plasmids. Although BoNT-producing C parabotulinum bacteria are distributed globally, they are more ubiquitous in certain specific geographic regions. Notably, northern hemisphere strains primarily contain ha gene clusters while southern hemisphere strains have a preponderance of orfX gene clusters. OrfX C. parabotulinum strains constitute a subset of this species that contain highly conserved bont gene clusters having a diverse range of bont genes. While much has been written about strains with ha gene clusters, less attention has been devoted to those with orfX gene clusters. The recent sequencing of 28 orfX C. parabotulinum strains and the availability of an additional 91 strains for analysis provides an opportunity to compare genomic relationships and identify unique toxin gene cluster characteristics and locations within this species subset in depth. The mechanisms behind the independent processes of bacteria evolution and generation of toxin diversity are explored through the examination of bacterial relationships relating to source locations and evidence of horizontal transfer of genetic material among different bacterial variants, particularly concerning bont gene clusters. Analysis of the content and locations of the bont gene clusters offers insights into common mechanisms of genetic transfer, chromosomal integration, and development of diversity among these genes.

Plastidial retrograde modulation of light and hormonal signaling: an odyssey.

The transition from an engulfed autonomous unicellular photosynthetic bacterium to a semiautonomous endosymbiont plastid was accompanied by the transfer of genetic material from the endosymbiont to the nuclear genome of the host, followed by the establishment of plastid-to-nucleus (retrograde) signaling. The retrograde coordinated activities of the two subcellular genomes ensure chloroplast biogenesis and function as the photosynthetic hub and sensing and signaling center that tailors growth-regulating and adaptive processes. This review specifically focuses on the current knowledge of selected stress-induced retrograde signals, genomes uncoupled 1 (GUN1), methylerythritol cyclodiphosphate (MEcPP), apocarotenoid and β-cyclocitral, and 3'-phosphoadenosine 5'-phosphate (PAP), which evolved to establish the photoautotrophic lifestyle and are instrumental in the integration of light and hormonal signaling networks to ultimately fashion adaptive responses in an ever-changing environment.